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Pathogenesis of coxasackievirus B3 induced myocarditis and systemic disease : viral localization, direct… Carthy, Christopher Michael 2002

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PATHOGENESIS OF COXSACKIEVIRUS B3 INDUCED MYOCARDITIS AND SYSTEMIC DISEASE: VIRAL LOCALIZATION, DIRECT INJURY AND ACTIVATION OF HOST CELL DEATH MACHINERY By CHRISTOPHER MICHAEL CARTHY B.Sc, Queen's University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pathology and Laboratory Medicine; Faculty of Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 2002 © Christopher Michael Carthy, 2002 11/07/02 10:54 FAX 604 822 9587 SPECIAL COLLECTIONS 12)003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P/rTthiCCl AiW) LMod/Wl fteoltifrC The University of British Columbia Vancouver, Canada Date N0^ 07/WL DE-6 (2/88) PATHOGENESIS OF COXSACKIEVIRUS B3 INDUCED MYOCARDITIS AND SYSTEMIC DISEASE: VIRAL LOCALIZATION, DIRECT-INJURY AND ACTIVATION OF HOST CELL DEATH MACHINERY Christopher M. Carthy University of British Columbia, 2002 Advisor: Bruce M. McManus, M.D., Ph.D. Summary Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology following infection. Work presented in this thesis will examine the nature of these cytopathic changes in vivo in myocarditis susceptible and resistant mice, and in vitro in HeLa cells, a common cell line used to dissect coxsackievirus lifecycle and host interaction. In mice, C V B 3 infection caused a widespread systemic disease including infection of the heart, pancreas, liver, brain, spleen, salivary glands, lung and kidney. Adjacent sections stained for viral RNA by in situ hybridisation and tissue injury by TUNEL have demonstrated that infected cells are directly injured prior to activation of the cellular arm of the immune system. In the lymphoid organs, there is increased apoptosis that may be responsible for the lymphoid involution characteristically noted early following infection. The association of virus with lymphocytes may interfere with the normal immune response. There is a depression of IL-2 and IL-4 protein levels in infected mice as compared to sham treated animals. In HeLa cells, multiple caspases are processed and activated following the release of cytochrome c from the intermitochondrial space to the cytosol. Caspase-specific substrates are cleaved, but these events are not solely responsible for the degenerative morphologic changes noted following infection. Using ZVAD.fmk or cells stably ii transfected with Bcl-2 and Bcl-xL, it was further demonstrated that cytopathic effect is not caused by caspase activation. Activation of caspases, however, plays a role in loss of cell viability and the release of the progeny virus from within the cell. Cytopathic effect is probably due to viral protease mediated degradation of host cell structural proteins including focal adhesion kinase that is cleaved by a protease not inhibited by ZVAD.fmk. cDNA microarrays were used to profile the host gene response in the myocardium at days 3, 9, and 30 post infection to dissect the various stages of disease progression, including, direct viral injury, host specific immune response and viral clearance, and late stage cardiac remodelling. From a limited repertoire of genes, we identified 169 known genes that were significantly up or down regulated at one or more of the three timepoints. This study was one of the first in vivo gene expression models examining cardiac disease progression in mice over time. Direct extensions of this thesis includes interrogation of direct host and viral protein-protein interactions, examination of mitogen activated protein kinase signalling pathways and relationship to cell viability and virus replication, and further dissection of the effect of viral proteases on degradation of various host cellular proteins and effect on host cell homeostasis. i i i TABLE OF CONTENTS Abstract ii Table of contents iv List of tables vii List of figures viii Abreviations xii Acknowledgements xvi 1 PICORNAVIRUSES AND VIRAL LIFE-CYCLE EVENTS 1 1.1. Family Picornaviridae, and genus enterovirus 1 1.2. Coxsackievirus B group: Host range and virulence factors 2 1.3. Viral life cycle 4 1.3.1. Viral receptors 5 1.3.2. RNA internalization, translation, and transcription 9 1.3.3. Disruption of host cell homeostasis 13 2 ROLE OF COXSACKIEVIRUS B3 IN MYOCARDITIS AND SYSTEMIC DISEASE. 18 2.1. Myocarditis 18 2.1.1. Viral myocarditis 18 2.1.2. Enterovirus myocarditis 19 2.1.3. Pathogenesis of enterovirus-induced myocarditis 20 2.1.4. Role of the immune system 21 2.2. The link between myocarditis and dilated cardiomyopathy 30 3 CELL DEATH 33 3.1. Apoptosis and necrosis: Perspectives, morphologic characteristics, ion regulation, and common Themes 33 3.2. Biochemical aspects of cell death 34 3.2.1. The caspases 36 3.2.2. Caspase activation 38 3.2.3. Cell surface signaling 38 3.2.4. Mitochondrial induced death 41 3.2.5. Effector caspases and cross talk between cell surface receptor activation and the mitochondria.... 41 3.3. Regulation of cell death 42 3.4. Caspase specific cleavage of substrates 54 iv 4 VIRUSES AND CELL DEATH SIGNALLING 58 4.1. Cytopathic effect: How is it defined? 58 4.2. Apoptosis as an innate and adaptive antiviral defense mechanism of the immune system 58 4.3. Virus interactions with host-cell death machinery 59 4.3.1. Viruses and viral proteins that inhibit apoptosis 60 4.3.2. Viruses and viral proteins that induce apoptosis 67 4.4. Apoptosis and Picornaviruses: Morphologic and biochemical evidence 73 5 CELL DEATH AND THE MYOCARDIUM 75 5.1. Cell death in the myocardium 75 5.2. The cardiac myocyte: Properties and morphology of death 75 5.3. Apoptosis and heart disease 77 5.4. Intrinsic inhibitors of myocyte apoptosis 83 6 RESEARCH FOCUS 89 7 HYPOTHESIS, SPECIFIC AIMS AND MAJOR QUESTIONS 90 8 EXPERIMENTAL DESIGN 91 8.1. Aim #1 91 8.2. Aim #2 91 8.3. Aim #3 92 8.4. Aim #4 93 9 RESULTS 94 9.1. Aim #1 94 9.2. Aim #2 106 9.3. Aim #3 117 9.4. Aim #4 139 10 DISCUSSION 154 10.1. Aim#1 154 10.2. Aim #2 162 10.3. Aim #3 165 10.4. Aim #4 170 11 SUMMARY AND MAJOR CONCLUSIONS 179 11.1. Aim#1 179 11.2. Aim #2 179 11.3. Aim #3 180 v 11.4. Aim #4 181 12 FUTURE DIRECTIONS 182 13 MATERIAL AND METHODS 186 13.1. Coxsackievirus B3 186 13.1.1. Propagation and handling of virus 186 13.2. HeLa cell culture 186 13.3. Generation and culture of stable-transfected cell lines 186 13.4. Collection of total cell lysate 187 13.5. Collection of cytosolic extracts 187 13.6. Caspase activation assays 187 13.7. Western blotting 188 13.8. MTT for cell viability 188 13.9. Morphology 188 13.10. Flow cytometry 188 13.11. Animal handling and sacrifice, and tissue distribution and evaluation 189 13.12. Tissue histopathology 189 13.13. Virus content of tissues and cells 190 13.14. Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end- labeling (TUNEL) 190 13.15. In situ hybridization 191 13.16. RNA isolation/cDNA probes 191 13.17. DNA microarray 191 13.18. Statistical analysis 191 14 REFERENCES 193 vi LIST OF TABLES Table 1 Evidence of Enterovirus genome in IDC Patients 31 Table 2 Myocarditis top 30: the 5 most up and down regulated transcripts at day 3, 9, and 30 post-infection 148 vii LIST OF FIGURES Figure 1 Schematic of the CVB3 lifecycle in permissive cells 6 Figure 2 Schematic of points of intersection between CVB3 and host protein partners during CVB3 replication in permissive cells 7 Figure 3 Schematic of CVB3 translation and polyprotein processing 10 Figure 4 Schematic of viral protease degradation of host cellular proteins: Disruption of normal homeostasis 15 Figure 5 Schematic of apoptosis induction: mitochondrial vs cell membrane signaling 35 Figure 6 Schematic of caspase processing 37 Figure 7 Schematic of the TNF receptor gene superfamily and the TRAIL receptors 39 Figure 8 Schematic of the Bcl-2 family of proteins 44 Figure 9 Schematic of cellular proteins that inhibit caspases - direct and indirect 47 Figure 10 Schematic of activation of NFKB pathway 49 Figure 11 Schematic of activation of Akt/PKB pathway 51 Figure 12 Schematic of p53 regulation of apoptosis 53 Figure 13 Rb protein regulation of proliferation and apoptosis 62 Figure 14 Schematic summarizing points of intersection where viral proteins can inhibit apoptosis 68 Figure 15 Schematic summarizing the potential intrinsic anti-apoptotic features of myocytes 88 Figure 16 Localization of positive strand viral RNA in A/J mice 24 h post-CVB3 infection 95 Figure 17 Localization of positive and negative strand viral RNA in A/J mice at day 4 post-CVB3 infection 96 Figure 18 In situ hybridization for CVB3 viral RNA localization and plaque assay for infectious virus in hearts of adolescent A/J and C57BL/6J mice 98 v i i i Figure 19 In situ hybridization for CVB3 viral RNA and TUNEL for tissue injury in spleen and thymus 102 Figure 20 TUNEL and flow cytometry in thymus following infection 103 Figure 21 IL2 and IL4 expression in murine hearts and spleens for CVB3 infected AJ and C57BL/6J mice at days 3 to 7 post-infection 104 Figure 22 In situ hybridization for CVB3 viral RNA and TUNEL for tissue injury in heart of infected mice 105 Figure 23 In situ hybridization for CVB3 viral RNA and TUNEL for tissue injury in liver, pancreas, and salivary gland of infected mice 107 Figure 24 In situ hybridization for CVB3 viral RNA localization and plaque assay for infectious virus in hearts of 4 and 10-week-old A/J mice following infection with CVB3 108 Figure 25 Infectious virus and ISH positivity in organs of 4 week and 10 week old AJ mice and 4W old mice at day 4 pi 109 Figure 26 Dynamics of viral infection in HeLa cells: CVB3 protein synthesis, infectious virus and morphology changes 111 Figure 27 Caspase 3 degradation following CVB3 infection of HeLa cells and increase in caspase 3 activity 112 Figure 28 Degradation of PARP and DFF in HeLa cells during CVB3 infection.. 114 Figure 29 ZVAD.fmk treatment of HeLa cells inhibits caspase activation, and substrate cleavage following CVB3 infection 115 Figure 30 ZVAD.fmk has no effect on cytopathic effect following CVB3 infection of HeLa cells 116 Figure 31 Activation of multiple caspases following CVB3 infection in HeLa cells 118 Figure 32 Activation of multiple caspases following TRAIL treatment of HeLa cells 120 Figure 33 Activation of multiple caspases following PDT treatment of HeLa cells 122 Figure 34 Biochemical events of cell death following CVB3 infection of HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells 123 Figure 35 Time course of caspase activity following CVB3 infection of ix HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells 125 Figure 36 Biochemical events of cell death following TRAIL treatment of HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells 126 Figure 37 Biochemical events of cell death following PDT treatment of HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells 127 Figure 38 7A6 expression in HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells following CVB3 infection, TRAIL or PDT treatment 129 Figure 39 Morphology of CVB3 infected HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells 131 Figure 40 Morphology of TRAIL and PDT treated HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells 132 Figure 41 Cell viability of HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells following CVB3 infection 133 Figure 42 Cell viability of HeLa/Neo, HeLa/Bcl-2, and HeLa/Bcl-XL cells following TRAIL or PDT treatment 134 Figure 43 ZVAD.fmk treatment of or Bcl-2 Bcl-XL overexpression within HeLa cells significantly decreased the amount of progeny virus released to supernatant in CVB3 infected cells and increases cellular virus.... 136 Figure 44 RasGAP and FAK degradation during CVB3 infection 137 Figure 45 Activation of ERK 1/2 and p38 during CVB3 infection of HeLa cells... 138 Figure 46 In situ hybridization of positive strand viral RNA at 3, 9 and 30 days post-infection in AJ infected mice. B) Direct injury in infected myocytes by ISH, Hematoxylin and Eosin, and Masson's trichrome staining 140 Figure 47 Histologic examination of CVB3 infected myocytes 3, 9, and 30 days post infection 143 Figure 48A Differential gene expression in myocardium of CVB3 infected mice at days 3, 9, and 30 post-infection... 146 Figure 48B Differential gene expression in myocardium of CVB3 infected mice at days 3, 9, and 30 post-infection 147 Figure 49 Gene clusters within Cell Defense functional group 149 Figure 50 Gene clusters within Cell Signaling functional group 150 x Figure 51 Gene clusters within Cell structure functional group 151 Figure 52 Gene clusters within gene expression functional group 152 Figure 53 Gene clusters within metabolism functional group 153 x i ABBREVIATIONS Apaf-1 apoptosis activating factor-1 AIF apoptosis inducing factor APC antigen presenting cell ANF atrial natriuretic factor ANT adenine nucleotide transporter AS anti-sense ASK-1 apoptosis signal-regulating kinase 1 BAG-1 BCL-2 athanogene-1 BH Bcl-2 homology CAR coxsackievirus and adenovirus receptor CARD caspase activation recruitment domain CAT chloramphenicol acetyltransferase Ced Caenorhabditis elegans death protein CHO Chinese hamster ovary CPE cytopathic effect CREB cAMP-responsive element binding protein CMV cytomegalovirus CTL cytotoxic T lymphocyte C V B 3 coxsackievirus B3 DAF decay accelerating factor DCM dilated cardiomyopathy DED death effector domain DFF DNA fragmentation factor DISC death inducing signaling complex dH20 distilled, Milli-Q™-filtered water DEPC diethylpyrocarbonate DPBS Dulbecco's phosphate buffered saline without magnesium DNA-PK DNA-dependent protein kinase EBV Epstein barr virus elF eukaryotic initiation factor ERK extracellular-signal regulated kinase xii ETC electron transport chain FAK focal adhesion kinase Fas fas receptor Fas L fas ligand FBS fetal bovine serum FITC fluorescein-isothiocyanate FLIP Flice inhibitory protein GAPDH Glyceraldehyde 3-phosphate dehydrogenase GSHPx-1 Glutathione peroxidase GPX-1 Glutathione peroxides 1 HBx hepatitis B virus X protein HBV hepatitis B virus HCC hepatocellular carcinoma hCMV human cytomegalovirus HIV-1 human immunodeficiency virus type-1 HSV herpes simplex virus Hsp heat shock protein IAP inhibitor of apoptosis protein IDC idiopathic dilated cardiomyopathy IE immediate early I FN interferons Ig immunoglobulin IGF-1 insulin growth factor-l I K B inhibitor kappa B IL-2 interleukin-2 iNOS inducible NO synthase ip intraperitoneal IRES internal ribosome entry site IRFs interferon regulatory factors ISH in situ hybridization KSHV Kaposi's sarcoma-associated herpesvirus LANA latency-associated nuclear antigen x i i i LMP-1 latent membrane protein mAb monoclonal antibody MAPK mitogen activated protein kinase MHC major histocompatibility complex NBT/BCIP .... nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt NFAT nuclear factor of activated T cells N F - K B nuclear factor kappa B NO nitric oxide O/N overnight ORF open reading frame PABP poly (A)-binding protein PARP poly (ADP-ribose) polymerase PBL peripheral blood lymphocytes PBR peripheral benzodiazepine receptor PCR polymerase chain reaction PDT photodynamic therapy PDTC pyrrolidine dithiocarbamate PE Phycoerythrin pfu plaque forming units pi post-infection PKB protein kinase B PKC protein kinase C PKR dsRNA-dependent protein kinase PML promyelocytic leukemia gene PTPC permeability transition pore complex PTB polypyrimidine-tract binding protein Rb retinoblastoma RT room temperature RT-PCR reverse transcription-polymerase chain reaction SCID severe combined immunodeficiency SD standard deviation xiv sRBC sheep red blood cell TBP tata box binding protein TFIIIC transcription factor III C Tg transgenic TNF tumor necrosis factor TRAIL TNF-related apoptosis inducing ligand TUNEL terminal deoxynucleotidyl transferase mediated nick end labelling UTR untranslated region VDAC voltage-dependent anion channel VEGF vascular endothelial growth factor vlRF1 viral interferon regulatory factor 1 VPg virus-encoded oligopeptide WHO World Health Organization wt wild-type XIAP X-linked inhibitor of apoptosis xv ACKNOWLEDGMENTS Without the love, support, and faith, during the good times and difficult times of a few key people in my life this thesis would have never materialized. First and foremost, I'd like to thank my parents, Susan and Michael Carthy. My beautiful mother, above all other accomplishments in life, only wanted one thing, to see me graduate. To my amazing father, who has always been a role model for dedication, hard work, honesty, and health. To the McManus family, who have demonstrated nothing but eternal support and generosity. Along the way, inside academics and outside in my industrial and entrepreneurial life I have had the opportunity to work with some world-class mentors. Bruce McManus, Poul Sorensen, Casey van Breemen, David Hunt, Timothy Triche, John Mendlein - each one shaping my mind and influencing me in a different way. Without these key people in my life I would not be writing this collection of research and knowledge. x v i 1 PICORNAVIRUSES AND VIRAL LIFE-CYCLE EVENTS 1.1. Family Picornaviridae, and genus enterovirus: Brief history and major features An understanding of microbiology and mechanisms of acquired disease remained elusive until the end of 19th century. In the late 1800's Robert Koch was able to identify bacteria responsible for some of these acquired diseases. In 1898, the first non-bacterial, animal-disease causative agent, the foot and mouth disease virus, was isolated by Loeffler and Frosch (441). This picornavirus was able to pass through Berkfeld filters and fueled the discovery of other viral agents responsible for many animal diseases. The first discovered human picornavirus, the agent responsible for poliomyelitis, a major dehabilitating disease in the 19* and 20th centuries worldwide, was identified in 1908 by Karl Landsteiner and Erwin Popper (412). They were able to transmit a bacterial free suspension from a 9-year old boy who had succumbed to poliomyelitis to monkeys while all other attempts to transmit the disease to laboratory animals including rabbits, guinea pigs, and mice had previously failed (412). Early attempts to propagate the poliovirus in cell culture systems were unsuccessful until John Enders, Thomas Weller and Frederick Robbins were able to demonstrate replication of the Lansing strain in human embryonic cell cultures (199) and first used the term 'cytopathic effect' (CPE). Picornaviruses are small RNA viruses that are encapsulated by an icosahedral protein coat. The encapsulated viral RNA is of positive polarity (therefore acts as a direct template for the translation of viral protein) and is approximately 7000-8000 nucleotides in length. The viral RNA contains both 5' and 3' non-translated regions that are involved in the regulation and initiation of viral translation and transcription. The protein coat is robust and serves to protect these viruses from harsh environmental conditions in nature and within their hosts (for a general review of picornavirus structure see (496))! Since 1898 there have been hundreds of picornavirus serotypes discovered. The family has been divided into six genera, the enteroviruses, the cardioviruses, the rhinoviruses, the apthoviruses, the parechovirus and the hepatoviruses (Hepatitis A virus, previously enterovirus 1 72, has been reclassified to genus hepatovirus instead of enterovirus), according to the Seventh Report of the International Committee on Taxonomy of Viruses ( The genus enterovirus was initially named for the ability of such viruses to survive acidic conditions and their propensity to infect the alimentary tract. As previously mentioned, poliovirus was the first discovered enterovirus (412). In 1947 Dalldorf and Sickles (164) isolated two unrecognizabled viruses from fecal specimens in Coxsackie, New York, during a poliomyelitis outbreak. These two viruses were able to induce paralysis in newborn mice and would eventually represent the Coxsackie A group viruses. About the same time, the first Coxsackie B group viruses were discovered. They caused spastic paralysis in mice rather than the flaccid paralysis seen with the Coxsackie A group viruses. Another member of the enterovirus genus, the echovirus, was initially isolated from stool samples. No apparent human disease associated with them and thus the name echo (ECHO: enteric, cytopathogenic, human, orphan (no disease)). 1.2. Coxsackievirus B group: Host range and virulence factors Between 1947 and 1950, Melnick and colleagues (497) identified Coxsackie group B viruses B2, B1, and B3. These viruses originated from Connecticut (B1 and B3) and Ohio (B2) from stool samples of patients with aseptic meningitis (B1), summer gripe (B2), and minor febrile illness (B3) patients (497). Currently, the B group consists of serotypes B1 to B6, although an almost limitless number of variants may exist. Each serotype are not neutralized to a significant extent by antisera to from another Coxsackievirus serotypes. Since this initial division of serotypes based on neutralizing antibody data, various molecular characterizations based on genomic RNA and protein amino acid sequence have revealed that Coxsackie B group viruses cluster well according to their respective serotypes (316). Unlike other enteroviruses with limited host range, the Coxsackie group B viruses can infect mice, primates and humans. Evidence also suggest that the virus can infect other hosts as reflected by infection of cultured rat cardiomyocytes (37, 776) and African green monkey 2 kidney cells (VERO-line) (175), among other cells used as a models for infection. This ability of Cosackieviruses to infect rodents (as demonstrated in Aim 1) has greatly aided in understanding the pathogenesis of enterovirus-induced disease. Differences in the course of infection and disease presentation among hosts can depend on both host and viral factors. Host species, age, immunogenetics, and sex all can influence the severity of disease and organ involvement during Coxsackie B group virus infection. The murine model of Coxackie B group virus infection serves as an excellent model to dissect various host factors involved in the pathogenesis of disease. In Section 9.1 it will be demonstrated that four-week-old A/J mice and ten-week-old A/J mice infected with the same 105 plaque forming unit (pfu) dose of virus have significantly different cardiac involvement. The younger mice have more infectious virus in their heart as determined by both plaque assay and in situ hybridization. Our experience thus far suggests that age-dependent differential cardiovascular susceptibility to CVB3 in the mouse is due to characteristics of heart muscle itself, and not to a generalized loss of susceptibility to Coxsackievirus replication in all organs. Thus, in certain other organs the virus appears to replicate to the same extent, if not more so, in adult mice as compared to adolescent mice, as will be demonstrated in Section 9.1. There may be age-related changes in receptor or accessory binding proteins on the surface of cardiac myocytes. Alternatively, other intrinsic proteins that may aid or inhibit viral replication may change their expression pattern with age thus affecting overall viral entry and replication within the myocytes (See Section 12, Future Directions). The basis of organ-to-organ differences in CVB3 susceptibility with host age is, in itself, intriguing, and may provide new clues to pathogenesis. Immunogenetics is another well-known host factor in differential susceptibility to CVB3-induced disease as will be demonstrated in Section 9.1. A/J mice (H-2a) and C57BL/6J mice (H-2b) infected with the same 105 pfu dose of CVB3 have distinctive cardiovascular disease progression. A/J mice contain more infectious virus in the myocardium and have greater viral RNA detectable by in situ hybridization as will be demonstrated in this thesis. Lower viral titres 3 and less tissue damage within the myocardium of C57BL/6J mice is not related to total systemic load as there is significant virus in the serum during peak viremia and the C57BL/6J mice show significant liver involvement as will be demonstrated in section 9.1.1. It is difficult to view any model as "resistant" to CVB3 infection because although a given murine strain may not replicate virus as well in the heart as another immunogenetically distinct host, other organs may be more vigorously infected. Gender is yet another host factor in CVB3 susceptibility examined by a few investigators. Male mice may be more susceptible to Coxsackievirus infections (311), however the question has not been studied in depth and predictions of susceptibility in humans on the basis of gender are not clear. Viral factors are also involved in Coxsackievirus disease pathogenesis. Mutations in different regions of the genome can influence virulence and tissue tropism (699). A mutation in the coding region may affect receptor binding as is the case with the H3 and H310A1 antibody escape mutants, where sequencing has shown a mutation at nucleotide 1442 corresponding to amino acid 165 of VP2 (387). There is emerging evidence that genetic variations which alter the virion capsid proteins can influence viral receptor or other binding protein attachment and play a role in pathogenesis (621) as will be discussed in Section 1.3.1. Other mutations in the 5' non-coding region have been shown to affect cardiovirulence. Tu et al (706) identified a single nucleotide change from 234C to 234U that yielded a cardiovirulent strain (706). Additional work on this mutation suggests that this 234C strain limits viral replication in multiple cell types and not just cardiocytes by inhibition at the level of positive strand viral RNA synthesis. Viral protein translation appeared normal, but ratios of positive to negative strand viral RNA were markedly diminished (755). Recent understanding regarding the molecular genetics of the 5' non-coding region offers the possibility that non-coding regions are important determinants of virulence, as will be discussed in Section 1.3.2. 1.3. Viral life cycle Coxsackie group B viruses are small RNA viruses that have an icosahedral protein coat 4 made up of 60 identical units, each of which consists of four structural proteins VP1-VP4. The viruses bind to protein receptors and attachment proteins on the surface of host cells and the viral RNA enters the cell if the appropriate host protein-capsid protein interaction occurs. An attachment protein differs from a receptor in that it cannot mediate viral entry to the cell cytosol. Various factors, both viral and host derived are responsible for the successful propagation of the virus (Figure 1). During the course of the viral life cycle, host cell homeostasis is altered as there are many points of intersection between the host and the virus (Figure 2). 1.3.1. Viral Receptors The interactions between viruses and constituents of the plasma membrane are critical components in the initiation of the viral life cycle. Host cell surface proteins are not constitutively expressed in all cell types or may not be localized at the cell surface in all cell types or during all phases of the cell cycle. Viruses may also require co-receptors and other factors on the surface of cells that aid in stabilization and interaction of the virus with the appropriate receptor. Viruses may also bind other proteins on the surface of some cells that play no role in viral infection, yet these interactions may aid in viral clearance or aberrant host cell signaling. For many years, the identity of the coxsackievirus receptor remained a mystery. Two monoclonal antibodies (RmcA (157) and RmcB (300)) were generated that were effective at protecting HeLa cells against infection by various CVB serotypes. Subsequently, the identity of the host proteins that the monoclonal antibodies were specific for were elucidated, and the 46 kDa (corresponding to RmcB) protein was determined to be the receptor for CVB3 (65, 698). The 46kD coxsackievirus and adenovirus (CAR) receptor, unlike previously discovered CVB3 cell-surface binding proteins such as decay accelerating factor (DAF (corresponding to RmcA)) (67, 632) and a 100kD protein in the nucleolin family (177), not only binds CVB3, but also mediates viral RNA entry into cells for viral replication. Both human and murine cells contain the receptor (hCAR and mCAR, respectively). The murine CAR was subsequently shown to also be a receptor for Coxsackie B group viruses by transfection studies using mCAR cDNA and non-permissive CHO cells (66). The high degree of genetic homology between hCAR and mCAR 5 Figure 1. A working model of the coxsackievirus life cycle. CVB3 can bind to the cell surface via decay accelerating factor (DAF) or directly with the virus receptor (usually located in caveolae or coated pits). Following CVB3 binding to the coxsackievirus and adenovirus receptor (CAR), the viral RNA enters the cytoplasm where it is translated into a single polyprotein by host translational machinery. The polyprotein is then processed by virus-encoded proteases into structural and nonstructural proteins. The virus-encoded RNA-dependent RNA polymerase (3Dp o 1) produces a negative-strand viral RNA which serves as a template for the production of multiple positive-strand virus genomes. Virions are packaged and the virus exits the cell. 6 (giViral capsid protein interaction with host cell surface protein JT ® Host protein modification I j)f surface permeability © Host protein interaction with ©strand vir^ aj, RNA • . © Viral protein interaction ® ^ P ^ f a 9 e .with host mRNA of cellular proteins ^ *| © Viral protein-host protein interactions © Host protein interaction ^ with Qstrand viral RNA A> Figure 2. Potential points of intersection between host and viral proteins. This schematic demonstrates the many sites where viral and host proteins may interact to disrupt normal host cell homeostasis. 7 (91% protein similarity) may partly explain why the mouse mirrors CVB3 induced-disease in humans, although distribution of these receptors on specific cells and tissues is yet to be clearly established. The primary sequence of CAR shows that it is a member of the immunoglobulin superfamily of proteins (66), consisting of two extracellular domains, a transmembrane domain and an intracellular domain. Work with truncated proteins has demonstrated that only the extracellular domain is required for viral infection (as truncated proteins that have the cytoplasmic or transmembrane domain deleted do not prevent viral infection (738)). This work suggests that the virus capsid-protein interaction with CAR 'S extracellular domain may cause release of viral RNA from the capsid that may enter the cell via endocytosis or by some other mechanism. The normal cellular role of CAR is not yet fully understood, but work suggests that it appears to be preferentially expressed at intercalated discs and cell-to-cell junctions of cardiomyocytes in failing myocardium (527), and may be involved in intercellular adhesion and growth inhibition in other cell types (539). CAR receptor mRNA expression has been demonstrated in a variety of murine and human tissues. By Northern blot analysis, Tomko et al. detected transcripts in pancreas, brain, heart, small intestine, testis, and prostate, lower expression in liver and lung, and no expression in kidney, placenta, peripheral blood leukocytes, thymus, and spleen (698). This expression profile will have implications to Section 9.1, Systemic Localization of Viral RNA. Some studies have shown that other cell surface factors may influence infection. The anti-CAR-Mab does not prevent infection by all CVB serotypes (467). CD-55 (DAF) is a glycoprotein that is anchored to the cell surface by glycoslyphosphotidylinositol (GPI) and limits complement-mediated cellular damage. It has been shown that CVB can bind DAF (which was considered the putative receptor in the mid 90's) (632). There are a variety of studies that demonstrate the ability of DAF and CAR mAb's to block virus infection. Anti-DAF antibodies can block infection of HeLa cells (466). Antibodies to DAF and CAR are both required for absolute inhibition of infection (633). Variants of CVB3 have been generated that can preferentially bind DAF and can have a different course of infection as compared to wild type strains. In primary human fibroblasts, the 8 CVB3 PD strain (where there are 6 amino acid substitutions in VP1 as compared to CVB3 Nancy P strain) binds strongly to DAF, but weakly to CAR (opposite to CVB3 P) and produces a lytic infection while the CVB3 P strain produces a persistent carrier state infection in the same cells (621). This is further supported by Selinka et al., who also suggest that a DAF binding strain of CVB3 induced a more aggressive cytopathic effect (629). Taken together, these studies suggests that although CAR is the protein required for viral RNA entry into the cell, DAF and other potential proteins may mediate viral attachment, viral presentation to the receptor, or host intracellular signaling that affects the intrinsic cell milieu and downstream viral replication. Viral variants that differ in their ability to bind surface receptors due to amino acid substitutions on their structural proteins may have different life-cycles and disease presentations. It should also be noted that murine DAF does not bind CVB3 like human DAF. Studies in murine and human DAF transfected CHO cells have demonstrated that only the human DAF cells bound virus (651). Murine DAF has approximately a 60% amino acid homology to human DAF, yet both transfected cells are able to inhibit complement activation (651). If DAF plays a role in CVB3 docking to the surface of cells and interacts with CAR for viral RNA entry into cells, there may be differences in the initiation of infection between different species. 1.3.2. Viral RNA internalization, translation, and transcription After binding to the Coxsackie and adenovirus receptor (CAR) (65, 698) present on myocytes and other host cells, viral RNA enters the cell and acts as a template for the translation of viral protein (Figure 3A). He et al. have demonstrated to 22 Angstrom resolution the binding of the CAR D1 domain to the canyon of CVB3 which would facilitate uncoating and viral RNA entry into the cell (274). Picornaviruses do not translate the viral mRNA into viral protein like most eukaryotic mRNAs. In eukaryotic cells, the primary mechanism of translation initiation requires the assembly of initiation factors at the 5' end of the mRNA where the 7-methyl guanosine cap structure is located. The 40S ribosomal subunit interacts with the 5'-cap structure and subsequently migrates to the AUG start codon (395). This process involves numerous 9 5' L PABP j PABPsJ A A A A A A A A A A A A A ^ 5' (S | Structural Proteins" Non-structural Proteins VP4J VP2| VP3 | VP1| 2A | 2B | 2C | 3A| 3B | 3CD Viral proteases 3CD 3C B B 5' i 742 3' The IRES Figure 3. A) Schematic demonstrating the internal ribosome entry of the 40S ribosome subunit and elF4G subunit to initiate translation. PABP binds to the poly(A) tail on the 3' non translated region of the CVB3 genome and can positively influence translation by interacting with the factors bound to the 5' non translated region. The poly protein is depicted along with the individual proteins that are processed by the viral encoded proteases. B) A schematic representation of the 5' non translated region of CVB3. Note that the IRES of CVB3 is located roughly at stem-loops G, H and I spanning nucleotides (nts) 529 and 630 l() translation initiation factors belonging to the eukaryotic initiation factor (elF)4 group. The complex of these factors, termed elF4F, allows for joining of the 40S ribosomal subunit to the mRNA, acting as an intermediate between the two. elF4F consists of three main polypeptides: elF4E, which binds to the 7-methyl guanosine cap structure (509); elF4A, which has RNA helicase activity to unwind any RNA secondary structure (579); and elF4G which seems to mediate joining of the components. Investigations (318, 410) have identified an elF4E-binding motif on elF4G and have also identified sites of interaction with elF4A and elF3 already bound to ribosomes. Thus elF4G plays an integral role in bringing together all the necessary factors for translation. In contrast to most host mRNAs, picornavirus RNAs are not capped with a 7-methyl guanosine cap, but instead are linked covalently with a virus-encoded oligopeptide (VPg) (418). Picornavirus 5'UTR RNA forms a highly ordered secondary structure and translation initiation is mediated by a novel mechanism involving internal binding of the ribosome on a sequence element of the 5'UTR, termed an internal ribosome entry site (IRES) (332). According to this model, the IRES directs binding of the small ribosomal subunit to viral RNA near the 3' border of the IRES, independent of a cap structure at the 5' terminus of the viral RNA (174) (Figure 3B). The importance of elF4G in eukaryotic translation initiation can be further demonstrated in the picornavirus infection model wherein the virus encoded 2A protease can cleave the protein, thus separating the elF4E binding domain from the elF3 binding domain (411) ultimately resulting in inhibition of eukaryotic cap-dependent protein translation (194, 277, 528). Eukaryotic initiation factor 4G is a 220kDa cytosolic protein expressed in all cell types. Degradation of elF4G has always been considered to inhibit cap-initiated translation, a prominent feature of picornavirus infection, however, more recent work using depleted and reconstituted reticulocyte systems is challenging this hypothesis and suggests that the inhibition of host translation is due to the viral RNA out-competing host cell mRNAs for limiting concentrations of 2A cleaved c-terminal or elF4G (9). The c-terminal fragment of 2A cleaved 11 elF4G binds to the IRES as efficiently as intact protein, and this interaction is critical to functional activity of the IRES element (443). In addition to the elF4F complex, the poly(A)-binding protein (PABP), which binds to the poly(A) tail found on most mRNA's, has been shown to increase the efficiency of eukaryotic mRNA translation. elF4G has been found to interact with PABP suggesting a circularization of the mRNA via protein interactions between 5' and 3' translation initiation factors (251, 416). It has also been shown that picornavirus protease 2A cleaves PABP (337) and the cleaved fragments are unable to stimulate eukaryotic cap-dependent translation in vitro (367) as the c-terminus poly A interacting domain is separated from the n-terminus elF4G interaction domain. Having said this, it has also been demonstrated that there is cooperation between the 5' and 3' factors during picornavirus mediated translation and attenuation of the poly A tail in picornavirus RNA's decreases translation by more than 10-fold (63), raising questions as to the molecular mechanisms if PABP is degraded. There have been eukaryotic mRNA's discovered that contain IRES-like domains and do not require cap-dependent translation. Many of these eukaryotic IRES-containing mRNA's are involved in cell regulation and stress pathways (207) as will be discussed in Section 1.3.3 disruption of host cell homeostasis. Like other Picornaviruses, the 5' untranslated region (5'UTR) of the CVB3 genome is unusually long (741 nucleotides). The presence of an IRES element in CVB3 RNA and the minimum sequences of c/s-acting translational elements within the 5'UTR were determined by a study of plasmid mutants from which bicistronic RNA transcripts, with all or part of the 5'UTR in the intercistronic space, were synthesized and translated in rabbit reticulocyte lysates (771). In the presence of an upstream cistron, the chloramphenicol acetyltransferase (CAT) gene, designed to block ribosomal scanning, the CVB3 5'UTR was capable of directing the internal initiation of translation of the truncated viral downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (771). This finding was further supported by data 12 on predicted secondary structures and the stability of each stem-loop within the 5'UTR (Figure 3B). Analysis of various deletion mutants demonstrated that the core sequence of the IRES of CVB3 is located roughly at stem-loops G, H and I spanning nucleotides (nts) 529 and 630. The region from nts 1 to 63 (stem-loop A) also appears important, and it may be an essential binding site for translation and transcription initiation factors derived from the host cell (771). Work on polioviruses has identified host proteins of different molecular weights that bind singly or in complexes to the 5' UTR (58, 265). Translation and transcriptional regulation of viruses has thus become an important area of study in relation to viral pathogenesis following infection. CVB3 mutations that affect the binding of host translation or transcription factors would potentially alter virulence. Age related changes in the susceptibility of the heart to Coxsackievirus infection may in part be related to changes in these interactions as will be discussed in Section 12, Future Directions. A number of host cytoplasmic proteins bind to the.5' and 3' non-translated regions to facilitate protein translation. The viral protein is initially synthesized as a large polyprotein (approximately 200kDa) that is further cleaved into individual structural, and non-structural proteins mediated by the virus encoded proteases 2A, 3C, 3CD (Figure 3A). One of the viral polyprotein products, 3D, an RNA-dependent RNA polymerase, is essential for transcription of the negative strand viral RNA intermediate. This negative strand then serves as a template for synthesis of multiple progeny genomes. The progeny RNA is packaged into a mature capsid and is released from the cell. The exact mechanisms of viral release are not known, but the steps in the lifecycle may be facilitated by host-encoded proteins and apoptosis as described in Section 9.3. 1.3.3. Disruption of host cell homeostasis During viral parasitization of the host-cell, numerous physical associations between host proteins and viral RNA and host proteins with viral proteins disrupt the regular homeostasis and natural milieu within cells. Disruption of normal cellular homeostasis may occur as early as CVB 13 binding to surface receptors or surface binding proteins as previously mentioned. Although the normal cellular function of CAR is only now beginning to be elucidated (539), it can be anticipated that CVB binding will induce a signaling pathway that may result in transcriptional activation within the nucleus. Unpublished preliminary observations by our group indicate dramatic changes in host transcriptional regulation at very early time points following infection using oligonucloetide cDNA microarrays and HeLa cell infection model. It is becoming apparent that CAR appears to play a role in cell-to-cell contact and growth inhibitory signaling (527, 539). Other work from our laboratory has documented virtually immediate activation of mitogen activated protein kinases (MAPK), an event potentially transduced via DAF and/or the src kinase p56lck (449). It is well established that the viral proteases can degrade host proteins in addition to cleaving the viral polyprotein (Figure 4). Like apoptosis (to be discussed in Section 3), cleavage of cellular proteins may produce active protein fragments or expose domains that were previously hidden. As mentioned, viral protease 2A can depress cap-dependent translation by disruption of elF4G (194) and PABP (367). It has also been shown that the 2A cleavage products of elF4G can stimulate cap-independent protein translation and thus increase viral translation efficiency (535, 536). Other cellular factors such as polypyrimidine-tract binding protein (PTB) (278) associate with virus RNA and stimulate cap-independent translation (249, 314). In addition to viral IRES, many host cellular proteins have also been shown to be able to be translated by IRES systems. Several of these proteins appear to be important regulatory or stress induced molecules (295) and also bind PTB protein such as Apaf-1 (147, 503), VEGF (313), XIAP (294), and BAG-1 (147). These cellular proteins might be translated during picornavirus infection and play an important role in determining the fate of the infected cell. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may work to oppose to the translation enhancing activity of PTB and decrease viral translation when present in large amounts (779). 14 Structural Proteins Non-structural Proteins Alteration of host cell signaling and cell survival Alteration of host cell structure and morphology Loss of function and alteration of normal homeostasis Figure 4 Schematic demonstrating currently known host proteins that are degraded by viral proteases during CVB3 infection and potential effect on host cell homeostasis 15 In addition to virus-induced modifications in translation, host-cell transcription is inhibited. Transcription-associated proteins CREB (766), Oct-1 (768), TBP (765), and TFIIIC (634) are degraded by viral proteases. Signaling pathways may be altered, including proteins like RasGAP (309), which are also cleaved by viral proteases and demonstrated in Section 9.3. Such cleavage favors activation of the MAPK pathway and may serve as a positive feedback loop that facilitates viral replication, induction of apoptosis and release of viral progeny, as inhibition of ERK1/2 by U0126 results in depression of viral replication and inhibition of apoptosis (449). Heart, or muscle specific proteins such as dystrophin may be cleaved by viral protease 2A (34, 37) which may enhance a dilated cardiomyopathy phenotype similar to dystrophin deficient mice (495). Data presented in Section 9.3 will demonstrate that focal adhesion kinase (FAK), a kinase that is involved in arrangement of actin filaments, is cleaved by viral proteases as well by caspases, and may be one of the proteins that may lead to cytopathic effect. Cleavage of many of these regulatory, signaling, and structural proteins alters normal functioning of the cells by either inhibiting their normal function or releasing active domains that alter cellular function or influence the viral replication cycle. Picornavirus RNA can bind multiple host proteins at both the 5' and 3' termini (58, 180). As previously mentioned, the PTB associates with viral RNA and stimulates cap-independent translation. We have preliminary data that more than 10 such proteins may exist that can bind CVB3 5' untranslated region (D. Yang, unpublished observations). These proteins may aid in translation of the viral polyprotein, aid in replication of the negative or positive strand viral RNA, or may be protective host factors in certain cell types that inhibit the viral replication process. Further, interaction of these cellular proteins with viral RNA may act as a sink thereby reducing the concentrations available in the cytosol to carry out normal physiological processes. It is clear that an adverse environment is created within an infected cell during the viral-replicative cycle. The adverse effects of viral infection on host cell function results in cytopathic effect and loss of cell viability in most cell types. There is certain evidence that persistent, carrier-state 16 cultures can be maintained in certain cell types such as human fibroblasts (621), and evidence in vivo that the viral RNA may persist for long periods following infection in specific tissues such as the myocardium (383). As will be shown in detail in Section 9.2, infection of HeLa cells results in caspase activation after the characteristic cytopathic effect (104). Caspases are key mediators of apoptosis (see Section 3.2), but it is interesting in the Coxsackievirus B3 model that the caspases are not responsible for the rounding up which is a key feature of the cytopathic effect. Inhibition of caspase activity with the synthetic caspase inhibitor ZVAD.fmk does not prevent CVB3-mediated cytopathic effect (104), suggesting that CPE and loss of viability may be separate events during infection. Work in this thesis will show that inhibition of caspases by either synthetic caspase inhibitors (ZVAD.fmk) or Bcl-2/Bcl-xL over-expression in HeLa cells protects against the loss of viability but not CPE. This would suggest that CPE is mediated by viral protease cleavage of a host structural protein or changes in ion concentrations. Work in this thesis also shows that caspase inhibition prevents the release of progeny virus from the cell. As mentioned and demonstrated in Section 9.3, the signaling protein RasGAP is cleaved in HeLa cells during the course of CVB3 replication (309), promoting activation of the extracellular-signal regulated kinase (ERK) 1 and 2 by dual phosphorylation. This work has been extended and we now have evidence that ERK is activated (449), but not p38 or JNK/SAPK. The interesting connection between death pathways and ERK is that inhibition of ERK results in decreased apoptosis, decreased production of viral progeny virus within the cell, and decreased release of virus to the extra-cellular environment. These observations would suggest that the virus is modulating ERK to facilitate viral replication, induction of apoptosis, and eventual release of virus from the infected cell. 17 2 ROLE OF COXSACKIEVIRUS B3 IN MYOCARDITIS AND SYSTEMIC DISEASE 2.1. Myocarditis Myocarditis is generally defined as myocyte necrosis/cell injury with associated inflammation of the heart muscle in absence of an ischemic episode (26). The incidence of myocarditis in the general population is difficult to determine since many episodes are preclinical and an optimal detection method is not fully agreed upon. The best estimates on incidence in the general population have originated from routine post-mortem evaluations (autopsies where appropriate). These studies have suggested that myocardial inflammation is present in 1 to 10 percent of cases (74, 248, 608). Applying the stricter Dallas criteria to post-mortem evaluations in 2 studies (12,747 cases with 1.06% myocarditis (256) and 17,162 cases with 0.53% myocarditis (556)) greatly decreased the number of positive cases, although it is generally accepted that the Dallas criteria may underestimate the true number of myocarditis cases (206). The introduction of the endomyocardial biopsy has allowed for 'live' determination of the incidence of myocarditis, but this method yielded highly variable results (0 to 80% (435)) as there are problems in patient sampling, diagnostic criteria, and limitations of the biopsy itself (206). ' ... ' 2.1.1. Viral myocarditis Although many pharmacological agents (eg. catecholamines, anthracyclines, cocaine) (493), toxins (arsenic, heavy metals), and many infectious agents (parasites, fungi, bacteria, and viruses) can lead to myocarditis, viruses are the most common cause of myocarditis in the Western world (490). Coxsackieviruses (Picornaviridae), influenza virus (Orthomyxoviridae), measles, mumps and RSV (Paramyxoviridae), adenoviruses (Adenoviridae), rubella virus (Togaviridae), human immunodeficiency virus-1 (Retroviridae), Hepatitis C virus (Flaviviridae), vaccinia virus (Poxviridae), herpes simplex virus, herpes zoster virus, and cytomegalovirus (Herpesviridae), among many others, have all been shown to be associated with some form of cardiac involvement and myocarditis (1, 145, 206, 756). The mechanisms of myocarditis following infection among the various viruses may differ. Some viruses may replicate directly within 18 myocytes and cause myocyte damage and incite an anti-viral immune response. Other viruses like HIV-1 may either directly infect the myocardium or allow for infection with secondary opportunistic viruses like hepatitis C virus that take advantage of the immunocompromised state and infect the myocardium (477). It has been suggested that "at least 70% of the general population has been exposed to cardiovirulent viruses, and no doubt half have had an episode of acute viral myocarditis, which was subclinical in the vast majority (530)." 2.1.2. Enterovirus myocarditis Global surveillance studies by the World Health Organization (WHO) from 1975 to 1985 demonstrated that Coxsackie B group viruses had the highest likelihood of causing clinical cardiovascular disease, with 30 - 40 cardiovascular diagnoses per 1000 documented virus infections (259). More recent molecular studies of otherwise unexplained, community-acquired myocarditis, corroborated the prominent role of coxsackieviruses in myocarditis (210, 336, 465). These data indicated that coxsackieviruses of the B group are the most likely of all viruses to present as cardiovascular disease. The incidence of myocarditis associated with enterovirus infections in humans has been evaluated by a number of methodologies including serology for neutralizing viral antibody titres, molecular techniques for presence of viral RNA in the myocardium, and immunochemical methods for the detection of viral protein in the myocardium. Serologic data, reviewed by Martino et al. (465), indicate that 38-50% of patients with clinical myocarditis have had or currently have an enteroviral infection. Similarly, data based on molecular techniques for detection of enteroviral RNA (RNA blotting, reverse transcription-polymerase chain reaction [RT-PCR] and in situ hybridization) in patients with myocarditis, have shown 8 to 70% of myocarditis patients are positive for viral genome (465). Improved immunochemical techniques to detect viral-antigen using an enterovirus group-specific antibody for viral capsid protein VP1 have been developed. In a recent study, VP1 positivity was detected in 80% of myocarditis cases and 75% of DCM cases with little or no positivity in the control cases (431). In worldwide experience (24, 81, 82, 348), considering biopsy material, explants at transplantation, and autopsies in a diverse group of 19 patients, and with a broad range of molecular techniques, the average frequency of myocardial positivity for enteroviruses in adult myocarditis patients is 25% (492). Such data indicate a clear association of enterovirus infection with myocarditis and IDC. 2.1.3. Pathogenesis of enterovirus-induced myocarditis Many competing hypotheses exist as to the pathogenesis of CVB3-induced myocarditis. Three distinct hypotheses have been proposed and investigated regarding the role of CVB3-induced inflammatory responses and the development of heart muscle disease. Perhaps the longest-standing hypothesis is that CVB3-induced immune responses are autoimmune in nature and that the cellular constituents of the immune system are lytic for uninfected as well as infected tissues (312, 597). A second major hypothesis is that the humoral limb of the immune response causes chronic tissue pathology through auto-reactive antibodies and molecular mimicry (226, 523). The third and most recently supported hypothesis is that CVB3 infection causes extensive direct injury in infected tissues prior to cellular infiltrates (382, 489), and that persistent low-level replication of virus elicits a chronic anti-viral inflammatory response. The autoimmune and molecular mimicry hypotheses of myocarditis have been reviewed in detail by others (160, 561, 596, 597, 756). Some of the more recent evidence to support and reject these hypotheses will be presented in Section 2.1.4, Role of the Immune System. Evidence to support the direct-damage-and-viral-RNA-persistence hypothesis has become available through re-evaluation of microscopic features in infected tissues and with the use of molecular techniques (346, 347). In situ hybridization for determination of presence, locale, and persistence of viral RNA in murine tissues following infection has yielded entirely new insight into the disease. Viral RNA localizes to cardiac myocytes as well as numerous other cells, tissues, and organs early following infection as will be demonstrated in Section 9.1. Klingel et al. have demonstrated persistence of the viral RNA in myocytes can occur until at least day 42 post-infection in immune-competent mice as determined by in situ hybridization (384). Other investigators have been able to detect CVB3 RNA in the myocardium using PCR in a proportion of animals 90 (586) and 98 days (235), respectively, after infection. 20 Light and electron microscopic cytological assessment alone of tissues from infected mice clearly indicates that cardiac myocytes are injured prior to immune cell infiltration of tissue, as determined by the distinctive histopathologic features including cytopathic effects, coagulation necrosis, and contraction band necrosis (28, 140, 489, 498) (see Section 9.1 for description of these types of myocyte injury). Beginning by day two post-infection cytopathic lesions are evident in ventricular myocytes, characterized by cell vacuolar changes, contraction bands and coagulation necrosis (489). This myocardial injury becomes more noticeable through days 3, 4, and 5, after which direct injury is obscured by inflammatory infiltrates, cellular calcification, and tissue edema. Use of terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL), which can be used to evaluate cell death by different pathological processes, has revealed mixed results as to the processes of cell death following CVB3 myocardial infection. Some cells that are TUNEL positive are positive for viral RNA as determined by in situ hybridization on adjacent sections (see Section 9.1). This injury appears largely non-apoptotic in nature, with diffuse TUNEL staining of the myocyte cytoplasm. Work by Huber (310) has identified TUNEL positive nuclei in certain strains of mice. As will be discussed in Section 5, the cardiac myocyte is a terminally differentiated cell with little or no capability to regenerate, therefore, intrinsic anti-apoptotic mechanisms must be present to protect myocytes against residual, low level apoptosis. Thus, apoptosis of cardiac myocytes following CVB3 infection may be a minor event, much less common than necrotic/oncotic cell death. Most apoptotic bodies in cardiac tissue are derived from immune cells; such cells have a short lifespan at the site of inflammation and die by programmed cell death (616). 2.1.4. Role of the immune system Further support for direct viral injury and the beneficial effect of the immune system comes from multiple experiments by numerous investigators using immunocompromised/immunomodified hosts (inbred, transgenic, and knockout mice), immunosuppressant therapy, and immunomodulators (138, 214, 289, 317, 380, 446, 675). In 21 general, these data have shown an enhancement of disease severity and animal mortality when the immune system is impaired or compromised, and a decreased severity of disease and mortality when the immune system is enhanced or when anti-viral agents are used. The issue of injury mechanism is confused by the different methods used to assay myocardial injury and disease. Simply examining for the presence, quantity, and quality of immune cell infiltrate is not a measure of myocyte damage and disease. Inbred mice containing spontaneous mutations in critical genes required for immune regulation gave early insights into the role of the immune system during CVB3 infection. Severe combined immunodeficiency (SCID) mice lack mature T and B lymphocyte functions caused by a defect in VDJ recombination, but other hematopoietic cell types appear to develop and function normally including NK and macrophages (79). Following infection with CVB3 there is increased mortality, RNA presence in the myocardium as determined by ISH, and foci of cardiomyocyte necrosis as compared to control mice (138). Infection using non-cardiovirulent CVB3 (234C) in SCID mice resulted in massive cardiomyocyte necrosis and reversion of the virus to a cardiovirulent strain (234U) (706). Athymic nude mice (nu/nu) possess a vestigial thymus and are incapable of producing mature T-cells (559). In 1984, Schnurr et al., demonstrated evidence of persistence of CVB3 to 94 days pi and the requirement for T-cells for viral clearance in N:NIH(S) II nu/nu mice (622). In other studies, infection of wt (+/+), euthymic (nu/+) and athymic (nu/nu) C3H/HeN (H-2k) mice with CVB3 showed increased viral titres and increased myocardial injury in the athymic mice (613). Further, there was persistence of viral RNA up to day 42 pi by in situ hybridization and up to day 92 pi by reverse transcriptase-PCR evident in the athymic mice while neither sense nor antisense RNA was detected after day 21 pi in control (613). Genetically modified mice by either knocking out genes of interest (knockout) or adding a gene of interest (transgenic) have allowed researchers to evaluate the roles of many genes, protein products and their signalling pathways, in normal development and physiology, and offer the ability to determine protein function in acquired disease. The use of these genetically 22 modified mice has demonstrated that the early innate immune response is critical for controlling viral replication and limiting end organ disease. Mice deficient in the IFN type I receptor and infected with CVB3 had 100% mortality by day 4 pi while mortality was significantly lower in IFN type II (-/-) receptor and wild type mice. There was also a dramatic increase of viral RNA in the liver of these IFN type I (-/-) receptor animals (748). CVB4 infection of mice lacking IFN-y resulted in hypoglycemia and rapid death while wild type mice were resistant. Further, selective expression of IFN-y in the pancreatic beta cells of Tg mice otherwise susceptible to lethal infection allowed for survival and protected them from developing the accompanying hypoglycemia (296). When these same transgenic mice expressing IFN-y in their pancreatic beta cells were infected with CVB3 they were protected from myocarditis. This work challenged the 'molecular mimicry' hypothesis as the viral antigens would still have been processed and presented by APC to CD4+ cells and emphasized the benefit of reducing early viral titres to reduce end-stage organ disease (297). Nitric oxide is another important cytotoxic effector of the innate immune system. The enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-a and TNF-a among other cytokines and agonists. Mice infected with CVB3 had increased titers of virus and a higher mortality when they were fed iNOS inhibitors (446). Similarly, CVB3 replicates to higher titers in NOS2(-/-) mice, and these mice clear virus much more slowly than wild-type mice. The NOS(-/-) mice also had more severe myocarditis than the wt mice (791). NO protects in other CVB models of infection as NOS2 (-/-) infected with CVB4 had higher mortality and the livers, pancreas, kidneys, and hearts and cleared virus more slowly than those of the wt group (213). In vitro, transfection of iNOS into HeLa cells inhibited viral replication (792) suggesting that this molecule had direct effects on inhibiting various components of the viral life cycle. NO has been shown to inactivate Coxsackievirus proteases. NO S-nitrosylates the cysteine residue in the active site of proteases 3C (615), and 2A (35) inhibiting protease activity and interrupting the viral life cycle. Generation of NO may also inhibit apoptosis by directly attenuating caspase activities and the effect of this on myocyte apoptosis will be 23 discussed in Section 3.3. In addition to inhibiting the viral life cycle, NO might prevent cytopathic effect and cellular death in some cells as viral proteases would be inactivated and unable to cleave host proteins as described in Section 1.3.3. Not all reactive oxygen species may be beneficial during early infection, as anti-oxidants may be other important regulatory molecules to control the innate immune system. Glutathione peroxides 1 (GPX-1) is an enzyme with antioxidant properties. Gpx1 (-/-) mice developed myocarditis after CVB3/0 infection, whereas infected wild-type mice (Gpx1 (+/+)) were resistant (53). Further, these normally-benign strains of Coxsackievirus B3 become virulent in either Se-deficient or vitamin E-deficient mice (52, 54), demonstrating that host levels of anti-oxidants are other critical factors in curtailing disease. It should also be mentioned that there is evidence that lack of antioxidant enzymes like GPX-1 makes cells more susceptible to apoptosis and that the aggravated injury in the GPX-1 (-/-) mice could also be do to increased susceptibility to apoptosis and cell death following noxious stimuli. Chemoattractants including the beta-chemokines MIP-1a and RANTES and their receptors mediate the trafficking and activation of a variety of leukocytes including lymphocytes and macrophages. MIP-1 alpha mutant (-/-) mice were resistant to Coxsackievirus-induced myocarditis seen in infected wild-type (+/+) mice (150). It should be noted here that disease was purely determined by the effect on inflammatory cell mobilization to the heart and not on other indicators of disease like myocardial viral titres and direct viral injury. Dissection of the roles of the specific immune response and effects on end organ disease and viral clearance are not as clear as are the roles of specific molecules of the innate immune system. B cell deficient mice (BcKO mice) infected with CVB3 established chronic infection in a variety of organs (heart, liver, brain, kidney, lung, pancreas, spleen). In most of these tissues the viral titers remain high for the life of the mouse, and in several there is severe pathology, particularly severe myocardial fibrosis with ventricular dilation, reminiscent of the dilated cardiomyopathy seen in humans with chronic enteroviral myocarditis (499). Similarly, IL-4 (-/-) mice developed a severe acute myocarditis on day 7 pi with severe myocardial damage 24 between day 7 and 21 pi and prolonged virus persistence in the heart tissue. IL-4 (-/-) mice were more susceptible to long-term heart muscle injuries as compared to wt mice after infection with CVB3 (422). Inappropriate MHC Class II presentation of antigen to CD4+ cells can also lead to aggravated disease as MHC class II (-/-) mice had fewer numbers of, but longer persisting inflammatory cells in the myocardium, persistent virus, enhanced fibrosis, and the mice displayed a weak IgG response with absence of virus neutralizing antibodies as compared to wt mice (421). These studies underscore the importance of the humoral limb of the immune system in clearing infectious virus from target organs and the serum. The role of the cellular arm of the immune system is the most controversial in understanding the pathogenesis of myocarditis. There is evidence supported by the SCID and athymic nude murine studies as mentioned above, that there is significant impairment of viral clearance and enhanced disease as compared to immune competent counterparts. Whether or not this is due to the absence of mature B-cells, or T cells or both is difficult to distinguish. There is evidence that compromising the CD8+ cytotoxic T cell response is beneficial to the host. Beta2-microglobulin (-/-) mice (thus inhibited MHC class I presentation of intracellular antigen to CD8+ cells) were less susceptible to early mortality showing a 180-fold increase in the 50% lethal dose as compared to wt mice (282). Opavasky et al. demonstrated that selective removal of CD4+, or TCR-beta cells by use of knockout mice afforded prolonged survival and minimized myocardial disease observed after CVB3 infection (545). CD8+ (-/-) mice had enhanced disease, while strikingly, a double knockout of both CD4 (-/-) and CD8 (-/-) molecules from T lymphocytes afforded the best protection mice from myocarditis (545). Further complexity of understanding the role of the cellular immune response is demonstrated in a study by Henke et al. who demonstrated a marked reduction in myocarditis and an increase in myocardial virus titers in CD4 (-/-) mice that have in vivo depletion of CD8+ cells (282). Cytotoxic effectors of CD8+ cells include ligands on the surface of cells including Fas L and TRAIL and granules that contain cytotoxic proteins including perforins and granzymes as will be discussed in Section 4.2. Perforin (-/-) (PKO) mice are completely resistant to a 25 normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis with rapid resolution, whereas their perforin+ littermates have extensive myocardial lesions with severe myocardial fibrosis. The PKO mice clear virus and the humoral arm is in tact as viral titers are indistinguishable between the two mice (228). In addition to modifying the host, investigators have created recombinant CVB3 viruses that encode for select cytokines (111, 284). Henke et al. created variants expressing IFNy, IL-10 and a control virus producing inactive IL-10. They demonstrated that the IFNy and IL-10 containing viruses showed less replication and dissemination of virus (as the viruses were constrained to the pancreas) as compared to the wt and inactive IL-10 recombinant virus. More importantly, secondary challenge with wt CVB3 in all three groups (IFNy, IL-10, inactive IL-10) lead to significant morbidity and mortality in the wt and inactive IL-10-infected groups, whereas little or none of the IFNy and IL-10 infected mice died and no pathological disorders were detectable (284). These findings suggest that the early viral replication is the most important factor in determining end organ disease and that modulation of a specific immune response is beneficial to the virus. These studies also seem to dispute the molecular mimicry and autoimmune hypotheses as there is little or no end organ disease in animals that are re-challenged with a virulent virus after the host has seen the viral antigen in the presence of proinflammatory cytokines. Prior to the wide availability of genetically modified mice, many experiments were conducted using immunomodulators such as recombinant cytokines and immunosuppressants such as cyclosporine and FK506 which were added prior to infection of mice. Similar to IFNy transgenics as described above, investigators have demonstrated that the addition of exogenous IFNy to animals greatly decreased the severity of end organ disease following CVB3 infection probably by limiting early viral replication and dissemination (378, 380, 478). In vivo administration of other cytokines including colony stimulating factor (290) and IL-2 (380) have demonstrated positive effects on limiting severity of myocarditis suggesting that enhancing the 26 immune system against viral infection is beneficial. It should be noted that IL-2 administration beyond the acute phase of the disease (day 7) increases the number of T cells infiltrating the myocardium (380). Depletion of NK cells by the injection of anti-asialo GM1 antiserum in mice increased CVB3 titers in the heart, exacerbated myocarditis with increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3 only (236, 237). Immunosuppression by the administration of compounds including cyclophosphamide (381), FK506 (287, 491), cyclosporine (377, 531) increased animal mortality and myocyte necrosis presumably by allowing unchecked viral replication and enhanced tissue injury. The relationship of these immune studies in mice to man can be highlighted by the Human Myocarditis Treatment Trial, which commenced before much of the experimental data supporting the direct viral injury hypothesis was available. This trial was a major multicentre, randomized study to evaluate the effectiveness of immunosuppressive therapy in the treatment of human myocarditis. No benefit was observed for immunosuppressives on patient survival or on ventricular function as evaluated by exercise times (473). It should also be noted that clinicians did not try to distinguish between viral myocarditis and myocarditis caused by other factors. It is still debated whether or not there are beneficial aspects to immunosuppressive therapy in humans who have episodes of myocarditis cause by unknown mechanisms. In mice, it has become clear that the extent of viral replication and dissemination of infectious virus to systemic organs early in disease is the best indicator of disease severity. Any attempt to inhibit the immune response or depress the generation of anti-viral antibodies is detrimental to the host. The role of the cellular immune response on viral clearance and immunopathologic tissue injury is more controversial and needs to be further dissected. One important question that has not been adequately addressed is whether or not the immune system is perturbed by viral infection. Early investigators noticed pronounced splenic and thymic atrophy by day 4 following infection (59, 479). Molecular techniques like ISH subsequently allowed investigators to note that there was significant localization of virus to the 27 white pulp regions of the spleen including germinal centres and the marginal zone (16, 384). In addition, a variety of investigators have noted that CVB3 can infect hematopoietic cells including T cells, B cells, and macrophages (16, 243, 499, 727, 728). Although the virus may only replicate to a limited degree in these cells, there is viral attachment to the cell surface. As described in Section 1.3.1, the virus can attach to DAF or CAR on the surface of cells, but CAR is required for productive infection. Studies on human hematopoietic cells have demonstrated that CAR is expressed on approximately 40% of all human bone marrow cells, including erythroid and myeloid cells, but not lymphoid cells (583). Potentially, infection of progenitor cells may lead to the marked involution of the lymphoid organs, perhaps due to apoptosis, as enhanced apoptosis is noted with in these organs as will be demonstrated in Section 9.1. There may be alternative mechanisms of infection of the lymphocytes including endocytosis of CVB3-DAF interactions and productive infection following Ag-CVB3 association with Fc receptors on macrophages and antigen presenting cells. These associations lead to the question of whether or not this association, and limited replication within these cells may alter normal immune surveillance or inhibit normal immune responses? Or whether these associations lead to dissemination of virus to end organs. In the B cell deficient mice there was delayed CVB3 dissemination to target organs (499), suggesting that B cells may play a role in early viral trafficking. The virus association with, and infection of hematopoetic cells may influence the systemic immune response to CVB3 antigen. Bendinelli et al. observed a decreased antibody response unrelated to third party antigens in infected mice and a depressed cellular mediated responses (60). Chatterjee et al. (114) observed dramatic decreases in B and T cell repertoires following CVB4 infection as compared to sham-infected controls in the spleen and periphery by 8 weeks pi. CD8+ cells decreased by 72 hour (h) pi and had decreased to 40% of the controls by 8 weeks pi. In the periphery at 8 weeks pi, there was a 74% increase in B lymphocytes while the T lymphocytes had decreased by 11% of control levels. These decreases in immune cell numbers contradict the typical virus-immune response paradigm as demonstrated by other 28 intracellular pathogens like LCMV where there is significant increase in immune cell numbers and lymphoid hypertrophy early following infection followed by long term return to normal state (582). Infection of monocytes by CVB3 resulted in the upregulation of mRNA for IL-1B, IL-6 and TNF-alpha, but challenge with LPS demonstrated that they were unreactive to further stimuli (283). Positive and negative strand RNA was found in monocytes and macrophages and this persisted for up to 10 days following infection (292). Additional studies determined that although mRNA for IL-10, IL-6 and TNF-alpha was present, little cytokine protein was released, whereas there was intense and persistent IL-10 expression (292). Importantly, the intense expression of IL-10 was confirmed in vivo in infected mice in the heart and myocardium through the acute phase of the disease (620). The cytokine profiles of the spleen and heart were comparable, with high expression of TNF-alpha, IL-1alpha, IL-10, IL-12, and IFN-gamma. IL-2 and IL-4 expression remained minimal (620). Another group confirmed the IL-10 expression induced by other enteroviruses including CVB4 and poliovirus type 1 in freshly isolated leukocytes (725). These finding suggest the possibility that IL-10 may play a role in acute and chronic myocarditis by down regulating the immune response (620). Work in Section 9.1 will show the massive apoptosis of the lymphoid organs during CVB3 infection of mice. Massive is defined as large numbers of TUNEL positive foci above the normal apoptosis as seen in sham-injected animals. The cortical CD4/CD8 double positive cells of the thymus are depleted as shown by flow cytometry. Further, examination of IL-2 and IL-4 cytokine protein within these animals demonstrates that there is a marked decrease in levels during the early acute phase as compared with sham treated animals. Overall, the role of the immune system appears to be beneficial for clearance of infectious virus. There is little controversy regarding the innate and humoral immune response. Greater clarity needs to be determined for the role of the cellular arm. It is also clear that the association of CVB3 with the cellular aspects of the immune system may directly or indirectly depress the immune response and may allow for greater persistence of infectious virus in systemic organs. 29 2.2. The link between myocarditis and dilated cardiomyopathy The hypothesis that myocarditis is an early indicator of, or subsequently leads to, dilated cardiomyopathy in humans, has been fuelled by work on viruses that induce myocarditis. Cardiomyopathy may be defined most simply as an illness of heart muscle leading to clinically significant dysfunction. Typically, heart muscle disease due to infarction, occurring secondary to coronary atherosclerotic disease, is excluded from such a definition. However, dysfunction of an acute, persistent, or chronic nature, arising on an idiopathic or secondary, nonischemic basis, includes a number of known cardiomyopathies. Dilated, hypertrophic and restrictive forms may be responsible for heart failure (178, 221, 593), however dilated cardiomyopathy is the leading cause of failure among these conditions. Evidence linking enterovirus infections, most notably CVB3 with dilated cardiomyopathy comes from several sources. The main two sources of evidence include molecular evidence of enterovirus genome in the myocardium of idiopathic dilated cardiomyopathy (IDC) patients and parallels between the phenotype of human cardiomyopathy and the phenotype of late stage CVB3 and encephalomyocarditis (EMC) virus-induced myocarditis. As mentioned, there is significant molecular evidence linking enterovirus infection to dilated cardiomyopathy in humans (Table 1). 30 TABLE 1: Evidence of Enterovirus genome in I DC Patients Study and year published Dilated cardiomyopathy Samples Control Samples #of patients positive for enterovirus # of patients % # of patients positive for enterovirus #of patients % Rey L et al 2001 (589) 21 55 38 0 55 0 Fujioka S et al 2000 (219) 9 26 34 N/A N/A Andreoletti L et al (17) 25 70 35 0 45 0 Grumbach IM et al 1998 (262) 7 23 30 N/A N/A Archard LC et al 1998 (25) 9 21 42 1 14 7 Satoh Metal 1994 (614) 17 35 48 0 10 0 Schwaiger A et al 1993 (623) 6 19 31 0 21 0 Crespo-Leiro MG et al 2000(154) 1 22 4 0 70 0 Li Y etal 2000 (431) 3 8 37 0 11 0 Giacca M etal 1994 (233) 4 53 7 0 21 0 Total 102 332 30 1 247 0.4 A cross-examination of various reports demonstrates that approximately 30% of clinical IDC cases contain enterovirus RNA in the myocardium as detected by PCR. Obviously, sampling and PCR issues must be taken into account in each report, but this rough estimate is enough to support a hypothesis that enterovirus infection may increase the susceptibility to, or lead to dilated cardiomyopathy in certain individuals. Using the murine model of enterovirus myocarditis, numerous investigators have noted significant changes in the myocardium during the chronic phase of the disease that resemble the cardiomyopathy state of either humans or genetically modified mice (as in disruption of the dystrophin gene). The acute and early chronic phase of infection results in significant myocyte loss due to direct viral injury and the cytotoxic branches of the immune system. The simple mechanics of myocyte loss, the subsequent remodeling of the myocardium, and increased workload of the remaining myocytes suggest that the heart muscle itself will undergo adaptive responses that may appear as dilated cardiomyopathy. Calcification and fibrosis in the myocardium begins around day 7 pi and may persist through the late chronic phases of the 31 disease in certain animals including more than eighteen months following infection in some studies (360, 407, 476). There is significant variation in the size and severity of lesions and amount of calcification and fibrosis with different murine strains or CVB3 variants (140). Other chronic changes indicative of dilative cardiomyopathy noticed in the murine model of myocarditis are increased heart weight/body weight ratios and ventricular dilation (345, 499, 514, 749), and extracellular remodeling (244, 379). In Section 9.4 we will examine host genes that change their expression following infection and which may play a role in the dilated cardiomyopathic phenotype. Many of the chronic changes indicative of dilated cardiomyopathy could be caused by the virus itself independent of an immune response. Researchers have found prolonged viral RNA presence in the myocardium of mice up to 90 days pi in some studies (384, 586). This persistent RNA may be residual RNA left in surviving myocytes, or may represent low grade, restricted replication in a limited number of cells. Transgenic mice that express the replication restricted CVB3 genome under regulation of the myosin light chain-2v promoter to induce myocyte specific expression had significant changes in heart morphology. These mice had marked interstitial fibrosis, myocyte degeneration, ventricular dilation, expression of ANF mRNA, all of which are characteristic of dilated cardiomyopathy and a failing heart (749). One important regulatory protein that is directly altered by the virus and may cause a cardiomyopathy phenotype is dystrophin. Dystrophin is a cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy (57). Following infection, dystrophin is cleaved by enterovirus protease 2A causing functional impairment and morphological changes of in the myocardium (37) as discussed in Section 1.3. This cleavage disrupts the dystrophin-associated glycoproteins interaction with the sarcolemma (417) in a similar fashion to the genetic dystrophin truncations. The absence of the glycoprotein associations with the sarcolemma via disrupted dystrophin can depress transmission of mechanical force from the sarcomere to the extracellular matrix (417). 32 3 CELL DEATH 3.1. Apoptosis and necrosis: Perspectives, morphologic characteristics, ion regulation, and common themes In 1965 using a model of hepatic ischemia, Kerr had the first definitive report of cells dying by a process different than necrosis (369). He noted that there were cells with parenchymal shrinkage and pyknotic chromatin (369) and intact lysosomes and organelles (368), which together he termed 'shrinkage necrosis' (370). Kerr, Wyllie, and Currie later called this process 'apoptosis' (greek for leaves falling from the tree), as a distinct form of cell death from necrosis (371). Morphologic and basic nuclear alterations of cells including chromatin condensation with DNA fragmentation, cell shrinkage, and maintenance of membrane integrity have been used to describe apoptosis (456). In contrast, necrosis/oncosis is defined by cell swelling, loss of membrane integrity with random non-specific cellular degradation (456). Consistent with release of cellular debris and lysosomal contents, an inflammatory infiltrate is attracted to the site of necrosis (624). Unlike apoptosis, the nucleus swells and remains somewhat intact until the later phases of necrosis when the DNA is degraded into a heterogenous mixture that can be visualized as a smear by agarose gel electrophoresis (664). In the mid 1990's, elucidation of the biochemistry of cell death pathways has defined apoptosis as caspase activation with cleavage of specific substrates, resulting in loss of cell homeostasis and distinctive morphological changes (526). Data suggest certain convergent common mechanisms and an overlap of features of death by apoptosis and necrosis (638, 704). It is becoming clear that the mitochondrion plays an important role in both apoptosis and necrosis. Apoptosis and necrosis share loss of mitochondrial membrane potential (470, 789), the generation of reactive oxygen species (100, 788), the inhibition of oxidative phosphyorlyation, the ETC and generation of dATP (257), and the release of apoptosis-inducing factor (AIF) (172). Apoptosis may simply involve further cell signalling that activates the caspase proteolytic cascade (AIF is caspase independent) which would result in rapid disruption 33 of normal homeostasis and marking of cells for rapid phagocytosis. Inhibition of the caspase cascade during apoptosis eventually results in necrosis. 3.2. Biochemical aspects of cell death In the mid to late 1990's, numerous cellular proteins were discovered to play important roles in cell death pathways. Some of these proteins were initially discovered as mammalian homologs to Caenorhabditis elegans proteins that were identified as key molecules in programmed cell death and development of the worm. In the worm three genes, Ced-3, Ced-4, and Ced-9 are intimately involved in controlling apoptosis during embryonic development. Ced-3 is a killer gene, Ced-9 is a survival gene that protects cells from inappropriate death, and the Ced 4 is the connecting element between the two proteins. Yeast-two hybrid technology, DNA databases (within and between species), antibody technology, and the generation of transgenic and knockout mice greatly facilitated the elucidation of the main cell death pathways. Many of the proteins discovered were new proteins, others were previously identified proteins with no known function, and yet others were surprisingly found to be known proteins that had newly recognized death functions in addition to their functions as previously described. The proteins identified can be arbitrarily divided into the following classes based on function; extracellular ligands and their cognate death receptors, adapter proteins, regulatory proteins and factors, cysteine proteases, macromolecule substrates, and phagocytosis markers. There are two main prototypical pathways for apoptosis that have been elucidated. Apoptosis may be initiated by ligand interaction with death receptors on the surface of cells, or by cytotoxic stimuli within cells that initiate apoptosis via the mitochondria (Figure 5). Independent of the inducing stimuli, apoptosis pathways converge on the activation of downstream cysteine proteases, now known as caspases (12), and in turn on the cleavage of specific substrates that result in the phenotype of apoptosis. While these are the core pathways, apoptosis mechanisms should not be considered black and white, since much attention is focused on the cross talk between the two apoptosis pathways, the interaction of apoptosis pathways with cell signalling pathways, and the difference in apoptosis susceptibility and signalling in different cell types. 34 Figure 5 Schematic demonstrating the two primary mechanisms of apoptosis induction. Apoptosis may be initiated by ligand interaction with death receptors on the surface of cells, or by cytotoxic stimuli within cells that initiate apoptosis via release of factors from the mitochondria. Regardless of which pathway is activated, there is a common executioner phase that results in the characteristic phenotypic features of apoptosis. 35 3.2.1. The Caspases Numerous mammalian caspases have been identified (526, 606, 692). These proteins are well conserved throughout evolution, originally identified as a homologs to the C. etegans death protein Ced-3 (784). Since this time, at least 14 caspases have been identified in mammals (694). The mammalian caspases are constitutively expressed in cell cytosolic and organellar fractions (109, 457) as catalytically inactive proenzymes. Following death stimuli and proenzyme processing, the catalytically active enzyme forms a heterotetramer, where two heterodimers (consisting of a large -20 kDa and small -10 kDa subunit) associate to form an active tetramer (Figure 6). Caspases have been arbitrarily divided into two groups based on their cellular function. Caspases involved in processing of proinflammatory cytokines and not required for apoptosis include caspase 1, 4, 5, and 11. Caspases involved in the promotion of cell death include caspase 2, 3, 6, 7, 8, 9, and 10. Caspases have either a short or long amino terminal pro-domain. Caspases containing long prodomains are important in the initiation of cell death either from death signaling from the surface of cells or through the release of death-inducing factors from the mitochondrion. Long prodomain caspases contain either a caspase activation recruitment domain (CARD) (caspase 2, 4 and 9) or a death effector domain (DED) (caspase 8, and 10). By using a substrate combinational library to determine the amino termini tetrapeptide specificities, amplification of the caspase cascade can be mapped out (693). Group 1 caspases (caspase 1, 4, and 5) all have high preference for the tetrapeptide sequence WEHD. Group 2 caspases (caspase 2, 3, 7, and Ced-3) prefer the tetrapeptide sequence DExD and group 3 caspases (caspase 6, 8, 9 and 10) prefer the sequence (l/L/V)ExD. Based on function and probable order of activation, group 1 caspases play a predominant role in inflammation (526), while those in group 2 are responsible for cleavage of macromolecular substrates. Those in group 3 are responsible for activation and amplification of group 2 caspases. 36 Caspases (Cysteinyl aspartate-specific protienases) Activation of proinflammatory cytokines Promotion of cell death Caspases 1, 4, 5, 11, 12, 13, 14 Caspases 2, 3, 6, 7, 8, 9, 10 Stabilization Destabilization Active enzyme Heterotetramer Figure 6 Schematic of the Caspases. Caspases involved in processing of proinflammatory cytokines and not required for apoptosis include caspase 1, 4, 5, 11, 12, 13, and 14. Caspases involved in the promotion of cell death include caspase 2, 3, 6, 7, 8, 9, and 10. Following death stimuli and proenzyme processing, the catalytically active enzyme forms a heterotetramer, where two heterodimers (consisting of a large ~20kDa and small ~10kDa subunit) associate to form an active tetramer. The cellular lAPs prevent formation of active heterotetramers. 37 3.2.2. Caspase activation Three distinct biochemical pathways can initiate apoptosis. First, death receptors of the TNF receptor gene superfamily bring together accessory proteins to form a death inducing signaling complex (DISC) at the cytoplasmic face of the plasmalemma following cognate ligand binding. Second, granzyme B, through cooperating with perforin, can enter a cell thorough the plasmalemma and activate caspase 10. Third, other apoptosis-inducing agents cause the release of mitochondrial factors from their normal site between the inner and outer membrane to the cytosol resulting in oligomerization of caspases in the cytosol and subsequent activation in the presence of dATP. 3.2.3. Cell surface signaling The TNF receptor gene superfamily includes a large number of surface proteins that when activated by specific ligand can induce a wide variety of cellular functions including activation, proliferation, differentiation, apoptosis or survival (Figure 7). The cell surface death receptors belong to the tumor necrosis factor (TNF) receptor gene superfamily and include Fas (Apo1, CD95), TNF-R1, DR3, TRAIL-R1 (DR4), and TRAIL-R2 (DR5), among others. The death receptors contain a homologous cytoplasmic death domain (683). Ligands belonging to the TNF gene superfamily such as Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL, APO-2L) and, TNF-a itself, bind to specific cell surface receptors (29) and have the ability to induce apoptosis. Following specific ligand binding to death receptors on the cell surface, a DISC is formed on the cytoplasmic face of the plasmalemma that recruits caspase 8 via a DED (510). Oligomerization and bringing caspases into close proximity is now understood as a key component of caspase activation. Experiments with transgenic and gene knockout mice have shown that caspase 8 and its adaptor FADD are needed for Fas and TNF-RI-induced apoptosis (which also requires TRADD as a second adaptor protein), but they are dispensable for mitochondrially-initiated apoptosis (778, 797) Fas-mediated apoptosis may be triggered by the binding of its natural ligand (FasL) or agonistic anti-Fas antibodies (662). Fas is expressed by many different cell types and its 38 TNF receptor superfamily Ligands TNF-alpha LT-alpha FasL TRAIL CD40L CD27L TNFR1 TNFR2 CD40 Fas TRAIL-R1 TRAIL-R2 TRAIL-R4 activation proliferation differentiation apoptosis survival Apoptosis Figure 7 A) The TNF receptor superfamily. The TNF receptor gene superfamily includes a large number of surface proteins that when activated by specific ligand can induce a wide variety of cellular functions including activation, proliferation, differentiation, apoptosis or survival. B) TRAIL can bind to at least five receptors in the TNF-receptor gene superfamily. TRAIL-R1 and TRAIL-R2 contain a death domain in their cytoplasmic domain while TRAIL-R3 does not contain a cytoplasmic domain and TRAIL-R4 does not contain a death domain and is not believed to initiate apoptosis 39 presence signifies that these cells may be receptive to apoptosis-inducing signals from FasL-bearing cells. In the periphery, Fas-FasL interactions serve to limit the proliferation and control senescence of activated T cells, promote the killing of virus-infected cells by cytotoxic T cells, and contribute to the maintenance of sites of immune privilege in different tissues by imperiling the survival of activated inflammatory cells (511). As will be discussed in Section 4.2 and 5.1, the expression and activity of Fas and Fas L which can be influenced by various viruses and in specific organs like the heart during states of viral infection, stress, injury, and disease. TNF may induce apoptosis in a wide range of cell types bearing the TNFR-1 (299). However, cells may be protected form the lethal effects of TNF through the nuclear translocation of the transcription factor, nuclear factor kappa B ( N F - K B ) (712, 732). N F - K B activates the transcription of genes encoding various pro-inflammatory and anti-apoptotic factors as will be discussed in Section 3.3 (141, 733). Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was identified as a protein having high homology to Fas Ligand (568, 754). TRAIL can bind to at least five receptors in the TNF-receptor gene superfamily (29). TRAIL-receptor 1 (TRAIL-R1) and TRAIL-R2 contain a cytoplasmic death domain while TRAIL-R3 does not contain a cytoplasmic domain and TRAIL-R4 does not contain a death domain and is not believed to initiate apoptosis (132, 452, 550, 551, 729) (Figure 7). TRAIL appears to preferentially induce apoptosis in transformed and virus infected, but not normal cell lines (30, 730). It was initially postulated that TRAIL susceptibility required TRAIL-R1 and/or TRAIL-R2 and the presence of TRAIL-R3 or TRAIL-R4 acted as decoy receptors and prevented apoptosis. However, there are cells that contain TRAIL-R1 or TRAIL-R2 but no decoy receptors but are resistant to TRAIL-mediated death and there are also cells that contain decoy receptors that are susceptible to TRAIL-mediated death (799). The susceptibility to TRAIL-mediated apoptosis may be a complex process that can be regulated at the surface receptors, death receptor regulatory proteins, and signaling molecules. 40 3.2.4. Mitochondrial induced death Various intracellular cytotoxic stimuli including ionizing radiation (117), staurosporine, high-dose Ara-C (HIDAC) (375), etoposide (468) or photodynamic therapy (103), among others, can induce the release of cytochrome c, Smac/Diablo, apoptosis-inducing factor (AIF) and other factors from the inter-mitochondrial membrane space to the cytosol. The release of these factors from the inter-mitochondrial space is under regulation of Bcl-2 family proteins (385, 774) and will be described in Section 3.3. Cyotchrome c and Smac/Diablo are both involved in the activation of procaspase 9. AIF triggers caspase independent apoptotic events in the nucleus (667). Mitochondrial cytochrome c can associate with apaf-1 in the presence of dATP and induce a conformational change that allows this complex to associate with the caspase 9 prodomain CARD sequence and induce auto-activation (652). Mice lacking functional apaf-1 die during development and have severe developmental malformations including craniofacial abnormalities, brain overgrowth, persistence of the interdigital webs emphasizing the importance of this adapter protein in programmed cell death (107, 782). Cells from these mice are susceptible to death receptor-induced death but resistant to many other inducers of cell death (107, 782). Mice with disrupted caspase 9 show similar embryonic lethality and brain malformations with cells showing resistance to a variety of apoptosis inducers (267, 406). Smac/Diablo is a negative regulator of the lAP's which are expressed in the cytosol of cells and inhibit the processing of caspase 9 and the activity of active caspase 3 and 7. Release of Smac/Diablo removes the lAP's from the caspases and allows for their subsequent activation or activity. 3.2.5. Effector caspases and cross talk between cell surface receptor activation and the mitochondria Active caspase 9 and caspase 8 can directly activate procaspase 3 and procaspase 7 (549). Activation of these caspases results in the degradation and inactivation (and activation in certain cases) of a variety of proteins that results in functional and phenotypic alterations as will be discussed in Section 3.4. 41 Amplification of the proteolytic cascade can involve mobilization of more than one pathway (cross talk). Active caspase 8 can directly activate effector caspases but it can also release apoptosis activating factors from the inter-mitochondrial membrane. Bid, a death agonist containing a Bcl-2 homology (BH) domain (735) has been shown to be cleaved by caspase 8 (427, 450). The degradation of Bid results in release of cytochrome c from the mitochondria and further amplification of the death cascade by activation of caspase 9. Bid degradation by caspase 8 cleavage results in the liberation of a protein localization signal and subsequent migration to the mitochondria where it directly interacts with Bak and Bax and facilitates release of cytochrome c from the intermitochondrial membrane space to the cytosol. This release occurs as Bid oligomerizes Bak and Bax and forms a pore in the outer mitochondrial membrane (743). Alternatively, caspase 8 can be rapidly mobilized in models of cell death originating from mitochondrial stimuli as previously described (103). 3.3. Regulation of cell death Regulation of apoptosis induction can occur at many levels including protein-protein interactions, protein localization, gene transcription, protein translation, phosphorylation and dephosphorylation of key proteins in the apoptosis pathways or associated pathways, among others. Three important aspects of anti-apoptotic signaling will be discussed: First, there are numerous proteins within the cell that directly or indirectly prevent caspase activation. Second, the expression profile of these proteins may be influenced by other signaling events that regulate gene transcription and protein translation. Third, proteins involved in the induction of apoptosis or the protection from apoptosis may be phosphorylated by signaling pathways that either stimulate or inhibit their function. Signaling by reversible protein phosphorylation is a major mechanism of signal transmission within the cell. Protein kinases add y-phosphate from ATP to target proteins and enzymes. Most protein kinases are those specific for serine/threonine or those specific for tyrosine residues. The classic example of regulation of cell death involves Bcl-2, the mammalian homolog to the C. elegans CED-9 protein. Bcl-2 was initially discovered as a chromosome 18 gene in a 42 14:18 chromosome translocation (558, 703) and was shown to be a prosurvival gene, not a proliferative gene (720). Multiple proteins containing Bcl-2 homology (BH1-4) domains have been discovered and are either grouped as pro-survival or pro-death proteins based on functional regulation of cell death (2, 585) (Figure 8). Mammalian Bcl-xL (77), A1/Bfl-1 (355, 356), Mcl-1 (396), Boo/Diva (320, 648), and many viral homologs (See Section 4.2.1) promote cell survival, while Bax (543), Bcl-xs (77), Bak (135, 205, 374) enhance apoptosis. The prosurvival Bcl-2 homologs have three or four regions with extensive amino acid sequence similarity to Bcl-2 while the death enhancers contain two or three similar domains. Another Bcl-2 subgroup of potent death inducers contain only a BH3 domain with no other resemblance to Bcl-2 or other known protein include Bad (773), Bik/Nbk (83), Bid (735), Hrk/DP5 (735), Bim/Bod (301), and Blk (275). Bcl-2 localizes to the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes (400). Similar subcellular localization has been reported for Bcl-xL, A1, Mcl-1, Bax, and Bak (398, 399). Over-expression of the Bcl-2 and Bcl-xL proteins protects against or delays the induction of apoptosis (110) by preventing the release of cytochrome c and apoptosis inducing factors from the inter-mitochondrial membrane space to the cytosol (386, 774), the generation of reactive oxygen species (350), and mitochondrial membrane permeability transition (179, 470). In contrast, over-expression of Bax induces apoptosis (543) and this Bcl-2 family member directly induces mitochondrial cytochrome c release (339). The mechanisms of cytochrome c release and Bcl-2 regulation have only recently emerged. Early hypotheses suggested that cytochrome c release could be direct through a specific membrane channel or through mitochondrial membrane rupture as a result of ion dysregulation (584, 718). Bcl-2 and BCI -XL may produce their anti-apoptotic effect by acting as pore forming proteins that regulate the flux of ions or proteins, through homo- or heterodimerization with other Bcl-2 family members near mitochondrial membranes or in the cytosol. As mentioned, it appears as though the pro-apoptotic members Bax and Bak form apoptotic clusters at the surface of the mitochondrial membrane (521): The BH3 only containing pro-apoptotic members Bik/Nbk, Bid, Bim, and 43 Anti-apoptotic members A BH4 BH3 BH1 BH2 B BH4 BH1 BH2 Pro-apoptotic members C BH1 BH2 BH4 Bcl-2 Bcl-x L Mcl-1 Bcl-w A-1 Boo Bax Bak Bok Bcl-x s Blk Hrk/DP5 Bim/Bod Bik/Nbk Bad Bid > g-Q. C/) 03 a a ; (ft m I I m p ° T | 03 O r-t-< 03 t—t-CD o Caspase 8 O o c w /-*• CD —1 03 CL —1 CD 0) 03 C/3 CD O >< o Figure 8 The Bcl-2 family of proteins. Multiple proteins containing Bcl-2 homology (BH1 -4) domains have been discovered and are either grouped as pro-survival or pro-death proteins based on functional regulation of cell death. Bcl-xL, A1/BA-1, Mcl-1, Boo/Diva promote cell survival, while Bax, Bcl-xs, Bak enhance apoptosis. The prosurvival Bcl-2 homologs have three or four regions with extensive amino acid sequence similarity to Bcl-2 while the death enhancers contain two or three similar domains. Another group of potent death inducers contain only a BH3 domain with no other resemblance to Bcl-2 or other known protein include Bad, Bik/Nbk, Bid, Hrk/DP5, Bim/Bod, and Blk. 44 Hrk/DP5 bind to other pro and anti-apoptotic Bcl-2 family members (83, 301, 735), and appear to antagonize or facilitate the respective anti or pro-apoptotic effects (126, 211, 813). The emerging model is that the anti-apoptotic members sequester the BH3 only containing pro-apoptotic members and prevent them from activating the multi-domain pro-apoptotic memebers (Bax, Bak, Bok) into forming large protein aggregates leading to the release of apoptosis-inducing factors from the mitochondria (126). Bax has been shown to directly induce cytochrome c release from mitochondria (339) and this is dependent on Bax association with the adenine nucleotide transporter (ANT), a component of the permeability transition pore complex (PTPC) (469). Others have suggested that Bax does not bind ANT, but requires the voltage-dependant anion channel (VDAC) (639, 641). VDAC, ANT, and the mitochondrial benzodiazepine receptor (mBzR) are located at the junctional site of the outer and inner membrane of the mitochondria to form the putative mitochondrial PTPC (487, 671, 672). Bax induction of apoptosis is associated with its movement from the cytosol to the mitocondrial membrane during apoptosis (260, 525), and subcellular redistribution is a mechanism of the regulating activity of other Bcl-2 including members Bid (261). As mentioned, redistribution of Bax, and the BH3-only members appears to facilitate the association of Bak and Bax into large clusters that are required for cytochrome c release. Cells deficient with both Bax and Bak are unable to release cytochrome c and fail to undergo apoptosis induced by a variety of stimuli (126). Bcl-2 and Bcl-xL may not only inhibit apoptosis at the mitochondrial membrane by sequestering pro-apoptotic members, but may also have other anti-apoptosis mechanisms. Evidence suggests that Bcl-xL can bind cytochrome c (373) and apaf-1 (307) thus having a dual roles in decreasing free cytosolic cytochrome c and preventing apaf-1 interaction with activating factors and caspase 9. In the C. elegans system of cell death Ced 4 (apaf-1 homolog) activates Ced 3 (caspase homolog), and is inhibited by Ced 9 (Bcl-xL homolog) binding to Ced 4 (131, 775). Microinjection of cytochrome c into cells results in apoptosis but over-expression of Bcl-xL can prevent this induction (426) suggesting a further cytoprotective role for Bcl-xL once 45 cytochrome c has been released to the cytosol. Work by Carthy et al. and summarized in Section 9.3, using photodynamic therapy and cells overexpressing Bcl-2 and Bcl-xL has clearly demonstrated that Bcl-2 and Bcl-xL can protect against caspase activation after cytochrome c has been released to the cytosol (103). With the large number of proteins identified with BH domains and the identification of phosphorylation sites on these proteins (described later in this section), more roles for Bcl-2 family in regulating apoptosis are being elucidated. Whether or not BCI -XL or Bcl-2 can prevent caspase interaction with Apaf-1 in the cytosol is disputed by different groups. Diva/Boo, a Bcl-2 homolog that is missing the BH3 domain was shown to bind to Apaf-1 and appears to be negative regulator of caspase activation (648). A number of proteins exist that share high homology to caspases, but cannot be processed into active enzymes, and other proteins exist that directly associate with active caspases and inhibit their function (Figure 9). A group of proteins, initially discovered as virus-encoded gene products (as will be discussed in Section 4.2.1) (305, 691) inhibits signaling through death receptors (322). These protein contains DED's and competes with caspase 8 and 10 for binding to the DED regions of FADD (322). Binding of these DED containing proteins to FADD prevent caspase 8 or 10 recruitment to the DISC and inhibiting subsequent autoactivation. Many names for these proteins exist including; FLIP, l-FLICE, FLAME-1, CLARP, casper, CASH, and MRIT (242, 270, 306, 321, 322, 645, 655). Other competitive intrinsic caspase inhibitors include ARC (apoptosis repressor with CARD) (393) which like FLIP'S, contains a CARD sequence and competes with caspase 2. Capase-9S/9b/CTD is a dominant negative inhibitor of caspase 9 as it is missing the catalytic site but is able to bind Apaf-1 and block Apaf-1-caspase 9 interaction (20, 631, 654). Several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. Hsp70 and Hsp90 bind to Apaf-1 and prevent the recruitment of procaspase-9 to the apoptosome, thereby inhibiting caspase-9 activation (55, 552, 605). Hsp27, a member of the small HSP family, has been shown to bind/sequester cytosolic cytochrome c from the 46 Caspase 2 Figure 9 Schematic of proteins that prevent caspase activation by either acting as decoys for apoptosis inducing factors, directly binding to caspases and preventing their processing, or directly associating with active caspases and inhibiting their activity. FLIP'S contain contain DED's and compete with caspase 8 and 10 for binding to the DED regions of FADD. ARC contains a CARD sequence and competes with Caspase 2. Capase-9S/9b/CTD is dominant negative inhibitor of caspase 9 as it is able to bind Apaf-1 and block Apaf-1-caspase 9 interaction. Hsp70 and Hsp90 bind to Apaf-1 and prevent the recruitment of procaspase-9 to the apoptosome. Hsp27, a member of the small HSP family, has been shown to bind/sequester cytosolic cytochrome c. lAP's prevent the processing of caspase 9 and inhibit the enzyme activity of caspase 3 and 7. 47 apoptosome and prevent procaspase-9 activation (90, 224). Of potential relevance here is my finding as discussed in Section 10.4, that Hsp 27 is upregulated in vivo during CVB3 infection of mice as determined by cDNA microarray analysis. This upregulation may serve to inhibit apoptosis within the myocardium of infected myocytes. lAP's (inhibitors of apoptosis) (191, 436, 711) can inhibit the activation of caspase 9, 3, and 7 by different mechanisms. It was shown that these proteins can inhibit the proteolytic processing of caspase 9 but not caspase 3 (182), but the lAP's could inhibit the active enzyme activity of caspase 3 and 7 (182). The defining structural motifs of the IAP protein family are the BIR (baculovirus AIP repeat) domain and a carboxy-terminal RING zinc-finger (436). Analysis with various truncated proteins has shown that the BIR domains are required for apoptosis inhibition by binding the active sites of 3 and 7 (108, 308, 590, 669). A negative regulator of these inhibitors is Diablo/Smac which is released from the intermitochondrial membrane space to the cytosol and prevents lAPs from suppressing caspase activity, thus demonstrating the importance of protein localization in regulation of cell death (190, 723). Activation of the N F K B pathway increases the transcription of proinflammatory and prosurvival genes including c-IAPI and C-IAP2, Traf 1 and Traf 2, A1/BA-1, and Bcl-xL(141, 733, 760), among others. Intracellular stimuli such as Ras-Akt/PKB kinase pathway cross-talk or death receptors in the tumor necrosis factor receptor superfamily such as TNF-R1, TRAIL-R1, TRAIL-R2 have the potential to activate the N F K B pathway. There are 5 known transcription factors of the N F - K B family: p50/p105 (N F - K B I ) , p52/p100 (NF-KB2), c-Rel, RelB, and p65 (RelA). Various inhibitory IKB proteins bind to the N F - K B proteins and act to retain them in the cytoplasm. The cytoplasmic complexes are prevent regulation of transcription until an appropriate stimulus (LPS, proinflammatory cytokines) activate the IKB kinases (IKKa and IKKB) which phosphorylate the IKB proteins on 2 serine residues. Phosphorylation of the IKB proteins results in ubiquitination and subsequent proteolysis by the 26S proteosome. Liberated N F - K B translocates to the nucleus where it activates various genes including the anti-apoptotic genes previously mentioned (Figure 10). The difference in the ability of a receptor to induce apoptosis 48 Figure 10. Schematic of NF Kappa B activation and subsequent upregulation of anti-apoptotic genes. Activation of NF Kappa B increases the transcription of proinflammatory and prosurvival genes including c-IAPI and C-IAP2, Traf 1 and Traf 2, AI/Bfl-1, and Bcl-xL, among others. Intracellular stimuli such as kinase pathway cross-talk (Ras-Akt/PKB) or death receptors in the tumor necrosis factor receptor superfamily such as TNF-R1, TRAIL-R1, TRAIL-R2 have the potential to activate the NF Kappa B pathway. There are 5 known transcription factors of the NF Kappa B family: p50/p105 (NF-Kappa B1), p52/p100 (NF-Kappa B2), c-Rel, RelB, and p65 (RelA). Various inhibitory I Kappa B proteins bind to the NF-Kappa B proteins and act to retain them in the cytoplasm. The cytoplasmic complexes are transcriptionally inactive. Phosphorylated I Kappa B proteins on 2 serine residues. Phosphorylation results in rapid ubiquitination and subsequent proteolysis by the 26S proteosome. Liberated NF-Kappa B translocates to the nucleus where it transactivates gene transcription. 49 or N F K B depends on the cell type, expression levels of different apoptosis inhibitors such as FLIP'S, and the inciting ligand. Many growth and trophic factors including insulin growth factor-l (IGF-1), vascular endothelial growth factor (VEGF) (230), nerve growth factor exert their anti-apoptotic action through PKB/Akt-mediated phosphorylation of specific apoptosis regulating proteins (Figure 11). Binding of these factors to receptor tyrosine kinases elicits the recruitment of PI3K to the plasmalemma and subsequent activation of Akt/PKB which can deliver direct or indirect anti-apoptotic signals to various substrates (204, 366, 562). PKB/Akt can phosphorylate the Bcl-2 family member Bad (171, 181) thereby preventing the pro-apoptotic function of this protein. Akt phosphorylation of BAD at serine 136 causes BAD sequestration in the cytoplasm by 14-3-3 proteins and prevents its heterodimerization and subsequent inhibition of anti-apoptotic protein Bcl-xL (181), which is then capable of binding other BH3 containing proteins to further inhibit apoptosis. Caspase 9, APAF-1 and neuronal lAP's are other apoptosis-related proteins that contain Akt phosphorylation sites. Caspase 9 phosphorylation by Akt may affect its cytochrome c-induced activation (101), but its role remains controversial as the Akt phosphorylation site is not present in rodent caspase 9 (220). Further work is required to determine whether APAF-1 and lAP's are bona fide Akt products and their mechanisms of potential apoptosis induction. Indirect inhibition of apoptosis by Akt includes activation of forkhead transcription factorl (FKHRL1), N F K B , p53 or other signaling pathways. Forkhead transcription factorl (FKHRL1) is phosphorylated by Akt and maintained in the cytoplasm and prevents its translocation to the nucleus where it can induce transcription of apoptotic genes such as Fas Ligand (91). Other death genes that contain a potential FKHRL1 promoter include TRAIL, T N F - a and its receptor and Fas, suggesting that these transcription factors upregulate death cytokines as well as their cognate receptors (92, 679). Other examples of cross talk between Akt and other pathways that would serve to inhibit apoptosis include N F K B induction by Akt (455) as previously mentioned, and potential Akt-inhibition of p53 (769) signaling. 50 Figure 11. PKB/Akt-mediated regulation of apoptosis proteins. Activation of Akt/Protein kinase B which can deliver direct or indirect anti-apoptotic signals to various substrates PKB/Akt can phosphorylate the Bcl-2 family member Bad, Caspase 9, APAF-1 and neuronal lAP's, Indirect inhibition of apoptosis by Akt by activation' of Forkhead transcription factorl, NF Kappa B, p53 or other signaling pathways. 51 The tumor suppressor p53 has been an intensely studied molecule for its critical role in the protection against cancer development. It has been estimated that approximately half of all cancers carry defects in p53. Many stresses activate p53 including DNA damage, oncogene activation, hypoxia, and loss of normal growth and survival signals. Activation of p53 can induce several responses in cells including differentiation, senescence, DNA repair, cell cycle arrest, and apoptosis. P53 is a sequence specific transcription factor that mediates many of its downstream effects by the activation or repression of target genes (Figure 12). Apoptosis induction by p53 appears to occur by multiple mechanisms, where some of which do, and some of which do not require new RNA synthesis (98). Non-transcriptional induction of apoptosis by p53 may be the result of the relocation of death receptors to the cell surface from the Golgi complex as shown after p53 activation (61) or the migration of p53 to the mitochondrial membrane prior to cytochrome c release in some models of apoptosis (462, 607). In contrast, p53 has been shown to activate the expression of many genes that participate in the apoptotic response including Bax (504), NOXA (532), PUMA (516, 783), p53AIP (533) among others. P53 is a potent inducer of cell death and consequently it is tightly regulated at numerous steps including transcription, translation, protein stability, subcellular localization, and activity (757) (Figure12). MDM2 can inhibit p53 transcriptional activity and target p53 for degradation via the ubiquitin pathway. In most cases, induction of p53 in response to cellular stress involves the inhibition of MDM2 function. This can be achieved by direct phosphorylation of p53 on serine 20 resulting in inhibition of MDM2 binding (118, 286). In other stresses, activation of the ARF protein directly binds to and inhibits the interaction of MDM2 with p53 (343, 570, 801). Cell localization can also affect p53 activation. Phosphorylation of nuclear export signal domains on p53 within the nucleus can prevent the export of the protein to the cytoplasm denying access to MDM2 (800). In some models, loss of p53 activity is associated with cytoplasmic accumulation. Other than subcellular localization and protein stability, other mechanisms exist that regulate function. Acetylation of the carboxyl terminus of p53 results in enhanced p53 activity (263, 603). 52 Figure 12. P53 regulation of apoptosis. P53 has been shown to activate the expression of many genes that participate in the apoptotic response including Bax, NOXA, PUMA, p53AIP, among others. P53 is regulated at numerous steps including transcription, translation, protein stability, subcellular localization, and activity. MDM2 can inhibit p53 transcriptional activity and target p53 for degradation via the ubiquitin pathway. In most cases, induction of p53 in response to cellular stress involves the inhibition of MDM2 function. The later may involve direct phosphorylation of p53 on serine 20 and interference with MDM2 binding. In other stresses, activation of the ARF protein directly binds to MDM2 denying access to p53. Cell localization can also affect p53 activation. Phosphorylation of p53 on a nuclear export signal domain within the nucleus of prevents its export to the cytoplasm and access to MDM2 53 Various phosphorylation sites on p53 can also influence the genes that are subsequently transcriptionally activated. For example, phosphorylation at ser46 by p38 or another associated kinase increases susceptibility to apoptosis while cell cycle arrest does not appear to be dependent on ser46 phosphorylation (95). A variety of other proteins including GADD45, p 2 1wafi/ci Pi p53R2, among others, are key modulators of growth arrest, DNA repair, and cell cycle arrest following p53 activation. 3.4. Caspase specific cleavage of substrates Effector caspases degrade a large number of cellular proteins resulting in a number of functional, structural, and morphological changes. Many of the morphological changes seen in apoptosis including cell rounding, cell shrinkage, membrane blebbing can be attributed to specific cleavage of cellular proteins at internal aspartic acid residues by effector caspases. Apoptosis is also characterized by the marking of cells for rapid phagocytosis with little or no inflammation (371). The markers for phagocytosis displayed on apoptotic cell surfaces such as phosphatidylserine are also the result of caspase cleavage of cellular proteins. In addition to the structural/morphologic alterations and targeting of cells for phagocytosis, normal homeostasis within cells is disrupted and key cellular processes including survival signalling, ionic regulation, DNA replication, repair, and transcription transcription may be disrupted. Poly (ADP-ribose) polymerase (PARP) wsa the first caspase substrate identified and was shown to be cleaved from a 112 kDa protein into 85 and 24 kDa fragments following induction of apoptosis by the chemotherapeutic etoposide (359). This degradation could be reproduced in a cell free system of apoptosis where apoptosis induced cytosolic extracts could cleave PARP (414). This cleavage was shown to be mediated directly by caspase 3, where specific inhibitors of activated caspase prevented the proteolysis of PARP. There has been a rapid advance in the identification of other substrates. Cleavage occurs only in certain proteins at discrete sites, in contrast to death by thousands of random cuts which may be seen during non-specific proteolysis as a result of necrosis. Caspases have 54 an absolute requirement for aspartic acid in the P1 position of substrates (678) and also require aspartic acid in the P4 residue, suggesting a DxxD-x motif for macromolecular substrates Nuclear changes including DNA fragmentation at inter-nucleosomal sites and DNA condensation were some of the first noted features of apoptosis (762). Since caspases are activated in the cytosol there are a series of proteolytic events that result in nuclear DNA fragmentation. A cytoplasmic heterodimer consisting of 40 and 45 kDa subunits was found to be associated with DNA fragmentation using cytoplasmic extracts and isolated nuclei (437). The 45 kDa subunit contains a caspase 3 cleavage site and is degraded upon induction of apoptosis in cells. In mice two proteins, caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) were identified and shown to be responsible for DNA fragmentation (198, 604). ICAD is a murine homologue to DFF 45kDa and degradation of this protein by caspase 3 allows CAD to enter the nucleus via nuclear-localization signal and degrade DNA at inter-nucleosomal sites (198). The 40 kDa subunit of DFF has since been shown to contain intrinsic DNase activity and ability to trigger chromatin condensation (437). Mice deficient in DFF45 do not exhibit DNA fragmentation and chromatin condensation but are still susceptible to apoptosis demonstrating that DFF45 is critical for these downstream events but other features of apoptosis are intact (798). Other than DNA fragmentation and DNA condensation, other nuclear processes including DNA replication, repair, and transcription can be inhibited during apoptosis. Some other nuclear proteins that are degraded by caspases include; DNA-dependent protein kinase (DNA-PK) (105, 271, 649, 687), PARP (414), the 70 kDa protein component of the U1 small ribonucleoproteins C (742), 140 kDa component of replication complex C (DSEB/RF-C140) (709), and the general transcription factor Sp1 (563). Accessibility of active caspases to the nucleus is required for substrate cleavage. Nuclear lamins can be cleaved by caspase 6 (280, 415, 676) which may decrease nuclear membrane integrity and facilitate nuclear localization of caspases. Another mecahnism for nuclear translocation of proteolytic cascade to the nucleus is 55 nuclear localization domains on apoptotic proteins. For example, caspases 1 and 2 prodomains contain nuclear localization signals (148, 459). The morphologic phenotype of apoptosis including cell rounding, cell shrinkage, membrane blebbing, and loss of cell adhesion can be explained by disruption of structural proteins. Some structural and structural regulatory proteins that are cleaved in apoptosis include actin (89, 364, 472), a-fodrin (158, 717), gelsolin (394), Gas2 (86), focal adhesion kinase (745), Rabaptin-5 (152), type I keratins (106, 402), and PAK2 (600). In most cases substrate degradation results in inactivation of protein but there are substrates that produce active peptide fragments with biological activity upon caspase degradation. Native gelsolin can severe actin polymers in a calcium dependent manner, while caspase generated gelsolin fragments preferentially cleave actin filaments independent of calcium. Microinjection of the actin cleaving fragment into fibroblasts results in rapid depolymerization of the actin cytoskeleton and change in cell morphology, including cell rounding and release from the monolayer and gelsolin -/- cells severely delay development of membrane blebs (394). Caspases can disrupt proteins that function to inhibit apoptosis or enhance cell survival thus removing any antagonistic forces that may be present once the caspase cascade has been initiated. Bcl-2 and Bcl-xL, as previously mentioned, are pro-suvival proteins that can prevent mitochondrial and some cytoplasmic death events during apoptosis. These two proteins can be processed by active caspase 3 and it has been shown that this may transform these proteins into apoptosis promoters (124, 143). N F K B pathway activation results in inhibition of apoptosis (56, 712). N F K B is usually sequestered in the cytoplasm by IKB alpha and beta (38). Degradation of phosphorylated-kBa by the proteosome results in the translocation of N F K B to the nucleus where it acts as a transcription factor for prosurvival genes including cellular IAP 1 and 2 (141, 733). kBa can be processed by caspases independent of its normal ubiquitin-proteosome cleavage site (45) and be more resistant to phosphorylation and subsequent cleavage by the proteosome. The tumor suppressor p53 is usually associated with the oncoprotein MDM2 resulting in continual degradation. Cleavage of MDM2 by caspases (122, 56 201) may enhance p53 stability and accumulation further enhancing apoptotic phenotype. Various components of other signaling pathways are disrupted during apoptosis. The 6-isoform of protein kinase C (PKC- 6) (232), PITSLRE kinases (71), RasGap (753), MEKK1 (752), Raf-1 (753), and Akt-1 (753) can all be cleaved by caspases. Activation of the PI-3 kinase and protein kinase Akt are known to interfere with the induction of apoptosis (650). Akt kinase can phosphorylate Bad and inhibit its pro-apoptotic function (171, 181). Further, Akt can phosphorylate caspase 9 on serine-196 and this phosphorylation prevents apaf-1/cytochrome c dependant-activation (101). Again degradation of Akt-1 by caspases would promote enhanced caspase cascade activation by removing apoptosis inhibition. The net effect of these multiple cleavage events may include; morphological changes, marking of cells for phagocytosis, cell cycle inhibition, loss of DNA repair mechanisms, genome fragmentation, inhibition of transcription, loss of cell attachment and cell adhesion, and removal of anti-apoptotic signalling. Of particular interest, the degradation of key cellular proteins in apoptosis is similar to picornarvirus protease degradation of key host proteins that disrupt normal cellular homeostasis. 57 4 VIRUSES AND CELL DEATH SIGNALLING 4.1. Cytopathic effect: How is it defined? In monolayers of cultured cells, infection with enteroviruses is generally associated with a visible cytopathic effect (CPE) using light microscopy (587). Since first described by Enders in 1949 (199), CPE has become a term used in multiple virus models where there is a characteristic change in morphology following infection. It is becoming evident that cytopathic effect in certain virus models may be caspase-dependent or caspase-independent (104). 4.2. Apoptosis as an innate and adaptive anti-viral defence mechanism of the immune system Apoptosis is used as a host defense mechanism against viruses and other intracellular pathogens. It is also clear that an integrated immune response involving both the innate and adaptive immune response is critical for maximal clearance of viral pathogens. The use of mice deficient in perforin (285) or mutations in Fas Ligand or its receptor (gld or Ipr mice) (677, 741), or a variety of soluble factors, have demonstrated the importance of these molecules in viral clearance. In the absence of a specific immune response, macrophages and NK cells can release TNF-a, TRAIL, and IFN-y (258, 363). As previously mentioned, TNF- a and TRAIL can induce apoptosis by signaling through their cognate death receptors (See Section 3.2). The downstream IFN-induced gene family is now known to comprise the death ligand TRAIL, the dsRNA-dependent protein kinase (PKR), interferon regulatory factors (IRFs) and the promyelocytic leukemia gene (PML), all of which have been reported to be mediators of cell death (43). Exogenous Exogenous IFN-y up-regulates the expression of TRAIL and TRAIL-Rs in cytomegalovirus (CMV) -infected fibroblasts, but not uninfected fibroblasts (627). Other studies showed marked upregulation of TRAIL on the surface of peripheral blood T cells following stimulation with IFN- y or anti-CD3 monoclonal antibodies (362). The cytolytic activity of both CD8+ and NK cells depends on perforins, granzymes, Fas-Fas L interactions, and soluble factors such as TNF-a, TRAIL, or combinations of these. The 58 mechanism of Fas L-induced apoptosis has been described in Section 3.2 and plays an important role in the clearance of virally infected cells. Perforin is essential for the induction of apoptosis in response to granzymes (636, 637). Perforin aids in the distribution of granzymes to the cytoplasm of the cell by playing a role in granzyme entry from the plasmalemma or by aiding in granzyme release from intracellular endosomes within infected cells (647, 700). Once free in the cytoplasm, granzymes induce apoptosis and cytolysis by a variety of mechanisms that appear to be caspase-dependent or caspase-independent. Granzymes have been shown to degrade Bid independent of caspase 8, thus allowing for its translocation to the mitochondrion and the release of death inducing factors (276, 668). The ability of various components of the innate and specific immune response to induce apoptosis during viral infection is an important mechanism to aid in the clearance of virally infected cells and reduce viral dissemination by inducing apoptosis at early stages of the viral life cycle prior to complete replication. As will be demonstrated in Section 4.3.1, this is not a passive process, as many viruses have evolved strategies to evade these anti-viral immune and pro-apoptotic responses or to otherwise utilize them to their advantage. 4.3. Virus interactions with host-cell death machinery Upon infection of a host cell, the virus and host compete for ultimate control and survival. Some viruses can live within a host cell and cause little observable changes in host cell function. Viral persistence and latency are two terms that are commonly used to describe the long-term survival of a virus within a host. Latency is the long-term survival of the viral genome in the absence of infectious virus, interrupted by periods of reactivation. Latency usually involves the incorporation of the viral genome into the host genome. Viral persistence is the long term survival of infectious virus within a host, usually involving a variety of strategies including; evasion of the host immune response, inhibition of host cellular death, manipulation of the host cell cycle, and restricted/regulated viral replication. In fostering cellular integrity, certain viruses find a favorable long-term niche in the host. Viral persistence can allow viruses the 59 opportunity to "optimize" replication, while controlling the release of viral progeny and the nature of immune perturbation. Other viruses attempt to hijack a host cell and use this to aid in the replication of the virus, subsequently killing the cell with release of progeny virus to infect new host cells or new hosts. Opposing this, the host immune system or the infected cell itself cell may intrinsically detect a foreign invader or a change in the natural cellular environment and attempt to commit suicide prior to viral replication, thereby limiting replication and dissemination. Both the host and the virus have evolved mechanisms to try and gain the ultimate advantage. Many viruses encode proteins that are homologous to, or directly interact with, host proteins that are involved in cell death or cell survival. Alternatively, mammalian host cells have evolved redundancy in many of the cell survival/cell death pathways to try and prevent loss of control of the cells. Virus modulation of cell death machinery and influence of survival is not one sided. Viruses not only inhibit or induce apoptosis, but may use multiple strategies at different points of the viral life cycle. In this regard, many viruses are known to induce apoptosis only during the late stages of virus life cycle (685). The classic example of a virus that has evolved numerous strategies, including inhibiting cell death and inducing apoptosis, to maximize conditions for viral replication is the adenovirus. As will be described in Sections 4.3.1 and 4.3.2, these DNA viruses use early strategies to parasitize a host cell and foster replication by inducing host cell cycle changes, inhibiting cell death, hiding from the innate and cognate immune responses which together maximize viral replication. This is followed by the induction of cell suicide by apoptosis to aid in the release of infectious virus while trying to minimize the immune response. 4.3.1. Viruses and viral proteins that inhibit apoptosis Many DNA viruses require a specific cellular environment for viral replication including active DNA synthesis. DNA viruses typically infect resting cells and induce transformation from G 0 or d into the S-phase of the cell cycle where host nucleotide pools and proteins involved in DNA synthesis become available for viral DNA replication (486). To induce transition into S-60 phases, these viruses typically manipulate cell cycle regulatory proteins, oncogenes, and tumor suppressor genes. Since manipulation of these cell cycle entry signals themselves can induce apoptosis in the absence of balanced-homeostasis, many of these viruses have evolved strategies to concomitantly inhibit apoptosis (486). The p53 and retinoblastoma (Rb) proteins are two important tumor suppressor proteins that regulate cell cycle progression and apoptosis, among other functions. The mechanisms of p53 induced apoptosis and regulation were described in Section 3.3. Unlike p53, the Rb protein is not a direct transcriptional transactivator, but instead acts as a negative regulator of transcription factor E2F-1, which when liberated results in the transcription of genes associated with proliferation and cell cycle entry (Figure 13). Another phenomenon associated with E2F-1 transactivation is induction of ARF, a protein which when expressed stabilizes p53 by binding to MDM2 (releasing p53) and thus inducing apoptosis in the appropriate conditions. These two proteins offer a unique ability of viruses to manipulate the cellular environment to inhibit cell death and maximize DNA replication. A general theme among some DNA viruses is to destabilize Rb (to stimulate DNA synthesis by E2F-1 activity) and p53 (to inhibit apoptosis) concomitantly during the viral replication cycle. Variations on this theme occur at different points in viral life cycle where it may be advantageous for the virus to activate p53 and induce apoptosis once maximal viral progeny has been produced. Adenovirus can utilize p53 to either inhibit or induce apoptosis depending on the stage of viral replication and expression of other anti-apoptotic viral proteins. Countering this apoptosis-promoting activity of E1a (as will be discussed in the next section), the adenovirus E1B 55K directly represses the p53 activity. E1B 55k can bind to p53 and inactivate its ability to transactivate various promoters (268). Kaposi's sarcoma-associated herpesvirus (KSHV) has evolved more than one strategy to inhibit p53 induction of apoptosis. At least three separate KSHV encoded proteins have been shown to directly associate with p53 and inhibit or repress its transactivating activity. KSHV encoded viral interferon regulatory factor 1 (vlRF1) (513, 630), the latency-associated nuclear 61 i replication L repair cycle progression Figure 13. Rb protein acts as an negative regulator of transcription factor E2F-1, which when liberated results in the transcription of genes associated with proliferation and cell cycle entry. Another phenomenon associated with E2F-1 transactivation is induction of ARF, a protein which when expressed stabilizes p53 by binding to MDM2 (releasing p53) and thus inducing apoptosis in the appropriate conditions. 6 2 antigen (LANA), (216) and K-bZIP (555) all interrupt p53-mediated host cell death to some degree. Indirect inhibition of p53 also occurs by KSHV open reading frame 50 (ORF50), which directly associates with CREB binding protein (CBP) (266) (a protein that acts as a p53 coactivator and potentiates its transcriptional activity (264)), and inhibits its co-activation of p53. Other herpesviridae proteins that interact with Rb and p53 and induce transformation and inhibition of apoptosis include EBV EBNA-5 interaction with Rb (673) and CMV IE2 which targets both p53 and Rb (136, 702). Human papillomavirus utilizes both the Rb and p53 pathway to transform cells and inhibit apoptosis. The E6 protein binds p53 (281, 618, 734, 747) and the E7 binds Rb (84) and targets them for degradation through the ubiquitin-proteosome pathway. Rb inactivation leads to liberation of transcriptional transactivators that induce cell cycle progression while p53 degradation prevents its apoptotic functions. E6 has also been shown to bind to CBP (557, 811) and depress its ability act as a co-factor for p53 transcription. The Simian Virus large T antigen (670) binds to the DNA-binding domain of p53 resulting in the accumulation of nonfunctional p53 complexes (747) and prevention of p53-mediated gene activation. The T antigen also associates with the Rb protein-E2F complex causing destabilization, and releasing E2F-1 to transactivate various cell proliferation genes (663). Hepatitis B virus X protein directly interacts with p53 (739) and inhibits its DNA consensus sequence binding and transactivator activity (701). This interaction also sequesters p53 in the cytoplasm, away from its DNA targets (196). It is becoming apparent that HBx is a pleiotrophic factor and may associated with various cellular factors that can induce as well as inhibit cell death as will be discussed further in this section. HTLV-1 Tax protein is an important protein involved in cellular transformation. This protein can interfere with p53 ability to induce apoptosis (565). Tax does not accomplish this by directly binding to p53, but rather by a unique mechanism that includes either constitutive phosphorylation of p53 at Ser-15 and Ser-392 that is dependent on Tax-activated NF-kappaB and subsequent transcriptional activity (566) or by direct Tax association with CBP which limits 63 CBP coactivator capabilities (27). The mechanism of p53 inhibition appears to be cell type dependent (567). Certain viruses are also known to contain Bcl-2 homology (BH) regions which can dimerize or interact with apoptosis regulators in the Bcl-2 family. The Epstein Barr virus-BHRF1 (279) and BALF-1 (463), adenovirus E1B-19 kDa (133, 578), Herpesvirus saimiri HVS-Bcl-2 (519), KSHV KSbcl-2 (125, 609), among others, are all examples of viral proteins that contain anti-apoptotic Bcl-2 homologs. Adenovirus E1B 19K can interact with Bax (120) and prevent Bax oligomerization to form a 500 kDa Bax complex (666). Human papillomavirus E6 protein can bind the pro-apoptotic member Bak (328, 690) as well as previously mentioned p53 (618, 747) and aid in its degradation. HBV HBx protein has been shown to decrease the expression of the pro-apoptotic protein Bid (121). Instead of direct viral homologs of Bcl-2 proteins, some viruses have been shown to directly or indirectly upregulate the expression of cellular Bcl-2. HIV-1Tat protein expression is able to transactivate Bcl-2 (740) and results in Bcl-2 upregulation in various cell types (793, 794). Some viral proteins can protect against apoptosis induction by directly inhibiting caspase activation or processing. The cowpox virus-encoded serpin, CrmA (222), is a potent inhibitor of caspases 1 and 8 (804) and would play a role reducing inflammatory cytokine processing (IL-1B) and inhibiting apoptosis induced by surface death receptor ligation (653). Other serpins include SPI-2 of rabbitpox and vaccinia virus (188, 372) and have similar function to CrmA. Another viral caspase inhibitor is baculovirus p35 (96) that appears to antagonize most known caspases, including initiation and effector classes of caspases (803). P35 is an active site inhibitor of the caspases that is cleaved by Ced-3 and most mammalian caspases (caspases 1, 3, 6, 7, 8, 10) (803). After p35 is cleaved it remains bound to the active caspase (764). Mutation of the caspase cleavage site in p35 abrogates its inhibitory function (70). Baculoviruses also contain another family of viral caspase inhibitors that appear to function upstream of p35 (which acts on active enzymes). These proteins are functional homologs to the cellular-IAPs (inhibitor of apoptosis proteins) (144, 156) which were described in 64 Section 3.3. These proteins can bind to and inhibit the activation of caspase 9 and directly inhibit active caspase 3 and 7 as previously described. As it is known that death receptor ligation is an important mechanism to clear virally infected cells by innate and acquired immune responses, it is not surprising that many viruses have evolved mechanisms to inhibit these pathways. For example, the gamma-herpesviruses, including KSHV, as well as in the tumorigenic human molluscipoxvirus contain FLICE (caspase 8)-inhibitory proteins (FLIPs). These FLIPs contain DED's and are able to interact with the adaptor protein FADD (69, 691) and thus block caspase-8 and caspase-10 recruitment and subsequent activation and transduction of apoptosis signals (691). Other strategies to manipulate this signaling include modifying death receptor density on the surface of the cell and the release of soluble factors that antagonize receptor-ligand interactions. Adenovirus RID complex (an E3-14.5K and E3-10.4K association) mediates Fas (695) and Trail R1 (696) internalization into cells and destruction by lysosomes. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections. Alternatively, some poxviruses encode soluble TNF receptor homologs that are secreted and compete for TNF binding. Cowpox CrmB and CrmC (302, 646), and myxomavirus T2 neutralize TNF (451) and play a dual role of competing for anti-inflammatory molecules and inhibiting apoptosis. Another way to disrupt receptor-mediated death is to manipulate expression of TNF superfamily genes. Activation of transcription factors of the nuclear factor of activated T cells (NFAT) family are implicated in the regulation of the immune response and other processes, including differentiation and apoptosis. NFATs normally reside in the cytoplasm, and a key aspect of the thier activation is regulation of nuclear import by the Ca(2+)/calmodulin-dependent phosphatase calcineurin. It has been shown that the African swine fever virus 5EL/A238L protein can bindi the catalytic subunit of calcineurin and inhibit its activity (502) which subsequently prevents NFAT-mediated gene transcription in vivo. It has also been shown that during early HSV infection, transport of NFAT transcription factors are efficiently blocked (626) by unknown viral proteins as translation of viral proteins. This mechanism of viral inhibition of 65 host signaling may play an important role in influencing immune regulation and cell death. Trail (737) and FasL (413) as well as many cytokine genes have a NFAT promoter sequence and would thus have impaired expression. In similar fashion to viruses that can inhibit death receptor mediated apoptosis, it is becoming clear that viruses can also inhibit mitochondrial-mediated apoptosis. In addition to the strategies of Bcl-2 like homologs as already mentioned, certain viruses contain proteins that appear to play roles in mitochondrial membrane permeability transitions. The myoxma poxvirus protein M11L localizes to mitochondria and inhibits mitochondrial membrane potential loss (203, 451) and the cytomegalovirus vMIA associates with the ANT subunit of PTPC and can prevent downstream apoptosis events (240). Activation or disruption of other cell signaling pathways that can either phosphorylate or regulate the expression levels of apoptosis-related proteins are other mechanisms that viruses use to inhibit cell death pathways. The HIV-1 vpr gene has been shown to inhibit apoptosis through activation of the N F K B transcription factor (31). Another HIV-1 protein, Nef, can inhibit ASK-1-dependent death signaling (229). The interaction of Nef with ASK1 inhibits both Fas- and TNFa-mediated apoptosis, as well as the activation of the downstream c-Jun amino-terminal kinase which inhibits apoptosis. Since, Nef has also been shown to upregulate Fas L expression (763), inhibition of ASK-1 may be a mechanism that non-infected, Fas expressing bystander cells are depleted while the infected cells are protected (229). The Epstein-Barr virus latent membrane protein-1 (LMP-1) has also been shown to activate N F K B signaling pathways (99, 184, 327, 365, 507). As will be discussed in section 4.3.2, there is evidence that HBx can directly induce apoptosis in some cell types (376) and this apoptosis is p53-independent (689). As mentioned above, HBx can also directly inactivate p53 and inhibit apoptosis. Further there is evidence that HBx can activate the JNK/STAT (186) and PI3-K-Akt (419) pathways in different cell types, both of which serve to inhibit apoptosis. Polyomavirus has also been shown to achieve protection 66 from apoptotic death through a middle T antigen-PI 3-kinase-Akt pathway that is at least partially p53-independent(162). As summarized in Figure 14, varied use of p53 inactivation, N F K B activation, PI3K-Akt activation, ASK-1 inactivation, expression of viral caspase inhibitors, and the expression of viral homologs of anti-apoptotic cell death proteins like Bcl-2 and FLIP'S, demonstrate the aried approaches that viruses use to delay cell death during infection. As will be demonstrated in the next section, many of these viruses that use anti-apoptosis strategies still induce apoptosis at later times in the viral life cycle. 4.3.2. Viruses and viral proteins that induce apoptosis Many viruses are now known to kill their host cells by apoptosis. Induction of apoptosis may allow for progeny viral release with minimal recruitment of proinflammatory mediators. Fewer viral proteins exist that directly induce apoptosis as compared to proteins that inhibit apoptosis. This is probably due to the fact that many viruses induce apoptosis by default following the decreased expression of their pro-apoptotic proteins. In many cases it is apparent that infection with viruses with deleted anti-apoptotic proteins results in the rapid induction of apoptosis. Many viruses regulate their protein expression during the course of their lifetime and many of the anti-apoptotic proteins appear to be expressed early during the replication cycle. Induction of apoptosis may be greatly facilitated simply by a decrease in viral expression of anti-apoptotic mediators. Many RNA viruses do not require active DNA synthesis in the infected cell, and many of the required host co-factors for viral replication are already expressed in many cell types. These viruses may not require as sophisticated mechanisms of apoptosis inhibition as they can produce viral progeny in relative short periods of time. Host cell E2F-1 activation, inhibition of protein transcription and protein translation, double stranded RNA mediated-IFNy activation and expression of IFN-induced genes, among others, are all known to induce apoptosis. It is therefore conceivable that the absence of anti-apoptotic viral proteins would be sufficient to send the cell into irreversible death. 67 Cowpox CrmB Cowpox CrmC 1 myxomavirus T2 Soluble receptors African swine fever virus 5EL/A238L HSV Plasmalemma KSHV Flice Molluscipoxvirus Flice Inhibited expression. 8M Death Ligands Death Receptors EBV EBNA5 CMV IE2 Papillomavirus E7 Simian Virus LTAg Adenovirus E1B55K KSHV proteins Papillomavirus E6 Simian Virus LTAg Hepatitis B X Caspase 8 • Internalization • T Adenovirus RID Cowpox CrmA Vaccinia virus SPI-2 Baculovirus p35 Baculovirus IAP Apoptosis DNA replication KSHV ORF50 — Papillomavirus E6 HTLV Tax EBV BHRF1 EBV BALF-1 HVS-Bcl-2 KSHV KSbcl-2 Adenovirus E1B 19K CATFJ Baculovirus IAP IUVII u o L I U I c r \ 1 ~ 1 Procaspase Cowpox CrmA Vaccinia virus SPI-2 Baculovirus p35 Baculovirus IAP 1 Active caspase Figure 14. General themes that viruses use to inhibit apoptosis and induce cellular proliferation. Viruses can liberate E2F, inhibit p53, inhibit cytochrome c release from the mitochondrion, inhibit caspase processing, regulate expression of surface receptors, produce soluble factors, or inhibit active caspases, among other mechanisms. 68 As mentioned above, adenovirus can both induce and inhibit apoptosis by p53 modulation. Ad E1A protein can increase p53 levels, drive cells into S phase and induce p53-dependent apoptosis (578). Expression of E1A has been shown to cause an increase in the level of p53 protein and induce p53-dependent apoptosis (445, 576, 686). This increase in p53-mediated apoptosis by E1A occurs by stabilization of p53 (512) and increased transcription of p53. E1A cannot directly bind to DNA motifs, but has been shown to associate with a variety transcription factor regulators including Rb (39, 751) and p53 transcriptional co-factors like CBP/p300 (23, 448). E1A interaction with Rb liberates E2F-1 (42, 119) and induces cell cycle progression (524, 744) and subsequent apoptosis. Activated E2F induces expression of tumor-suppressor protein p14ARF, which neutralizes HDM2 (human homologue of MDM2) and thereby stabilizes the p53 protein and induces p53-mediated apoptosis (51). E2F mediates p53-independent apoptosis by induction of p73, a homologue to p53 (323). E1A can also interact with the cyclin-dependent kinase inhibitor p21 and restore Cdk2's activity, and thus drive cells out of cell cycle arrest and in turn induce apoptosis (116). An adenovirus with deleted E1B 55k has been produced (Onyx Pharmaceuticals; ONYX-015) with the hypothesis that it will selectively kill p53 deficient tumor cells (as discussed above, E1B 55K degrades and inactivates p53). In the absence of p53 in cells, E1A can induce apoptosis in a p53-independent fashion, a process which may involve products of early region 4 (E4) of Ad (461). The early region 4 transcription product can produce at least 7 protein products from 7 different open reading frames. The Ad5 E4orf4 protein plays a role in down-regulation of virally induced signal transduction. This down regulation of signal transduction is hypothesized to induce apoptosis (643). Ad5E4orf4 was shown to specifically interact with protein phosphatase 2A (PP2A) B-alpha subunit and induce apoptosis in a p53 independent manner (460). Treatment with antisense RNA that inhibited PP2A-B alpha subunit transcription inhibited apoptosis induced by Ad5E4orf4 (644). Additional studies on this E4orf4 suggest that this protein can engage death receptor mediated induction of apoptosis as dominant negative mutants of caspase 8 and FADD inhibited E4orf4 apoptosis and that the mechanism of death 69 induction can differ depending on the cell type (440). Dissection of the various adenovirus proteins has demonstrated that this virus uses multiple pathways to modulate cellular transformation, inhibition or activation of cell death. Another strategy of p53 activation involves the direct interaction with p53 by viral proteins. HCV core protein has been shown to activate p53 (447). This protein augments the activity of p53 by directly interacting with it and enhancing its DNA binding affinity and transcriptional activity (547). Alternatively, it has been shown that the core protein can bind to the cytoplasmic domain of the TNFR-1 and sensitize them to TNF-induced apoptosis in certain cell types (805). HCV core protein has been shown to interact with the DD of FADD and TRADD and it was hypothesized that this facilitated FADD recruitment to TNF-R1 and thus preferential activation of the death pathway (806). Other studies have shown that HCV core protein inhibits TNF-alpha mediated apoptosis (580, 581) by preferentially activating the N F K B survival pathways (642, 781). This differential activation may depend on cell type as in some cells the apoptosis signaling pathway will be preferentially activated while in others the N F K B signaling pathway will be preferentially activated. Infection of cells with mutants of the baculovirus Autographa califomica nuclear polyhedrosis virus (AcMNPV) lacking a functional p35 gene results in apoptosis (573). Transfection with viral gene EI-1, a transactivator of baculovirus gene expression, was able to induce apoptosis in transcription dependent manner (573). Another baculovirus gene PE38 was shown to dramatically enhance EI-1 induced apoptosis (572). A third baculovirus protein, the product of the AcMNPV orf92 p33 formed a stable complex with p53 and induced apoptosis when no IAP or p35 was expressed, yet enhanced the susceptibility to apoptosis when IAP and p35 were co-expressed (574). Apoptosis is an important mechanism involved in chronic liver disease caused by HBV. As mentioned before, HBx appears to have multiple functions including the inactivation of p53 and induction of survival signaling. In other states, the HBx is associated with transactivation and induction or sensitization to cell death (62, 134, 272, 376). This apoptosis-induction appears to be p53-independent as p53 null cells are sensitized to apoptosis in HBx transfection 70 (62, 689). HBx has been shown to stimulate the expression of Myc (661, 688) protein that in turn can sensitize cells to killing by TFN-a (660). Gene expression studies utilizing microarray analysis and HBx expression in primary adult human hepatocytes (liver samples from HBV-infected chronic active hepatitis patients when compared with normal liver samples), and in hepatocellular carcinoma (HCC) cell line (SK-Hep-1) ectopically expressing HBx via an adenoviral system demonstrated consistent alteration of many cellular genes including a subset of oncogenes (such as c-myc and c-myb) and tumor suppressor genes (such as APC, p53, WAF1 and WT1) (759). Other viral strategies exist for inducing apoptosis. The African swine fever virus encodes a viral homologue (5EL/A238L) of the human inhibitor K B (IKB) (522). Cells expressing the A238L gene inhibited N F K B binding to DNA (571) and it was shown that the viral homologue co-immunoprecipitated with p65 N F K B (588, 674). By such a mechanism the virus may prevent NFKB-mediated transcription and upregulation of proinflammatory cytokine production (571) and sensitize cells to apoptosis, since N F K B activation is also responsible for the transcription of anti-apoptotic genes (141). Clinically significant CD4+ T lymphocyte depletion occurs in association with human immunodeficiency virus-1 (HIV-1) infection. Regulation of cell surface death receptors and ligands, as well as regulation of the internal signaling pathways play a role in depletion of infected, as well as uninfected CD4+ lymphocytes. As mentioned, HIV infection can upregulate Fas Ligand expression on infected cells by Nef association with the zeta chain of the T cell receptor (TCR) complex (763). HIV has also shown to upregulate Fas expression in infected cells, yet the cumulative action of increased Fas L and Fas may not be sufficient for death induction as other mechanisms like Nef inhibition of ASK-1 signaling can inhibit death by these pathways. Tat expression can also upregulate Fas L and caspase 8 expression, producing apoptosis, and sensitize cells to apoptosis induction by other agents (50, 392, 750). Another contributing pathway may be the HIV envelope glycoprotein (gp120) binding to surface receptors CXCR4 which has shown to induce apoptosis independent of Fas signaling pathway (68, 73, 71 594). Vpr can directly associate with the ANT of the PTPC and its expression has been shown to facilitate loss of mitochondrial membrane potential, release of cytochrome c, and AIF (330, 331). Therefore, HIV appears to be another virus that uses a complex strategy of both inhibiting and inducing apoptosis at various points of the viral lifecycle. The cumulative effects of the competing pathways, as well as cell type, will determine cell fate. The HTLV-1 tax protein appears to be similar to the HIV tat protein in its ability to sensitize cells to apoptosis and upregulate Fas L and TRAIL expression. As previously mentioned, tax can activate N F K B (710) which can induce the expression of a variety of cellular genes. It was further shown that Tax-induced TRAIL expression and death susceptibility is dependent on N F K B signaling (591). The complexity of tax-mediated apoptosis is demonstrated by other groups who have shown PI3K activation (439), increased Bcl-xL expression (506), and interference with p53-mediated death as previously mentioned. Another HTLV-1 protein, p13(ll) can selectively target the mitochondria and induce specific changes in mitochondrial morphology suggestive of altered inner membrane permeability and swelling (161). The chicken anemia virus encodes a nuclear binding protein (apoptin) that is capable of inducing p53-independent apoptosis that is not inhibited by Bcl-2 (165, 808-810) . Interestingly, apoptin induces apoptosis in transformed or malignant cells, but not in normal cells (165). In normal cells, apoptin is found predominantly in the cytoplasm, whereas in tumor cells it is located in the nucleus (166), yet this localization alone cannot explain the dichotomy between normal and malignant cells. The exact mechanism of apoptosis induction is still not known, but apoptin causes mitochondrial membrane potential loss, cytochrome c redistribution, and caspase activation (167). It has been demonstrated that reovirus infection results in the induction of apoptosis that is dependent on the sigmal viral protein (708). This model has identified a autocrine signaling loop where viral attachment to the cellular receptor junction adhesion molecule (JAM) (49) results in cellular signaling, including N F K B activation (149), and the subsequent expression of TRAIL, DR4, DR5 (142), and TRAIL-mediated death (142). An interruption along any part of this 72 pathway including inhibition of sigmal binding to surface receptor, inhibition of N F K B activation, or inhibition of TRAIL activity will inhibit reovirus induced apoptosis. There are a variety of other viral protiens that have been shown to induce apoptosis following expression in different cell types, yet their mechanisms of activation are not completely understood. 4.4. Apoptosis and Picornaviruses: Morphologic and Biochemical Evidence Unlike many of the DNA viruses that require DNA synthesis and cells to enter the cell cycle for infection, picornaviruses only require host translation and transcriptional machinery (see Section 1.3.2.). As will be discussed in more detail in Section 10, there is evidence that many picornaviruses, including poliovirus, coxsackie B group viruses, Theiler's murine encephalomyelitis viruses, and hepatitis A virus induce apoptosis as demonstrated by the characteristic morphologic and biochemical changes. In vitro, poliovirus infection induces characteristic cytopathic morphology including DNA fragmentation and biochemical evidence as demonstrated by activation of the DEVD-specific caspases (4, 5, 14, 444, 697) in a variety of cell lines such as HeLa, CaCo-2, and U937. Further, independent transfection of the 3C (44) and 2A (241) induced apoptosis over a period of 2-4 days. In vivo evidence of poliovirus induction of apoptosis was demonstrated by intracerebral infection of transgenic mice expressing the human PV receptor (234). Coxsackievirus B group virus (104, 424, 595) and Theiler's murine encephalomyelitis virus (333, 334) induced apoptosis in cell lines and mice (310, 705), where the apoptosis is probably a combination of direct viral and immune response-mediated as will be discussed in Section 10. Despite the similar gene organization shared with other picornaviruses, hepatitis A virus (HAV) is characterized by its slow-growth phenotype (155, 390), the inability to shut off host macromolecular synthesis, and, in general, lack of cytopathic (cp) effects in permissive cell cultures. Variants that induce cytopathic changes in cells are known to induce apoptosis as determined by morphologic examination and DNA laddering gels (85, 250). Picornaviruses have relatively small genomes and usually replicate rapidly as compared to 73 many other types of viruses. The interplay between host and viral proteins and the determination of cell fate will be demonstrated in Section 9 and discussed in more detail in Section 10. 74 5 CELL DEATH AND THE MYOCARDIUM 5.1. Cell death and the myocardium Ischemia, ischemia followed by reperfusion, hemodynamic overload, myocardial infarction, dilated cardiomyopathy, viral infection, and allograft rejection, among others, all involve the loss of myocytes by apoptosis or a quasi-form involving apoptosis and necrosis. This section will revisit the classical types of cell death in the myocardium: coagulation necrosis, coagulative myocytolysis (also known as contraction band necrosis), and colliquative myocytolysis (vacuolar degeneration). The biochemical processes of myocyte apoptosis will be summarized with an emphasis on the intrinsic mechanisms that inhibit apoptosis and the structural and functional consequences of myocytes once the apoptosis cascade has been initiated. 5.2. The cardiac myocyte: properties and morphology of death There are three alternative destinies of cardiac myocytes during prenatal development. Cardiac myocytes have the option to survive as a single cell, to replicate into more than one cell, or to undergo programmed cell death. All pathways are crucial for normal cardiovascular development in the embryo. After birth, cardiac myocytes have several other options: survival, migration, proliferation, hypertrophy or aberrant signaling, and death. Many of these options may be related to one another. The differentiated myocyte expresses a protein repertoire that is specialized for numerous functions: metabolism and energy production, cell-to-cell signaling and contraction. As myocytes become terminally differentiated they are unable to proliferate/regenerate under normal conditions that makes cell death in the myocardium a critical process; loss of myocytes results in decreased efficiency of myocyte function where the remaining myocytes have an increased workload to compensate for the overall decrease in cell number. Loss of myocytes and the increased workload of remaining myocytes begins a feedback loop that enhances the failing heart phenotype. The final common pathway of different models of myocyte death and compensation is cardiac myocyte heterogeneity: changes in myocyte size, shape, nucleic acid complement, protein content, and energetics. 75 Cardiac muscle is composed of interwoven bundles and bands of muscle fibers anchored to the fibrous framework of the heart. Myofibrils, the contractile elements of cardiac myocytes, are formed by a repeating unit known as the sarcomere. The sarcomere is composed of inter-digitating thick protein filaments and thin protein filaments, composed of myosin (thick), and actin and some tropomyosin B (thin). The state of myofibril contraction is important in defining classical cell death. Coagulation necrosis, coagulative myocytolysis (contraction band necrosis), and colliquative myocytolysis (vacuolar degeneration) are descriptive forms of myocyte cell death originated based on histology (46-48). Coagulation necrosis is myocyte death in a relaxed state. No evidence of interstitial edema or exudation is detectable during this early stage. The myocardial cells maintain their myofibrillar structure, and registered sarcomeres are recognizable, with no contraction bands or other changes being visible. Coagulative myocytolysis, also known as contraction band necrosis, is the opposite of that seen during coagulation necrosis, and occurs when the myocardial cell arrests in hyper-contracted state, which results in a reduced length of the sarcomeres with respect to the normal limits. The first detectable sign is hyper-contraction of sarcomeres, involving the entire myocyte (holocytic) or one portion of the cell, usually in the region of the intercalated disc (paradiscal). The thickness of the Z-lines is also increased. Unlike coagulation necrosis, where the myofibrillar apparatus remains visible and in register, the sarcomeres become hypercontracted and distorted. The linear arrangement of myofibrils is disrupted, and in some regions eosinophilic transverse bands appear between zones, which may be clear or contain fine granules. By electron microscopy, the myofilaments can be seen to be out of register and translocation of mitochondria occurs. The sarcomeres appear to be torn apart, and dehiscence of the intercalated disc may be seen. In colliquative myocytolysis (vacuolar degeneration), the myocyte retains its ability to relax and contract, however, it does so with a progressive reduction in the contraction force and velocity. The cell has intracellular edema and myofibrillar dissolution with progressive vacuolization and maintenance of membrane integrity. The basic changes that occur include the disappearance of 76 the myofilaments and swelling and degeneration of the sarcotubular system and mitochondria. The cellular events that lead to the types of cell death outlined above are not completely known. Previous investigators have suggested changing calcium levels, ATP status, and pH as predominantly responsible for the morphologic alterations. The discovery of a proteolytic pathway in apoptotic cell death has redefined the cause of morphologic alterations seen in dying myocytes. 5.3. Apoptosis and heart disease Apoptosis in the heart can be determined by pathologic examination of cardiac tissues from clinical cases of heart disease. The use of clinical myocardial tissue samples has significant limitations to examine apoptosis in heart disease; First, access to tissue via autopsy can result in a significant delay in time from death to harvesting of tissue. The normal degradation of tissue following death including non-specific degradation must be taken into consideration when determining the proper assays to detect apoptosis. Good quality tissue may be obtained from cardiac biopsies with sufficient co-ordination between surgical teams and researchers. Second, access to sufficient tissues at different temporal periods of heart disease may be difficult. Most of the tissue that is accessible may be very late stages of heart disease where the heart has already undergone significant myocyte apoptosis, myocyte hypertrophy, and ventricular remodelling. Several detection methods to distinguish apoptotic cells in fresh or fixed human tissue have been developed based on the distinct morphological and biochemical characteristics of apoptosis. These techniques primarily involve the detection of DNA fragmentation in tissue by DNA laddering on agarose gels, the in situ labeling of DNA free 3'-hydroxyl ends (TUNEL) or active caspases and/or degraded substrates, or looking for cell surface markers indicative of apoptosis including phosphatidylserine (PS) that translocates from internal to the external leaflet of the cell membrane during apoptosis and has high affinity for annexin V. Both electron and light microscopy, have been important tools in determining morphologic changes associated with apoptosis. 77 DNA fragmentation is associated with ultrastructural changes in cellular morphology during apoptosis. High molecular DNA fragments of approximately 300 and 50 kb can be detected using pulse-field gel electrophoresis (88, 529). This pattern of DNA fragmentation precedes caspase activated DNase (CAD) mediated inter-nucleosomal (180 bp) DNA cleavage (665) as previously described. When DNA is extracted from tissue undergoing apoptosis and run on an agarose gel, the multiple DNA fragments are recognized as a characteristic ladder composed of multiple bands at 180 bp intervals (11). Similar to agarose gel detection of DNA laddering, terminal deoxynucleotidyly transferase mediated dUTP nick end labelling (nick end labelling or TUNEL) method is based on the inter-nucleosomal fragmentation of DNA by CAD. This technique allows for in situ tissue localization of DNA fragmentation. Briefly, DNA strand fragmentation is determined by enzymatically labelling the 3'-hydroxyl termini with modified nucleotides (usually digoxigehin, fluorescein, or biotin) using the enzyme terminal deoxynucleotidyl transferase (TdT) (247). TdT-added dUTP's are detected by the addition of immunochemical, fluorescent, or affinity labeling. There are disadvantages in using these DNA fragmentation assays to determine apoptosis in animal or human samples. These two techniques identify apoptotic cells dependent on the caspase degradation of the inhibitor to the DNase, ICAD. Since this is only one out of a large number of substrates that can be degraded by effector caspases there might be cells that can undergo apoptosis but are resistant to DNA fragmentation (146). For example, MCF7 cells are deficient in caspase 3, and although ICAD can be cleaved by more than one caspase, a caspase 3 specific cleavage site is required for DNA fragmentation (682). DNA agarose gels also do not allow for the localization of apoptotic cells. This technique can only determine if apoptotic cells are present in the sample or not. It is well known that there is normal recycling of inflammatory cells at sites of inflammation (616). Inflammatory heart disease, regardless of cause, would probably be associated with DNA laddering, even if the myocytes were not dying. TUNEL has undergone extensive criticism as a method to label apoptotic cells in situ. Necrotic cells, proliferating cells, or excessive RNA may be labelled by 78 TUNEL (112, 255, 388). Differentiating between non-specific DNA degradation, as seen in necrosis, and caspase-ICAD/CAD-specific degradation as seen in apoptosis is the largest difficulty in using TUNEL as an assay for apoptosis. In apoptosis there is nuclear condensation and maintenance of the nuclear envelope and TUNEL staining appears as dense nuclear staining. Diffuse cytoplasmic staining may be more necrotic where the nuclear membrane has been degraded. In either case, contiguous 4 uM sections stained with haematoxylin and eosin or Masson's Trichrome is used in combination with TUNEL to try to identify morphologic criteria of cell death. Another method that may be more attractive to detect apoptosis in the myocardium is to detect the phospoholipid phosphatidylserine that translocates from the internal to the external leaflet of the cell membrane. This process may occur earlier than DNA fragmentation (482) and can be detected in vivo by labeled-annexin V (either radionucleotide, fluorescent, or biotin), which has high affinity for this phospholipid (714, 715, 796). Various animal models have been established that allow for better examination of the importance of apoptosis during the progression to heart failure. During the past seven years these animal models have lead researchers along a path towards therapeutic intervention, as it has been determined that cell death in the myocardium is a significant component of end stage disease. From the first phenotypic descriptions of apoptosis in the myocardium in these animal models, including the controversy over apoptosis versus necrosis, to understanding the biochemical pathways of cell death in the myocardium, and the role of survival signaling in the myocardium, researchers have generated numerous potential therapeutic targets to minimize cell death and maintain myocardial integrity. Progress is demonstrated by dissecting a classic animal model of myocyte cell death, occlusion/ischemia and reperfusion-oxygenation in the rat that models myocardial infarct injury. The ischemia and reperfusion model in the rat has been used to dissect the method of cell death in myocardial infarcts. Cell apoptosis as demonstrated by TUNEL staining and DNA agarose gel assays are evident as early as 2-3 h after occlusion and are still evident for long periods after occlusion (342). This detection of apoptosis can be noted earlier if different 79 detection methods are used such as the exposure of phosphatidyl serine on the outer leaflet of the plasmalemma, as DNA damage is a late event in apoptosis and the degree of DNA fragmentation is debated in the heart. Both ischemia and reperfusion contribute to the apoptosis, as ischemia alone induced apoptosis and reperfusion accelerated the process (212). It was also noted that preconditioning by short periods of ischemia followed by reoxygenation can reduce the extent of apoptosis and myocyte loss during longer periods of ischemia and reperfusion (485, 564). In the affected area, was that endothelial cells experience apoptosis at times prior to apoptosis in myocytes (617) and the lesions appear to spread with time from the affected endothelial cells suggesting that these cells may release soluble proapoptotic mediators that influence apoptosis of surrounding myocotyes. As in other models of myocyte apoptosis, many investigators have noticed changes in expression of certain apoptosis or regulatory proteins. There is an increase in the expression of Bax and decrease in the expression of Bcl-2 in rat ischemia/reperfusion injury (128). H9c2 cells that are exposed to periods of hypoxia significantly decrease ARC expression, which is know as an inhibitor of caspase 2 activation (195) as will be discussed in Section 5.4. During protective preconditioning, there is an upregulation of Bcl-2 and activation of N F K B (480, 481), and a decrease in AP-1 binding activity (611). It has been suggested that there is a role for PKC in preconditioning as its inhibition using calphostin C dramatically decreased the protective role of preconditioning (537). Cultured rat myocytes in prolonged hypoxia led to increased p53 transactivating activity and expression of p21/WAF-1 (442) and in rat models significant induction of the expression of p53 occurred after ischemia and reperfusion in isolated working hearts (483). Angiotensin II type I receptor (AT1) receptor expression increase in the rat myocardium immediately following a period of ischemia and reperfusion (770). The ATI receptor partially mediates activation of myocardial JAK-STAT pathway in acute myocardial ischemia (544). It has been shown that components of the JAK/STAT signaling pathway (STAT5a, STAT6) can bind to the promoter of the angiotensinogen (ANG) gene and consequently upregulate the level 80 of ANG mRNA (471). Additional evidence of the importance of the AT-1 receptor and ANG II in mediating cellular integrity come from experiments using losartan, an AT-1 receptor blocker, and HOE 140, a bradykinin B(2) receptor blocker. Bradykinin B(2) receptor activation leads to inhibition of ANG II formation. Treatment with losartan significantly reduced myocardial ischemia/reperfusion injury by blocking AT(1) whereas HOE 140 increased injury, presumably by allowing for ANG II generation (612). Conflicting reports demonstrated increased expression and transcriptional activity of STAT-1 and antisense STAT-1 vector decreased cell death in cardiac cells (658). It was also reported that the Fas/FasL genes and apoptosis are activated by STAT-1 in cardiac myocytes exposed to ischemia/reperfusion and these effects are dependent on the Ser-727 phosphorylation of STAT-1 (659). Death ligands and their cognate receptors may also influence the cell viability following ischemia and reperfusion. Soluble CD95 ligand/Fas ligand, TNF-alpha, and TRAIL were released by the postischemic hearts early after the onset of reperfusion in isolated hearts (335), suggesting the potential for an autocrine/paracrine phenomena. In addition to the presence of the ligands, in primary adult rat myocyte culture, hypoxia and reoxygenation caused a marked increase in sensitivity to the apoptotic effects of CD95 ligand and isolated hearts from mice lacking functional CD95 (Ipr) display marked reduction in cell death after ischemia and reperfusion compared with wild-type controls (335). The MAPK signaling pathways all can influence the eventual survival of the myocytes and the severity of the disease. All three pathways have been shown to be induced during the ischemic and reperfusion episodes. Using various inhibitors, transgenics, and knockout mice, the influence of these pathways on eventual survival of the cells can be elucidated. The extracellular signal-regulated kinases (ERK) 1/2 are activated by oxidative stress in cardiac myocytes and protect cardiac myocytes from apoptosis. Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of ischemia/reperfusion-induced ERK activation and the number of apoptotic cells was increased to 33.4%. It should 81 also be noted that MEK1/MEK2 inhibitor increased p38 and JNK activities by 70.3% and 55.0%, respectively (785). In cultured neonatal rat cardiac myocytes p38 MAPK activation was observed during ischemia and SB 203580, a p38 and JNK2 inhibitor, reduced activation of caspase-3, a key event in apoptosis (453). Further, pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, in ischemia/reperfusion culture where ERK was concomitantly inhibited by PD98059, the number of apoptotic cells were reduced by 42.8% and 63.3%, respectively (785). Another study using H9c2 cardiac cell line and oxidative stress demonstrated that inhibition of the JNK1 pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress (707) and additional studies revealed that apoptosis induced by other JNK-activating stimuli, including ceramide, heat shock, and UV irradiation, was partly suppressed after treatment with JNK1 AS but not JNK2 AS (298). Taking a different approach, the use of the tyrosine phosphatase inhibitor, vanadate, to prevent p38 inactivation resulted in higher susceptibility to cell death from ischemia (454). These studies clearly demonstrate a protective role for ERK while p38 and JNK activation result in increased susceptibility to apoptosis. The protective role of ERK1/2 activation may be associated with COX-2 induction (3). A variety of growth factors, antioxidants, and caspase inhibitors that can influence signaling pathways have been shown to limit injury and apoptosis following ischemia/reperfusion. Growth factors including FGF-1 (159) HGF (515), IGF-1 all mediate anti-apoptotic effects, presumably through PI3-kinase and Akt activation. The oxidative state and presence of free radical scavengers are important factors in determining the extent of injury. Glutathione peroxidase (GSHPx)- experiments using isolated hearts from GSHPx-1 knockout mice and transgenic mice overexpressing GSHPx-1 demonstrate significant number of apoptotic cells present in GSHPx-1 knockout mice after 30 minutes of ischemia while very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-1 (484). In the isolated heart model, preperfusing in the presence of SOD and catalase greatly 82 decreased the presence of apoptotic cells and DNA fragmentation in the myocardium (223). In vivo treatment with PDTC in the ischemia/reperfusion model also had significant effects in the decrease in lesion size and number of apoptotic cells (430). ZVAD.fmk treatment of cells has been shown to limit the induction of apoptosis after infarct in the rat, but whether or not these inhibitors prevent eventual cell death is not completely known. Cells may already be committed to die as some studies have demonstrated that ZVAD.fmk inhibited myocyte DNA fragmentation and caspase activation without reduction of the infarct size in ischemia-reperfused rat hearts (538). The study of the ischemia/reperfusion model in rats using isolated myocytes, isolated and perfused hearts, and in vivo models has demonstrated that cell death by apoptosis is a significant contributor to heart dysfunction. These studies have also demonstrated that no single pathway can be implicated as being most important, or that all cells do not die by the same signaling mechanisms. It is most likely a heterogenous-state where cells are receiving different stimuli depending on where they are situated in the infarct, how long they are under ischemic conditions for, the status of their neighbor cells, the local hormonal and soluble mileu, the oxidative state and nutritional status of the host, among many others. 5.4. Intrinsic inhibitors of myocyte apoptosis Cardiac myocyte survival is of central importance in the maintenance of cardiac function as myocytes are terminally differentiated and typically deemed not able to proliferate. It is also well known that various cell type expresses its own distinct repertoire of proteins that will influence all cellular aspects including cell death. No two different cell types will expect to undergo the same signaling pathways with the same consequences following the same stimulus. The use of gene expression arrays has demonstrated that there are distinct expression profiles within each cell type and these expression profiles will change during the life of the organism and following a variety of stimuli. There is growing evidence to suggest that the myocytes may be more resistant to apoptosis as compared to other cell types that are able to undergo cell division or regeneration. 83 Mammalian FLIPs, as previously described in Section 3.3, inhibit death signaling via the death receptors. These proteins contain DED's and compete with caspases and prevent recruitment to the DISC and subsequent autoactivation (322). It is interesting that these proteins are highly expressed in muscle tissue including the heart (322). It is assumed that this over-expression in myocytes would depress sensitivity to TNF-a, Fas-ligand, Trail, and other death receptor activation mechanisms of apoptosis induction under-normal conditions. During systemic infection or other states of immune activation the cardiac tissue could be surrounded by activated T cells and humoral factors capable of initiating the death cascade and it would be beneficial to inhibit any non-specific activation of apoptosis. In many inflammatory diseases of the heart, including myocarditis, there is an abundance of activated T cells dispersed throughout the network of connecting myocytes, but by light microscopic examination the myocytes appear relatively healthy. Similar to FLIP'S, the apoptosis repressor with CARD (ARC) protein contains a competing CARD sequence similar to that is present in RAIDD and not in capase-9. This protein interacts with caspase 2 and 8, and remarkably is apparently restricted to skeletal and cardiac myocytes (393). Transfection of ARC into H9c2 cells results in additional resistance to ischemia-mediated apoptosis and this works upstream of mitochondrial events as cytochrome c is not released from the mitochondria (195). Capase-9S/9b/CTD is dominant negative inhibitor of caspase 9 as it is missing the catalytic site but is able to bind Apaf-1 and block Apaf-1-caspase 9 interaction (20, 631, 654). Casp-9-CTD is expressed in multiple tissues, with the relative highest expression observed in ovary and heart. Cells that overexpressed this protein were severely compromised in their ability to induce apoptosis after cytochrome c release from the mitochondria (20). Myocytes have adapted other methods to delay or inhibit caspase 9 activation. Evidence suggests that myocytes and other types of terminally differentiated cells including neurons sequester caspase 9 in the inter mitochondrial membrane space, where it is released in response to stimuli that release other apoptosis factors from this space (397). Sequestered caspase 9 would prevent apoptosis induction if there was a potential cytochrome c leak into the cytoplasm. Examination 84 of human myocytes in dilated cardiomyopathy has demonstrated a large amount of cytochrome c present in the cytoplasm (518). Several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. Hsp70 and Hsp90 bind to Apaf-1 and prevent the recruitment of procaspase-9 to the apoptosome, thereby inhibiting caspase-9 activation (55, 552, 605). Hsp27, a member of the small HSP family, has been shown to bind and sequester cytosolic cytochrome c from the apoptosome and prevent procaspase-9 activation (90, 224). Upregulation of HSP70, 90 and 27 are all associated with preconditioning of the heart and various stress inducing conditions (170, 200, 520). Beta-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3 (344). Beta-crystallin is constitutively expressed in many tissues, and it is particularly abundant in the heart and skeletal muscle (358). Cardiotrophin-1, a cardiac myocyte cytokine related to interleukin-6, is capable of inhibiting apoptosis in cardiac myocytes (635). CT-1 mRNA is expressed in the heart and upregulated in a variety of conditions including hypertrophy (560), hypoxic stress (291), congestive heart failure (338), myocardial infarction (22), and coxsackievirus-induced myocarditis (540). This cytokine can act via the luekemia inhibitory factor (LIF) receptor and gp130 signaling subunit (127, 560). gp130 signaling can activate PI-3 kinase in cardiac myocytes following stimulation with LIF, a factor closely related to cardiotrophin-1 (534). It was demonstrated that cardiotrophin-1 activates Akt (408), a target of PI-3 kinase, and can deliver anti-apoptotic signals to cells as previously described in Section 3.3 (204, 366, 562). In addition to PI3K/Akt, cardiotrophin-1 has also been shown to activate p38 and ERK pathways and to induce the translocation of N F K B to the nucleus in cardiac myocytes (153). Using inhibitors of N F K B activation, it was demonstrated that the N F K B translocation to the nucleus and subsequent transactivation functions were required for its cytoprotective effect (153) in myocytes, and has also been shown in neurons (500). 85 Other growth factors and survival factors that are known to inhibit apoptosis via PI3K/Akt in myoctes include insulin growth factor-l (94, 429, 736), and VEGF (230). Activation of the PI3K/Akt is cytoprotective in myocytes against a variety of conditions including hypoxia and ischemia (218, 474, 475). In addition to various growth factors and cytokines, selective P(2)-AR stimulation protected myocytes from apoptosis through increased ERK activation as well as PI3K/Akt phosphorylation (129, 807). Neuregulins (NRG) can promote survival and growth of cardiac myocytes as well as inhibit apoptosis induction (802). ErbB2 and ErbB4 are present in adult cardiac myocytes and stimulation with NRG-1 activated the p42/p44 mitogen-activated protein kinase (MAPK) in neonatal rat ventricular myocytes (41). In other cells, including Schwann cells, the NRG's activate the PI3K/Akt pathway (432) suggesting that this pathway may also play a role in the cardioprotective effect of NRG stimulation. It appears as though ERK activation is protective while p38 and JNK1 activation can enhance apoptosis in a variety of myocyte models of apoptosis. When H202-induced activation of ERKs was selectively inhibited by PD98059, the number of cardiac myocytes that showed apoptotic death was increased (7). Apoptosis induced by other JNK-activating stimuli, including ceramide, heat shock, and UV irradiation, was partly suppressed after treatment with JNK1 AS, but not JNK2 AS. These findings demonstrate that the JNK1 isoform plays a preferential role in apoptosis induced by ischemia/reoxygenation as well as diverse JNK-activating cellular stresses (298). The Bcl-2 family of proteins are important regulators of apoptosis induction as previously mentioned. In transplant rejection and end-stage heart disease there is evidence of enhanced expression of Bcl-2 (501, 542). Bad and Bax were present at high levels in neonatal hearts, but they were barely detectable in adult hearts (151). As mentioned the mammalian lAP's (inhibitors of apoptosis) were discovered as homologs to a baculovirus viral protein (156) that prevents cytochrome c-mediated caspase 9 activation, and directly inhibits the protease activity of active caspase 3 and 7 (182, 183, 599). The expression and cell-specific regulation of the lAPs including their regulator molecules 86 Smac/DIABLO in the myocardium are not completely understood, but activation of the N F K B pathway is now known to increase the transcription of pro-survival genes including c-IAPI and C-IAP2 (733). Considering TNF-a apoptosis induction could be inhibited by FLIP'S, the N F K B pathway may induce enhanced IAP transcription in the heart. TNF-a can be produced by myocytes (353) and the autocrine-paracrine actions of this cytokine on heart failure are an intense area of study. Serum TNF-a levels are greatly increased in patients with chronic heart failure (423). TNF-a stimulation of isolated cardiac myoctes results in a significant increase in gene transcription and N F K B translocation to the nucleus (176). Other studies have shown that TNF-a stimulation of isolated cardiac myocytes results in apoptosis (401). Transgenic mice with TNF-a over-expression in the heart (93, 404) had dilated hearts with chronic inflammation and depressed cardiac function. Cell death was evident but not abundant. TNF-a may be protective against death in cardiac myoctes, but the pro-inflammatory and negative inotropic effects of this cytokine may out-weigh its benefits. As summarized in Figure 15, enhanced expression of a variety of apoptosis inhibitors and an aptitude to signal via NFkappaB, ERK1/2, and PI3-K/Akt pathways suggests that cardiac myocytes may be less susceptible to apoptosis than other cell types. This natural resistance may play an important role in the persistence of viral RNA within the myocardium as compared to other organs as will be discussed in Section 10. 87 Figure 15. Overview of intrinsic, anti-apoptotic features of the cardiac myocyte. There appear to be numerous proteins expressed within the cardiac myocyte that enhance resistance to unchecked apoptosis. These features may contribute to persistence of viral genome following infection with CVB3 8 8 6 RESEARCH FOCUS The life or death decisions of a cell following virus infection are critical in determining the fate of the virus and host. Viruses have evolved mechanisms to inhibit early cell death to allow for viral parasitization and maximal viral replication. Conversely, in order to limit viral replication and dissemination, the host has evolved mechanisms to rapidly induce apoptosis, either directly by the infected cell or indirectly in response to factors released by non-infected interacting cells. Too much apoptosis or too little apoptosis are critical features of many diseases including heart disease and cancer. Further, terminally differentiated cells like myocytes, where extensive death is detrimental to the overall health of the host, may have intrinsic expression profiles of proteins to prevent unchecked apoptosis, but may create an environment that is optimal for persistence of virus. In the following sections of the dissertation I will present data which address three issues: 1) CVB3 is a systemic disease with widespread dissemination and localization in multiple cell types, and this infection results in direct cell mediated injury in multiple organs 2) The death of infected cells is a regulated process involving host proteins and viral proteases, allowing for maximal replication and release of progeny virus. 3) The infection in myocytes and other cells initiates a complex cascade where various cellular proteins change their expression profiles. Infected cells, neighboring cells, immune cells, and humoral factors all contribute to this heterogeneous milieu where the fate of the host cell is determined. 89 7 HYPOTHESIS, SPECIFIC AIMS AND MAJOR QUESTIONS The central hypothesis of this work is that the pathogenesis of Coxsackievirus B3 induced disease in multiple organs including the heart is determined to a significant degree by the direct activation of host cell death machinery within the infected cell. The following aims are addressed experimentally: 1. To determine the extent of direct virus induced injury in the murine host. 2. To determine the role of host cell "death machinery," including caspases and regulatory molecules in virus-induced cytopathic effect in transformed cell lines, and myocarditis-susceptible mice. 3. To determine the effect of caspase inhibition on cell viability, viral modification of host cellular events, and virus replicative cycle in transformed cell lines. 4. To identify key cellular genes and pathways that are activated during the acute, sub-acute, and chronic stages of CVB3 infection in mice and understand their influence on viral replication and host cell viability and host phenotype. Major questions relevant to the hypothesis and aims are: 1. Does CVB3 replicate in multiple organs following infection? 2. Does replication in multiple organs induce apoptosis? 3. Is the decrease in immune cell numbers noted during viral infection due to viral activation of apoptosis? 4. Is cytopathic effect apoptosis? 5. Does host cell apoptotic machinery participate in the death of the infected cell? 6. What is the effect of inhibiting host cell death machinery on viral replication and host viability? 5. What are the protein expression profiles in the myocardium during acute and chronic infection? And their relevance to host and virus death and survival? 90 8 EXPERIMENTAL DESIGNS Note that the detailed methodologies of the techniques utilized in the following experimental designs, are outlined in Section 13. 8.1. Aim #1. To determine the extent of virus induced injury in the murine host 8.1.1. Time course AJ and C57BL6J mice Adolescent A/J and C57BL/6J mice were infected with 1x105 pfu of myocarditic CVB3 or PBS and euthanized on days 1, 2, 2.5, 3, 3.5, 4, 5, 6, and 7 post-infection. Four mice per group per time-point were euthanized and heart, liver, spleen, lymph nodes, pancreas, salivary glands, testis, lung, kidney, brain, and skeletal muscle were fixed in 4% paraformaldehyde. These organs were evaluated for direct viral damage at the light microscopic level and concentration of virus by plaque assay. Virus RNA was evaluated by in situ hybridization (ISH). Apoptosis was evaluated by TUNEL. IL-2 and IL-4 levels and caspase activity were assayed in the hearts and spleens of mice at day 4 and 7 post-infection and compared to sham infected animals. Animals that died naturally after infection were not included in experimental analysis. 8.1.2. 4 week vs. 10 week old animals Adolescent (4 week old) and adult (10 week old) A/J mice were infected with 1x10s pfu of myocarditic CVB3 or PBS sham and euthanized on days 4 and 7 post-infection. Eight mice per group per time-point were euthanized and heart, liver, spleen, lymph nodes, pancreas, salivary glands, testis, lung, kidney, brain, and skeletal muscle were fixed in 4% paraformaldehyde. These organs were evaluated for direct viral damage at the light microscopic level and concentration of virus by plaque assay. Virus RNA was evaluated by ISH. Apoptosis was evaluated by TUNEL. Animals that died naturally after infection were not included in experimental analysis. Yang et al. (772), in the McDonald Research Laboratory, St. Paul's Hospital, performed differential display analysis on the myocardium of these mice (discussed in Section 10). 91 8.2. Aim #2. To determine the role of host cell death machinery in virus induced cytopathic effect HeLa cells (human cervical carcinoma cells) were infected at a multiplicity of infection (MOI) of 5 with coxsackievirus B3 or sham treated with minimum essential medium (MEM) without fetal bovine serum (FBS) for 45 minutes. Cells were washed with phosphate buffered saline (PBS) and complete MEM containing 10% FBS was replaced. A positive apoptosis control consisted of treating HeLa cells with the photosensitizer benzophorphyrin derivative monacid ring A (BPD-MA, verteporfin) for one hour followed by exposure to visible light (103). Caspase inhibition studies utilized ZVAD.fmk treated (0-200 uM) HeLa cells in conjunction with viral infection or BPD-MA and light treatment. Cells were harvested at 0, 1, 3, 5, 6, 7, 8, 9, 10, and 12 h post-infection (CVB3) or 2 h following light treatment. Cell lysates were collected at each time-point and stored at -20°C for further biochemical analysis. 8.3. Aim #3 To determine the role of caspase inhibition on cell viability, viral modification of host cellular events, and virus replicative cycle in transformed cell lines HeLa cells (neo or overexpressing Bcl-2 or Bcl-xL) were infected at a multiplicity of infection (MOI) of 10 with CVB3 or sham treated with minimum essential medium (MEM) without FBS for 45 minutes. Cells were washed with phosphate buffered saline (PBS) and complete MEM containing 10% FBS was replaced. As a comparative model, HeLa cells were exposed to concentrations of TRAIL including 0, 10, 25, 50, 100, 500, and 1000 ng/ml or incubated with 200 ng/ml verteporfin for 60 min at 37°C and exposed to blue fluorescent light. Cells were maintained at 37°C with 5% C0 2. Cells were harvested at 0, 1, 3, 5, 6, 7, 9, 10, and 12 h post-infection (CVB3), or various timepoints following PDT or TRAIL treatment depending on apoptosis kinetics. 92 8.4. Aim #4. To identify key cellular genes and pathways that are activated during the acute, sub-acute, and chronic stages of CVB3 infection Adolescent A/J mice were infected with 1x105 pfu of myocarditic CVB3 or PBS sham and euthanized on days 3, 9 and 30 post-infection. Fifteen mice per group per time-point were euthanized and small pieces of heart, liver, spleen, and pancreas were fixed in fresh 4% paraformaldehyde. These organs were evaluated for direct viral damage at the light microscopic level. The remainder of the heart was fresh frozen for mRNA isolation. SCIOS, Inc. (Sunnyvale CA) performed cDNA microarray analysis on the isolated mRNA as described in Section 12. Animals that died naturally after infection were not included in experimental analysis. 93 9 RESULTS 9.1. Aim#1 Viral localization to multiple organs early post-infection. Myocarditis-susceptible A/J (H-2a), and myocarditis-resistant C57BL/6J (H-26) mice were used as models to study the systemic localization of viral RNA following CVB3 infection. In both strains of mice examined following ip infection, CVB3 positive-strand viral RNA could be detected in many organs by 24 h post-infection using ISH (Figure 16). At this early time-point, viral RNA localizes to rare individual cells in the heart, liver, brain, fat and kidney. There is a greater amount of viral RNA localized in the pancreas at 24 h as compared to other organs. By day two post-infection, the virus exhibits enhanced replication with more viral RNA positivity in each organ, including clusters of cells; negative strand viral RNA can be seen. The presence of negative-strand viral RNA as detected by using a sense-strand riboprobe is important in understanding the viral life cycle in tissue. The presence of positive-strand viral RNA is not an indicator of replication, but only an indication that the host cell presumably contains a receptor for coxsackievirus B3 and the viral RNA is able to enter the cell. Once in the cell, the viral RNA must be translated by host proteins for production of the viral-encoded RNA-dependent RNA polymerase, which facilitates the production of the negative strand viral RNA. Although not a perfect indication of viral replication, the presence of negative strand viral RNA indicates that all the materials required for virion packaging are present in the cell, as long as there has been high fidelity of the RNA-dependent RNA-polymerase. Maximal viral RNA localization to multiple tissues during peak viremia. In murine enterovirus infections, peak viremia occurs between days 3 and 4 post-infection (16, 225). During this period there is maximal infection of systemic organs with corresponding maximal localization of positive and negative-strand viral RNA. The heart, liver, spleen, exocrine pancreas and endocrine pancreas, brain, lung, kidney, thymus, salivary glands, skeletal muscle, lymph nodes, and visceral fat all contain cells that are positive for viral RNA as determined by in situ hybridisation (Figure 17). Organs more permissive for viral replication have widespread 94 Figure 16. Coxsackievirus B3 RNA localization in A/J mice at 24 hours pi. A/J mice were infected with 1 x10 5 PFU of CVB3 and tissues were harvested at 1,2,2.5, 3,3.5,4, 5,6, and 7 days post-infection. Virus RNA was evaluated by in situ hybridization. At 24 hours post infection positive strand viral RNA could be identified in various organs including A) heart, B) liver, C) fat, and D) pancreas. N= at least 3 animals per group. (Magnification 20X) 95 Figure 17. Coxsackievirus B3 RNA localization in A/J mice at 4 days pi. A/J mice were infected with 1 x105 PFU of CVB3 and tissues were harvested at 1,2,2.5,3,3.5,4,5,6, and 7 days post-infection. Virus RNA was evaluated by in situ hybridization. At 4 days post infection, maximal loads of positive strand viral RNA could be identified in various organs including A) heart, B) liver, C) fat, D) brain, E) exocrine and endocrine pancreas, F) spleen, G) salivary glands, H) lung, and I) testis. N= at least 3 animals per group (Magnification 20X). 96 positivity; such permissiveness is observed in heart, pancreas, liver, and brain wherein viral RNA is prominent. The lymphoid germinal centres and marginal zones are positive for positive strand viral RNA. Other organs including lung, kidney, thymus, and skeletal muscle have fewer cells with viral RNA positivity. C57BL/6J mice, classically used as a resistant model for coxsackievirus induced myocarditis, have a similar pattern of systemic disease as in the A/J model. Although the organ and tissue distribution among cells similar between both mouse models, except for the lymphoid organs as previously demonstrated by Anderson et al. (16), the total amount of viral RNA and infectious virus in organs differs between models. Comparison of ISH scores and infectious virus in the myocardium between A/J and C57BL/6J mice between days 3 and 7 post-infection demonstrates that the virus is rapidly but not completely cleared from the myocardium of C57BL/6J mice (Figure 18). Cellular localization of CVB3 in various organs and histologic features. Heart (Figure 17A) - Heart muscle is progressively infected in A/J mice between 1 and 4 days post-infection. There is a clear lack of infection in endothelial cells, either those lining the ventricular cavities or those in small arteries, veins, or lymphatics within the tissue. As well, there does not appear to be a detectable preference for infection of interstitial cells, such as fibroblasts. Indeed, the most visibly and clearly infected cell type is the cardiac myocyte. The pattern of apparent cell-to-cell spread is suggested as previously indicated by Klingel et al., (382) by the adjacency of cells that are positive for viral genome. However, equally striking, are the cardiac myocytes that are quite apparently singularly infected. There is a clear demarcation at the level of the intercalated discs that can be appreciated in certain longitudinally-cut myocytes. The involvement of the ventricular septum, left ventricular free-wall, and right ventricular free-wall appear generally similar in pattern, although the extent of right ventricular free wall involvement may be somewhat less. Clearly the atrial tissue has more limited viral genome than the ventricular cardiac myocytes. There is an interesting coexistence of cardiac muscle cells that have numerous coalescent vacuoles indicative of cytopathic effect, often 97 A 1E+05 C57BL/6J 1E+00 A/J C57BL/6J day 3 pi J day 4 pi | day 5 pi II day 6 pi Figure 18 In situ hybridization for CVB3 viral RNA localization and plaque assay for infectious virus in hearts of adolescent A/J and C57BL/6J mice. Animals were sacrificed at days 3,4,5, and 6, and plaque assay and in situ hybridization were performed as previously described in Section 12. Note the increase in detectable viral RNA in the hearts of A/J mice (A) as compared with C57BL/6J mice (B). Over the time couse of day 3 to day 6 post-infection there is significantly more viral RNA (C) and infectious virus (D) in the hearts of the A/J mice as compared with the C57BL/6J mice (*p<0.05). N= at least 4 animals per group. (Magnification 20X) 98 immediately adjacent to cells which have apparently high-titre viral replication, but which have not undergone cytopathic effects. Pancreas (Figure 17E)- Pancreas tissue is exceedingly permissive for coxsackievirus B3 replication. There is near 100% infection of exocrine cells in the pancreas by the time there is prominent, but less dramatic infection of other tissues such as heart muscle and fat. The cells of the exocrine pancreas are preferentially infected as compared to both ductular cells and the endocrine acinii. When the infection is observed in hybridized tissue, there is a retention of the lobular architecture of the pancreas and no evident "outside-in" or "inside-out" patterns, but rather a virtual wall-to-wall infectious process proceeding almost simultaneously beyond 24 h post-infection. There is a trivial interstitial and interlobular amount of inflammation up to day 7. The endocrine acinii have infrequent cells at their margins that are positive for both positive and negative viral RNA. Thus there is evidence of replication of the virus, not only dramatically in the exocrine pancreas, but in a more limited fashion within endocrine acinii. Liver (Figure 17B) - The pattern of hepatic infection is one where the most susceptible mouse in this regard is the C57BL/6J. The hepatocytes themselves are dramatically- affected in a somewhat random scattered pattern. As the infection becomes more prominent and overwhelming, there is wide-spread involvement of hepatocytes. This is concurrent with infection of kuppfer cells in the liver. With less dramatic infection, there is a clear preference for virus to infect hepatocytes that are in the periportal to mid-zone region, generally sparing the region surrounding the central vein (central lobular). The basis of this pattern is unclear, however the drainage of virus through the portal vascular system may be partially reflected in the pattern of hepatocyte involvement. When hepatocytes become infected, there is clearly a cell cytoplasmic clearing, coalescent vacuoles representing cytopathic effects, as well as more densely infected small cells that can be visualized. Salivary glands (Figure 17G) - Of the three distinguishable types of salivary glands, the serous type of gland is preferentially infected, the adeno-serous is rarely infected, and the mucous gland appears to be non-permissive to CVB3. The glands themselves as opposed to the ducts 99 appear to be preferentially involved. There is also a pattern of RNA localization suggesting that the lobules of the glands are infected from the "outside-in". The infection within the preferentially infected subset of salivary glands is one of cell-to-cell, thereby infecting clusters as well as individual cells. The infection of the adeno-serous type of gland is widespread in distribution with an interesting spacing between individual cells that are infected or small clusters of cells. It would appear that the preferential cell of infection within the adeno-serous gland is the adeno cell. The prominence of positive strand viral RNA is strikingly greater than the negative viral RNA, even in the most permissive of salivary tissue. However, there are clearly salivary gland cells that are replicating viruses. The ratio of detected positive strand to detected negative strand viral RNA is in the range of 10:1 as compared to the exocrine pancreas which is more like 2:1. Fat (Figure 17C) - Mediastinal as well as mesenteric fat has a similar pattern of infection. There is a definite preference by the virus for adipose cells. Thus, there is prominent infection of fat, in the "outside-in" pattern mentioned for salivary glands, wherein there is almost a complete infection of the most superficial fat with relative sparing of the central adipose cells during the early days following infection with complete infection of the tissues at later time points. Cells that are infected appear enlarged and show evidence of cell death or cytopathic injury. There is not only prominent positive strand genome detected by in siu hybridization, but also very striking negative strand viral RNA Negative strand viral RNA in permissive tissue. By day seven viral RNA has been cleared from numerous organs. The induction of a specific immune response to viral antigens, including humoral and cytotoxic T cell mechanisms (CTL's) (340, 494, 541), is primarily responsible for viral clearance. Other mechanisms including non-specific immune responses, such as the generation of nitric oxide (792), or burnout where the virus kills numerous cells which are can no longer support viral replication may also be responsible for the clearance. Persistence of CVB3 RNA has been shown previously to be an important factor in the chronicity of viral myocarditis (18, 349, 382). In A/J mice, cells of heart, pancreas, salivary glands, lymphoid organs, and 100 brain contain both positive and negative strand viral RNA at day 7 post-infection (data not shown). Other organs including liver, kidney, lung, and testis are not positive for viral RNA by this time suggesting a more effective immune response or low permissiveness to infection. By day 7 the C57BL/6J mice have cleared viral RNA from most organs except for occasional foci of infected myocytes and pancreatic exocrine cells. Direct tissue injury in multiple organs by CVB3. Depending on the pattern of staining by TUNEL assay, cells undergoing different mechanisms of cell death could be identified. When contiguous sections were probed for viral RNA by ISH and tissue injury by TUNEL, CVB3 was seen to directly injure cells both by apoptotic and oncotic mechanisms. This demonstrated that cells that contain viral RNA are, indeed, preferentially injured in this disease process. In the spleen there is widespread TUNEL positive cells throughout the white pulp regions (Figure 19). The positive viral RNA localizes to germinal centres in A/J mice and germinal centres and the marginal zone macrophages in C57BL6J mice as we have previously demonstrated (16). The TUNEL positive cells extend beyond the zones of viral localization and localize to throughout the white pulp regions. In the thymus, there is massive apoptosis of the cortical zone T cells (Figure 19), but little or no presence of viral RNA positive strand as determined by ISH. The loss of the thymocyte double positive CD4CD8 positive cells population following infection is confirmed by flow cytometry using dual staining with CD4+ and CD8+ monoclonal antibodies (Figure 20). Interesting, in related studies that were mentioned in Section 2.1.4, examination for the presence of IL-2 and IL-4 in the heart and spleen of infected A/J and C57BL/6J mice between days 4 and 7 post-infection suggested that in addition to the massive apoptosis noted in the spleen, there was a per mg decrease in cytokine protein below sham infected levels as determined by ELISA (Figure 21). In the heart, staining of adjacent sections by TUNEL and ISH demonstrated that virally infected myocytes were TUNEL positive, but the staining was not focal within nuclei, but diffuse throughout the cytoplasm (Figure 22). This diffuse and rare staining of myocytes will be 101 Figure 19 . Splenic involution may be caused by apoptosis as demonstrated by co-localization of viral RNA by in situ hybridization (A,C, and E) and tissue injury by TUNEL (B,D,F,G,H) in adjacent tissue sections in A/J mice (A,B) and C57/BL6J (CD) murine spleens and A/J thymus (E,F) at day 4 post infection. Spleen (G) and thymus (H) from sham treated A/J animals at day 4 post infection represent residual apoptosis. (Magnification 20X) 102 Sham Infected FITC 1 2 : 4.2% I 5 % 1 mk 43.1% 4 n i l , m i I I M i l l I I . i 1009 FITC 60 ? 50 C "<5 40 4 - 1 ( 0 g> 30 +S "jg 20 O Q_ N « 10 0 s CD4 fluorescent intensity • Sham • Infected CD8+ CD4+ CD4CD8++ Figure 20 . Thymic apoptosis and decrease in CD4CD8 double positive cell population following CVB3 infection in A/J mice. A/J mice were infected with CVB3 and harvested at day 4 pi. Thymocyte apoptosis was confirmed by TUNEL staining of (A) sham and (B) infected mice. Single cell suspensions of splenocytes or thymocytes were prepared by delicately homogenizing the spleen or thymus in a Wheaton™ glass tissue homogenizer. Cells were labelled for surface antigens CD4, CD8, with antibodies conjugated to FITC or R-PE. After labelling, cells were analyzed on a FACStar°'u s flow cytometer. Results show flow cytometry data from one animal in each group and graph shos data obtained from 4 animals. (X10 Magnification) Error bars indicate standard deviations. 103 IL-2 e x p r e s s i o n for CVB3 - i n f e c t e d C57 a n d A J hear ts IL-2 e x p r e s s i o n in CVB3 - in fec ted C57 a n d A J s p l e e n s 10 N 4 sham T i m e ( d a y s ) 0.2 sham T i m e (days ) IL-4 e x p r e s s i o n h CVB3 - i n f e c t e d C57 a n d A J hearts 10 3 7 £ 5 o> 3 sham IL-4 e x p r e s s i o n h CVB3 - in fec ted C57 a n d A J sp l eens sham T i m e ( d a y s ) T i m e (days) Figure 21. Expression of IL-2 and IL-4 in hearts and spleens of A/J and C57BL/6J mice. Animals were sacrificed at days 3, 4, 5, 6, and 7 and hearts and spleens were gently homogenized in PBS. IL-2 and IL-4 protein expression was determined by using ELISA kits. Note the decrease in IL-2 and IL-4 protein levels during the early acute immune response below basal levels in infected mice. N= 4 animals per group. Some groups had 3 animals due to animal mortality. Error bars indicate standard deviations. 104 Figure 22. Contiguous staining of myocardial sections for viral RNA by ISH (A) and cell death by TUNEL (B). Select cells positive for viral RNA (arrow) also stain positive TUNEL reaction. (20X Magnification) 105 discussed further in Section 1.0, but is probably related to Section 5.4 which outlines why myocytes may be more resistant to caspase activation than other cells. In other organs, including the pancreas, liver, and salivary glands, there was a direct concordance where viral RNA positive cells were TUNEL positive (Figure 23). In all organs, the TUNEL positivity appeared to coincide with positive strand viral presence within the cells. Looking at organs where there is no viral positivity as determined by ISH, we could find little or no evidence of apoptosis, other than the rare cells that are probably undergoing residual cell death. Age related changes in viral RNA positivity and infectious virus in 4 week old and 10 week old A/J mice As outlined in Section 2.1.2, there is experimental evidence that adolescent mice become more resistant to viral myocarditis. We wanted to determine if this state of resistance occurs in all organs, or just the myocardium following infection. We used 4 and 10 week old A/J mice to directly compare the viral localization and infectious virus in multiple organs at days 4 and 7 post infection. In the myocardium there was clearly less positive strand viral RNA and infectious virus in the 10 week old A/J mice at 4 days following infection as compared to 4 week old A/J mice (Figure 24). Paradoxically, there was increased mortality of the 10 week old mice during the early acute phase of the disease (days 2-5) as compared to the 4 week old mice. By examination of organs of 4 and 10 week old mice, it was clear that other organs were equally involved with CVB3 infection as determined by ISH localization and concentration of infectious virus (Figure 25). There are numerous potential reasons for why the myocardium may become less susceptible to CVB3 infection in adult mice as will be discussed in Section 10. Other than concentration and amount of localization, there were no distinct differences in the patterns of localization and TUNEL concordance between both age groups. 9.2. Aim #2 At time zero, HeLa cells were infected with CVB3 or sham-treated (no virus) with MEM containing no FBS. Cultures were examined and harvested at 0, 1, 3, 5, 6, 7, 8, 9, 10, and 12 h 106 Figure 23. Direct viral injury in the (A,B) pancreas, (C,D) liver, and (E,F) salivary glands as demonatrated by by co-localization of viral RNA by in situ hybridization (A,C,E) and tissue injury by TUNEL (B,D,F) in adjacent tissue sections (Magnification 15X). 107 Heart Pancreas 4 week-old A/J 110 week-old A/J Figure 24 In situ hybridization for CVB3 viral RNA localization and plaque assay for infectious virus in hearts of 4 week-old A/J and 10 week-old A/J mice following infection with 1x105 PFU of CVB3. Animals were sacraficed at day 4 post-infection, and plaque assay and in situ hybridization were performed as described in Section 10. Note more detectable viral RNA in the hearts of 4 week-old A/J mice (A,B) as compared with 10 week-old A/J mice (D,E). The decreased susceptibility of 10 week-old A/J mice was myocardium specific as other organs such as the pancreas (C,F) were equally infected. There was significantly more viral RNA (G) and infectious virus (H) in the hearts of the 4 week-old mice as compared with the 10 week-old mice (*P<0.05). N=at least 4 animals per group. 108 (0 3 (A tfl (0 Cfl (0 © D) CO 3 Q.'S. 1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 • 10W # ^ ^ ^ £ o o CO c o ^ '*= + CO if) <fl c • 10W Figure 25. Plaque assay for infectious virus and ISH scores in organs of 4 week-old A/J and 10 week-old A/J mice following infection with 1x105 PFU of CVB3. Animals were sacraficed at day 4 post-infection, and plaque assay and in situ hybridization were performed as described in Section 10. N= 6-8 animals per group depending on mortality. Experiment was repeated twice. Note that in organs beyond the myocardium, there is more infectious virus and viral RNA localization 109 post-infection. Cells were treated with verteporfin and light as a positive control for apoptosis. The temporal pattern of production of CVB3 viral proteins, progeny virus, and the evolution of HeLa cell degenerative morphology changes were considered in conjunction with an examination of host cell death proteins. Significant increases in viral protein synthesis can be detected between 3 and 5 h post-infection by immunoblot analysis using a goat polyclonal anti-CVB3 antibody (Figure 26). The viral proteases cleave viral as well as host proteins early following infection. Eukaryotic initiation factor 4G (elF4G) is cleaved by viral protease 2A beginning within 1 hour following infection with the complete loss of the 220 kDa protein by 5 to 6 h following infection (Figure 26). A faint band can be seen appearing at the bottom of the elF4G gel at 12 h post infection. As will be demonstrated later, and subsequently confirmed by other groups (787), caspases also play a role in elF4G degradation during CVB3 infection and is responsible for this late degradation product. Progeny virus in the supernatant (corresponding to packaged virus which has been released from the cell cytoplasm) is present at basal levels between 1 and 5 h. Between 6 and 8 h post-infection there is a detectable increase in virus supernatant levels and exponential virus production after 9 h post-infection as determined by plaque assays (Figure 26). HeLa cells exhibited marked changes in morphology including cellular condensation, rounding up as noted by phase contrast microscopy between 6 and 7 h following infection and release from the monolayer around 10 to 12 h pi (Figure 26). To determine whether the host cell death machinery is activated following CVB3 infection, immunoblot analysis of lysate collected at specific time-points was performed. Caspase 3, a principal downstream effector of apoptosis, is normally present in cells as a 32 kDa precursor protein. Following CVB3-infection, levels of the 32 kDa precursor protein began to diminish between 7 and 8 h post-infection and it was almost completely undetectable by 12 h post-infection (Figure 27). This antibody did not pick up the cleavage product, so to determine whether or not the depleted procaspase 3 had been proteolytically processed from a single-chain zymogen to an active two-chain enzyme, a caspase activation assay using fluorescent substrates, which gain fluorescent capabilities when specifically-cleaved was employed. Using 110 10000000000 & c £ 1000000 5) fi 10000 3 o I D u_ CL B . 100 1 .1 i i • nm CVB3 elF4y —220kDa —100kDa 0 1 3 5 6 7 8 9 10 12 hours post-CVB3 infection 12 hours post-CVB3 infection Figure 26. Release of progeny CVB3 virus, host cell production of CVB3 viral protein, shut-off of host cellular translation, and cell morphology changes following CVB3 infection. A. There was an increase in the amount of infectious virus released over the 12 hour experiment (PFU/ml). B. Cell lysate was collected from CVB3 infected HeLa cells and immunoblot analysis with a CVB3 polyclonal antibody that recognizes major viral proteins was performed. Note the marked increase in viral protein production occurring between 3 and 5 hours post-infection. C. Cytosolic extract was then analyzed for the presence of the 220kDa eukaryotic initiation factor 4 gamma (elF4G) component of the translation initiation complex. Note the rapid cleavage of elF4G beginning within 1 hour following CVB3 infection. The protein was not detectable by chemiluminescence by 5 hours post-infection. D. Phase contrast morphology of HeLa cells at 0,6,7, and 12 hours post-infection. Note the extensive cytopathic changes occurring between 6 and 7 hours post-infection. 111 32kDa 0 1 3 5 6 7 8 9 10 12 I I hours post-CVB3 infection Figure 27. Caspase 3 activation and cleavage of 32 kDa proform following CVB3 infection in HeLa cells. A. 10 ug of cell lysate was incubated in 150 ul of reaction buffer containing the caspase 3 specific substrate, Ac-DEVD-AMC. After incubation at 37°C for 1 hour, fluorescence levels were determined with an excitation wavelength of 380 nm and emission wavelength of 460 nm. Note the increase in fluorescence, representing caspase activity, beginning after 7 hours post-infection and increasing to maximum levels by 10 hours post-infection. Error bars represent standard deviation on triplicate runs. Experiments were repeated at least 10 times with similar results. B. HeLa cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblotting for the presence of the 32 kDa proform of caspase 3 demonstrates that this protein was processed between 7 and 12 hours post-infection. 112 this approach, caspase 3-activity was evident around 8 h post-infection, with an increase in activity to 10-12 h post-infection, where caspase 3 reached a maximum level of activation (Figure 27). This protease assay demonstrated that caspase 3 was in an active form in infected cells, and capable of proteolytically processing other caspases and substrates. Caspase 3 cleaves specific substrates at aspartic acid residues as mentioned in Section 3.4. In CVB3-infected HeLa cells, PARP degradation and the appearance of a 85 kDa fragment was detectable by 9 h post-infection with further reduction of levels of the 116 kDa peptide by 10 and 12 h following infection (Figure 28). DFF is cleaved from a 45 kDa protein producing a 30 kDa fragment beginning 9 h following infection with continued processing and loss of the 45 kDa protein between 10 and 12 h post-infection (Figure 28). To determine whether caspases contribute directly to the characteristic cytopathic effect that occurs following picornavirus infection, cells were treated with the pan-caspase inhibitor ZVAD.fmk in increasing concentrations. This peptide is has been shown to inhibit caspase-induced morphologic alterations induced by multiple apoptotic stimuli. ZVAD.fmk concentrations of 25 to 100 uM blocked caspase activity, and cleavage of PARP and DFF in BPD-MA and light treated HeLa cells (positive control for apoptosis) (Figure 29). In CVB3 infected HeLa cells, the cleavage of PARP and DFF was partially prevented with inhibitor concentrations of 25 and 50 uM (Figure 29). There was no evidence of PARP or DFF cleavage fragments in CVB3 treated cells at 10 h following infection at the highest concentration of the inhibitor (100 uM). To determine if inhibition of caspases influenced cytopathic effects, morphology was examined at various intervals following infection. At 2 h following photoactivation of BPD-MA, HeLa cells were condensed, had extensive membrane blebbing, and were releasing from the monolayer (Figure 30). At increasing concentrations of ZVAD.fmk, this apoptotic phenotype was not apparent, with cells maintaining morphology similar to the control cells (Figure 30). Conversely, CVB3 infected HeLa cells were condensed, releasing from the monolayer, but exhibited no membrane blebbing at 10 h following infection. Blockade of caspase activity by 113 hours post-CVB3 infection Figure 28. Caspase 3 specific cleavage of PARP and DFF substrates following CVB3 infection. A. Cell lysates were collected from CVB3 infected HeLa cells and immunoblot analysis was performed with an anti-PARP Ab which recognizes a 85 kDa cleavage fragment. Note the cleavage of PARP beginning 9 hours following infection, with marked loss of the 116 kDa native protein by 10 hours post-infection. B. Cell lysates were similarly analyzed by immunoblot analysis for DFF cleavage following CVB3 infection. Note the change in DFF status beginning 9 hours post-infection with the appearance of a 30 kDa fragment. 114 Figure 29. ZVAD-fmk inhibits caspase activation, and cleavage of PARP and DFF following CVB3-infection or induction of apoptosis by BPD-MA and light treatment of HeLa cells. Cell lysates were incubated in reaction buffer containing the caspase 3 specific substrate, Ac-DEVD-AFC. After incubation at 37°C for 1 hour, fluorescence levels were determined by excitation wavelength of 400 nm and emission wavelength of 505 nm. Note the lack of caspase activation in HeLa cells treated with ZVAD-fmk (50 - 200 uM) at 10 hours following CVB3 infection and at 2 hours following BPD-MA and light treatment. B. Cell lysates were collected from treated HeLa cells and immunoblot analysis was performed with an anti-PARP Ab. Note the equivalent cleavage of PARP in the CVB3-infected HeLa cells without ZVAD.fmk and the BPD-MA and light treated HeLa cells without ZVAD.fmk. ZVAD.fmk treatment (50 - 200 uM) of HeLa cells prevented PARP processing in the BPD-MA and light treated HeLa cells, while in CVB3-treated HeLa cells the PARP processing was limited, but not completely inhibited at ZVAD.fmk treatment at 50 and 100 uM. C. Immunoblot analysis for DFF cleavage at 10 h following CVB3 infection and 2 h following BPD-MA and light treatment was similar in pattern to PARP cleavage. 115 10 hours post-infection sham I 0 nM 0 |xM 50 (.iM 100 200 irM ZVAD-fmk no light ' I 2 hours post-light treatment (2 J/cm2) Figure 30. ZVAD.fmk treatment effects on morphologic changes following CVB3 infection or BPD-MA and light treatment of HeLa cells HeLa cells were treated with ZVAD.fmk at 0-200 uM and then infected with CVB3 or treated with BPD-MA and light. HeLa cells were examined at 10 h following CVB3 infection or 2 hours following PDT. Note the difference in morphologic appearance between CVB3-infected and photodynamically-treated HeLa cells. 116 ZVAD.fmk did not alter the cytopathic phenotype at concentrations up to 100 uM even though cleavage of substrates (DFF and PARP) was inhibited. Multiple repeated time course experiments demonstrated no ability of ZVAD.fmk to prevent, delay or inhibit the cytopathic effect that occurred around 6 to 7 h post infection in this model. This result suggested that caspases were not the primary reason for the cytopathic effect noted following CVB3 infection. 9.3. Aim #3 Next we examined the biochemical events of CVB3 activation of host cell death machinery in more detail and compared and contrasted the events using two other distinct models of cell death (using TRAIL ligand or BPD-MA plus light (PDTP)). We also examined cell death events in HeLa cells over expressing anti-apoptotic factors Bcl-2 and Bcl-xL as well as viral replicative events in more detail. Finally, we did some initial studies into the activation of cell signaling pathways (p38, ERK, JNK) that might cross talk with the cell death pathways, and serve as indicators as to how viral infection was inducing apoptosis. HeLa cells were CVB3- or sham-infected at an MOI of 10 and examined for processing and activation of caspases and downstream targets at 0 to 14 h (h) following infection. Concurrently, other cultures were treated with optimal concentrations of TRAIL ligand or BPD and light to induce apoptosis. Consistent with earlier experiments (104) in Section 9.2, cells exhibited cytopathic effect including cell shrinking and rounding up from the monolayers between 6 and 7 h following infection in a similar fashion to cells infected with an MOI of 5. Using a panel of anti-caspase antibodies, we demonstrated that capases- 1, 2, -3, -4, -6, -7, 8, and 9 and substrate processing could be detected between 9 to 14 h following CVB3 infection of HeLa cells (Figure 31). Caspase 8 processing was delayed as compared to caspase 9 and the effector caspases suggesting that it is downstream of the caspase 9. The caspase 1 blot demonstrated some early processing events noted by cleavage products as early as 3 h pi. This cleavage could be the result of the normal host cell processing of IL18 in the face of a viral infection, or potential a viral protease mediated event used to inhibit IL-1 production. In examining mitochondrial events, the apoptosis-related factor cytochrome c was released from 117 S-100 cell lysate (cytosol) cytochrome c procaspase 1 procaspase 2 procaspase 3 procaspase 6 procaspase 7 procaspase 8 procaspase 9 _ . , lamin A / C — — — «-P A R P Bid > 3 6 8 9 10 12 14 14 i I I | J5 hours post CVB3 infection •= o Figure 31. Temporal relationship between cytochrome c release, caspase processing, and substrate cleavage in HeLa cells treated CVB3. HeLa cells were infected with CVB3 (MOI 10) and harvested 0-14 hours post infection. Cytosolic extracts were separated by SDS-PAGE, immunoblot analysis was performed using antibodies directed against the different caspases and their substrates. Caspase processing can be detected by either disappearance of the proform or appearance of a cleavage product, or both. Timecourse repeated four times. 118 the inter-mitochondrial membrane space into the cytosol (s-100 cell lysate) beginning 8 h following infection (Figure 31). Cytosolic cytochrome c can bind to Apaf-1 and associate with the caspase-9 CARD sequence in the presence of dATP resulting in oligomerization , resulting in caspase-9 autocatalysis and activation as is observed in CVB3 infection of HeLa cells. Cytochrome c release can also be initiated by caspase-8 degradation of Bid. Caspase-8 activation is normally associated with cell surface death receptors, but activation also occurs following intracellular cytotoxic stimuli such as chemotherapy or photosensitization (208, 252). Caspase-8 activation occurs downstream of cyt c release and after effector caspase activation. This is in contrast to the TRAIL model of cell death as demonstrated in Figure 32 where caspase 8 was clearly processed prior to caspase 9 and caspase 3. In the viral model, caspase-6 activation was confirmed by evidence of procaspase-6 processing beginning 10 h post-infection and by degradation of lamin A/C, a specific substrate for caspase-6 (Figure 31). In comparative studies, TRAIL or PDT were elected as controls for apoptosis induction. HeLa cells were treated with 150 ng/ml of TRAIL with or without 100 |aM ZVAD.fmk and cells were collected at 0 to 5 h following ligand addition. Cells exhibited apoptotic morphology, including shrinking, rounding up, and blebbing between 1 and 2 h following ligand treatment (data not shown). Caspases 2, 3, 6, 7, 8, and 9, PARP, elF4G, and lamin A/C processing could be detected by 2 h following treatment (Figure 32). As mentioned, in contrast to CVB3 induction of apoptosis, caspase 8 processing could be demonstrated within 1 hour post TRAIL treatment and prior to cytochrome c release and activation of the other caspases. Bid degradation in the total cell lysate was evident by the disappearance of the Bid protein between 2 and 5 h following TRAIL treatment and cytochrome c in the cytosolic fraction was also detectable between 2 and 5 h following TRAIL treatment. ZVAD.fmk prevented apoptotic morphology (data not shown), caspase processing, substrate degradation, and the appearance of cytochrome c in the cytosolic fraction of the cells (Figure 32). In studies using BPD plus light, HeLa cells were incubated with photosensitizer (200 ng/ml) with or without ZVAD.fmk (100 uM). Following exposure to red light (2 J/cm2) cells were 119 cytosolic fraction cytochrome c —' procaspase2 m procaspase 3 mm —• procaspase 6 procaspase 7 procaspase 8 procaspase 9 t 5 time (h) - TRAIL (150 ng/ml) + ZVAD(100 uM) Figure 32 . Temporal relationship between cytochrome c release, caspase processing, and substrate cleavage in HeLa cells treated with TRAIL. HeLa cells were incubated with TRAIL (150 ng/ml) with or without ZVAD.fmk (100 uM). Cells were collected at 0 to 5 h and cytosolic extracts were examined for cytochrome c and total cell lysates were separated by SDS-PAGE and immunoblotted with antibodies to caspase 2, 3,6,7,8,9, elF4G, PARP, lamin A/C, Bid. Cleavage products are indicated by arrows. 120 collected at 0, 0.5, 1, and 2 h following irradiation. By 2 h following red light illumination, HeLa cells treated with porphyrin-like photosensitizer at 200 ng/ml exhibited characteristic signs of apoptosis including cell rounding, shrinkage, and membrane blebbing (data not shown). Strikingly, in this model, cytochrome c release appeared to be maximal immediately following light irradiation with no apparent change between 0 and 2 h following PDT. In contrast, caspase and substrate processing were not apparent until 1 hour following exposure (Figure 33). The pan caspase inhibitor, ZVAD.fmk did not block immediate cytochrome c release like in the TRAIL treatment model, but did prevent caspase processing as previously demonstrated in Figure 32. HeLa cells over expressing Bcl-2 and Bcl-xL were used to evaluate the characteristics of apoptosis in the three different models. First, HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells were used to determine the influence of Bcl-2 or Bcl-xL over-expression on CVB3-induced apoptosis. Cells were either sham or CVB3 infected and cell lysates were collected 10 h following infection. Overexpression of Bcl-2 and Bcl-xL was confirmed by immunoblot analysis. Bcl-2 and Bcl-xL overexpression prevented the release of cyt c, processing of caspases 2, -3, -7, -8, -9 and PARP at 10 h following CVB3 infection (Figure 34). Equivalent levels of infection in the three transfected cell types was confirmed by measurement of translated and processed VP1 protein at 10 h post infection in HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells. elF4G is rapidly degraded by viral proteases within 3 h following infection as a mechanism that participates in the inhibition of host protein translation. It has also been demonstrated that caspases can degrade elF4G by activated caspase 3 (97). Both viral protease and caspase-mediated degradation of elF4G were obeserved during CVB3 infection as demonstrated by the MW cleavage product at 10 h following infection in the HeLa/neo cells (Figure 34). Results shown in Figure 34 also show that processing of procaspases and caspase substrates was inhibited in HeLa/Bcl-2, and HeLa/Bcl-xL cells. Under the later conditions, cytochrome c was not released into the cytosol, suggesting that it is required for induction of apoptosis in CVB3 infected HeLa cells. DEVDase-like cleavage activity in total cell lysates, which represents both 121 B cytochrome c Bax caspase 9 +- 36 kDa +- 30 kDa caspase 3 « - 1 2 kDa PARP * - 85 kDa caspase 8 Bid 0 0 0 . 5 1 2 2 2 2 — + + + + + — + + + + + + + tmmm + + photosensitizer light ZVAD.fmk Figure 33 Temporal relationship between cytochrome c release, caspase processing, and substrate cleavage in HeLa cells treated with PDT. HeLa cells were incubated with photosensitizer (200 ng/ml) with or without ZVAD.fmk (100 uM). Following exposure to red light (2 J/cm2) cells were collected at 0,0.5,1, and 2 h. (A) Cytosolic extracts were examined for cytochrome c and Bax levels by immunoblot analysis. (B) Total cell lysates were separated by SDS-PAGE and immunoblotted with antibodies to caspase 3,8,9 PARP and Bid. Cleavage products are indicated by arrows. 122 O — J . O — — 0 ) 0 0 § o u z co m z o co Bcl-2 Bcl-x L C V B 3 Vp1 cytosolic fraction — cytochrome c procaspase 2 procaspase 3 procaspase 7 procaspase 8 procaspase 9 PARP elF4G 1 0 1 0 1 0 1 0 1 0 1 0 — — — + + + h C V B 3 Figure 34. Caspase processing and substrate degradation in HeLa cells over-expressing Bcl-2 or Bcl-x, following CVB3 infection. HeLa/neo, HeLa/Bcl-2, and Hel_a/Bcl-xL cells were infected with CVB3 or sham treated and cell lysates were prepared 10 h following infection. Cell lysates were separated by SDS-PAGE and immunoblotted using antibodies various proteins. Cytosolic extracts were assessed for presence of Bcl-2 and Bcl-xL, cytochrome c release from the inter-mitochondrial membrane space to the cytosol, multiple caspase activation, cleavage of PARP and elF4G, and CVB3-Vp1 expression at 10 hours following CVB3 infection 123 caspase -3 and caspase -7 activity, was observed in neo cells peaking at 10 to 12h post infection and decreasing in activity thereafter. In contrast, caspase 3 and 7 activity was barely detectable in cells expressing either Bcl-2 or Bcl-xL (Figure 35). HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells were then used to determine the influence of Bcl-2 or B C I - X L over-expression on the biochemical events of TRAIL-induced apoptosis. HeLa/neo, HeLa/Bcl-2, HeLa/Bcl-xL cells were treated with 150 ng/ml of TRAIL and collected at 0 to 5 h following ligand treatment (Figure 36). At 2 h post treatment there was evidence of apoptosis in the HeLa/neo cells, as evident by cytochrome c presence in the cytosol and degradation of caspase 2, 3, 6, 7, 8, and 9 and cleavage of substrates. At 2 and 5 h post TRAIL treatment of HeLa/Bcl-2 cells there was no evidence of cytochrome c in the cytosolic fraction even though caspase 8 had been processed and Bid had been degraded (Figure 36). HeLa/Bcl-XL cells had blocked cytochrome c release partially and by 5 h post TRAIL treatment there was evidence of caspase processing and substrate degradation. It is interesting to note that although caspase 8 was processed, apoptosis was still delayed suggesting that HeLa cells signal through the mitochondria during TRAIL mediated death. This can probably be accounted for by factors such as SMAC/DIABLO that are released with cytochrome c to the cytosol and remove any inhibitory effects mediated by the lAP's as discussed in Section 3.4. HeLa/Bcl-2 and HeLa/Bcl-XL cells treated with verteporfin and red light exhibited a delay in the onset of and lower DEVDase-like activity following light treatment in comparison to HeLa cells containing the neo construct (Figure 37). DEVDase-like activity was highest at 1 h for the HeLa/neo cells, while maximal activity was observed at 2 and 3 h post-PDT respectively for HeLa/Bcl-xL and HeLa/Bcl-2. However, the maximal DEVD-ase like activity was only about one-third that observed for HeLa/neo lysates. Cytochrome c release, the state of different caspases, substrates and Bcl-2 and Bcl-XL were determined for cell lysates prepared at 0 and 2 h following PDT (Figure 37). Cytochrome c was detectable in the cytosolic fractions prepared from verteporfin-treated (200 ng/ml) HeLa/neo, HeLa/Bcl-2 and HeLa/Bcl-xu cells immediately following light-irradiation. As observed previously in response to treatment with CVB3, evidence 124 Figure 35. Neo cells, and HeLa cells overexpressing Bcl-2 or Bcl-xL overexpressing HeLa cells, and Bcl-xL overexpressing HeLa cells were infected with CVB3 (MOI 10). Cells were harvested 0-14 hours post infection and caspase-3/7 activity was measured by DEVDase-like cleavage activity 125 X X o 1 1 o o 1 o u u o o o z CQ CQ z CQ CQ z CQ CQ — «— — — m — 3 (1 2 «P Bcl-2 apaf-1 cytosolic fraction _ cytochrome c caspase 2 — — caspase 3 * ~ • caspase 6 caspase 7 • — ^ caspase 9 Bid • • - • lamin A/C elF4G PARP 0 0 0 2 2 2 5 5 5 — — — + + + + + + h TRAIL Figure 36. Caspase processing and substrate degradation in HeLa cells over-expressing Bcl-2 or Bcl-xL following TRAIL. HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells were treated with 150 ng/ml TRAIL ligand. Whole cell lysates were prepared from 0 to 5 h following treatment, separated by SDS-PAGE and immunoblotted using antibodies to cytochrome c, caspase 2-9, PARP, lamin A/C, elF4G, Bcl-2 and Bcl-xL. Cleavage products are indicated by arrows. 126 HeLa/neo T3 1 2 3 5 hours post-irradiation 10 C N X C M X O — — O — — CD O O ( D O U Z CO CO Z CQ CO B — Bcl-2 — Bcl-xL — cytochrome c I 0 I 01 01 01 01 01 " ™ Caspase 9 <-Caspase 3 4-12kDa PARP •«-85kDa red light photosensitizer light UVB light Figure 37. Caspase processing and substrate degradation in HeLa cells over-expressing Bcl-2 or Bcl-xL following PDT. HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells were treated with photosensitizer (200 ng/ml) and red light (2 J/cm2). (A) Whole cell lysates prepared from 0 to 10 h following verteporfin and light treatment were incubated with DEVD-AMC (100 LIM) and fluorescence was measured at 460 nm with a cytofluorimeter. Error bars indicate standard deviations. (B) Cell lysates were separated by SDS-PAGE and immunoblotted using antibodies to caspases 9, 3, PARR Bcl-2 and Bcl-xL. Cleavage products are indicated by arrows. 127 was obtained to indicate processing of caspase 9, caspase 3, and PARP in HeLa/neo cells treated with PDT for 2 h. For Hel_a/Bcl-xL and HeLa/Bcl-2 these changes were less pronounced. These data suggest that Bcl-2 and Bcl-xL can exert their anti apoptotic effects after cytochrome c has been released from the intermitochondrial space to the cytosol. HeLa/neo, HeLa/Bcl-2 and HeLa/Bcl-xL cells were infected with either CVB3, treated with 150 ng/ml of TRAIL, or 200 ng/ml verteporfin plus light, in the presence or absence of 100 uM ZVAD.fmk. At 2 and 5 h (TRAIL treatment), 10 h (CVB3 infection), or 0 and 2 h following verteporfin and light treatment, cells were stained with the monoclonal antibody AP02.7 to detect the mitochondrial epitope 7A6, which has been previously shown to correlate with apoptosis induction (389, 795) (Figure 38). Enforced Bcl-2 and Bcl-xL expression prevented the appearance of 7A6 at 2 h following TRAIL addition in contrast to HeLa/neo cells. At 5 h (data not shown) following ligand addition a small number of the Bcl-2 cells expressed 7A6 while the B C I - X L cells had an intermediate number of 7A6 expressing cells between neo and Bcl-2 cells. Enforced Bcl-2 and Bcl-xL expression in CVB3 infection severely depressed expression of 7A6 antigen at 10 h following CVB3 infection. ZVAD.fmk prevented 7A6 expression in TRAIL mediated apoptosis, but had no discernible effect in CVB3-mediated apoptosis (Figure 38). This was similar to the inability of ZVAD.fmk to prevent release of cytochrome c from the intermitochondrial space to the cytosol in CVB3 infected HeLa cells. Comparatively, the mean channel fluorescent intensity values of the 7A6 antigen were present in PDT-treated HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells immediately following light treatment (Figure 38). By 2 h following photo-irradiation 7A6 levels were further increased for all 3 transfected cell lines. Therefore the Bcl-2 and Bcl-XL were able to depress or delay the 7A6 antigen in the CVB3 and TRAIL treated HeLa cells, but were ineffective in preventing expression in PDT treated HeLa cells. As demonstrated in Section 9.2 caspase inhibition with ZVAD.fmk prevented processing of caspases and substrate degradation but did not prevent CPE following CVB3. Similarly, overexpression of Bcl-2 or Bcl-xL in HeLa cells did not prevent CPE as determined by DIC 128 neo neo + ZVAD.fmk Bcl-2 Bcl-x, control CVB3 (10 h) TRAIL 2 h (150 ng/ml) 1 L LLL LLL Lt 1 ^ B neo log fluorescence intensity (AP02.7) Bcl-2 Bcl-xL neo neo + Bcl-2 Bcl-xL ZVAD. fmk L L L L + + Log fluorescent intensity (AP02.7) 0 h 2 h verteporfin light Figure 38. 7A6 Expression after CVB3 infection, TRAIL or PDT Treatment in HeLa cells. A) Neo cells, neo cells treated with ZVAD.fmk, Bcl-2 overexpressing HeLa cells, and Bcl-xL overexpressing HeLa cells were either infected with CVB3 (MOI 10) for 10 hours, exposed to TRAIL (150ng/ml) for 2 hours, or control treated, and flow cytometry with AP02.7 was utilized to detect the mitochondrial epitope 7A6. B) 7A6 expression was assessed 0 to 2 h following PDT. Cells were permeabilized and exposed to PE conjugated mouse anti-AP02.7 (line tracings). Parallel staining was performed with a PE-conjugated isotype-matched antibody (shaded areas). 129 microscopy (Figure 39). CPE was not delayed and there were no apparent differences in morphologic appearance between the different cell types. The only discernible difference under light microscopy was that caspase inhibition by enforced Bcl-2 or Bcl-XL or ZVAD.fmk treatment under conditions that prevented the release of cytochrome c from the intermitochondrial membrane space reduced the release of rounded of cells from the monolayer at late stages (>14 h) following infection (data not shown). In the PDT treated HeLa cells (Figure 40), HeLa/Bcl-2 and HeLa/Bcl-xL cells demonstrated delayed kinetics of the apoptotic phenotype as compared to their neo counterparts as assessed by DIC microscopy following treatment with 200 ng/ml verteporfin and light (2 J/cm2). Morphological changes were fulminant by 2 h following the photodynamic treatment of HeLa/neo cells, while the majority of HeLa/Bcl-2 and HeLa/Bcl-xL cells exhibited normal morphology at this time. However, by 5 h following light treatment a majority of HeLa/Bcl-xL were non-viable in appearance while HeLa/Bcl-2 cells required up to 10 h to exhibit these changes. In TRAIL treated HeLa/neo cells (Figure 40), apoptotic morphology as determined by DIC microscopy was clearly evident by 2 h. HeLa/Bcl-2 and HeLa/Bcl-xL cells treated with TRAIL had morphology that was similar to untreated cells at 2h. By 5 h following treatment some of the HeLa/Bcl-xL cells had apoptotic morphology (data not shown). Cell viability may not be directly related picornavirius CPE. It appeared that numerous cellular processes were still occurring cells showing CPE including activation of apoptotic proteolytic machinery. MTT assay 6 h following infection when cell rounding was evident, showed that cells appeared viable as there was little or no decrease in their propensity to utilize MTT as a mitochondrial substrate (data not shown). Further examination of cell viability over 18 h during CVB3 infection demonstrates that ZVAD.fmk, and HeLa/Bcl-2, and HeLa/Bcl-xL individually, or in combination inhibited and delayed the loss of viability following infection (Figure 41). These data suggest a direct role for activation of caspases in the loss of viability following CVB3 infection. In other models of cell death, TRAIL was titrated at 0 to 1 ug/ml in the cells and cell viability was determined 24 h later by MTT assay (Figure 42). There was little or 130 neo + neo neo Z V A D . f m k B c l " 2 B C I " X L C V B 3 10h pi Figure 39. Morphologic changes in HeLa cell transfectants following CVB3 infection. Neo cells, neo cells treated with Z V A D . fmk, Bcl-2 overexpressing HeLa cells, and B C I - X L overexpressing HeLa cells were either infected with CVB3 ( M O I 10) for 10 hours or sham infected. Cells were evaluated for apoptotic morphology by D I C microscopy. 131 transfectant neo Bcl-2 Bcl-x L Figure 40. Morphologic changes in HeLa cell transfectants treated with PDT or TRAIL. HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells were incubated with verteporfin (200 ng/ml) and then irradiated with red light (2 J/cm2) or exposed 150 ng/ml of TRAIL ligand. Cells were evaluated for apoptotic morphology by DIC microscopy at increasing times following treatment. 132 120 i Figure 41. Cell viability following CVB3 infection. HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL, and HeLa/neo plus 100 uM ZVAD.fmk were either infected with CVB3 (MOI 10) for 18 hours or sham infected. Cell viability was measured by MTT dye reduction assay. MTT was added to each culture well. The reaction was stopped 2 h later by the addition of acidified isopropanol and color development was measured at 590 nm with a microtiter plate reader. Relative cell viability units are determined by OD at 590 nm. Each group was assayed in 6 wells per group, repeated for 3 separate experiments. Error bars indicate standard deviations. 133 0 1(1 25 SO 100 500 1000 verteporfin (ng/ml) Figure 42. Cell survival following PDT or TRAIL treatment. HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells cells were incubated in microtitre plates with either verteporfin (0-200 ng/ml) and then irradiated with red light (2 J/cm2) or TRAIL (0-1000 ng/ml). At 24 h following treatment, MTT was added to each well. The reaction was stopped 2 h later by the addition of acidified isopropanol and color development was measured at 590 nm with a microtiter plate reader. Relative cell viability units are determined by OD at 590 nm. Error bars indicate standard deviations. (n=4) 134 no loss of cell viability in HeLa/neo, HeLa/Bcl-2, or HeLa/Bcl-xL treated up to 25 ng/ml of TRAIL. At 50 and 100 ng/ml TRAIL, there was a loss of viability in the HeLa/neo cells but no significant change in the HeLa/Bcl-2 or HeLa/Bcl-xL cells. At 500 and 1000 ng/ml TRAIL, there was complete loss of viability in HeLa/neo cells while protection against loss of viability could be demonstrated in the Bcl-2 cells and intermediate for Bcl-xL HeLa cells. In the PDT model, at 24 h following treatment, there was no discernible cytoprotective effect afforded by Bcl-2 or Bcl-xL over-expression at the 200 ng/ml dose whereas there was a small degree of protection evident at 50 and 100 ng/ml of verteporfin plus light treatment. Since activated caspases can disrupt normal cellular architecture it may be hypothesized that this disruption of structure may facilitate viral progeny release from the host cell. Treatment with the general caspase inhibitor ZVAD.fmk, or Bcl-2 or Bcl-xL expression significantly decreased the amount of progeny virus released into supernatants at 10 h following infection as determined by plaque assay (Figure 43). Plaque assay for infectious virus on cytoplasmic extracts confirms that the decrease in released virus is accounted for by an increase in cell-associated virus (Figure 43). The persistent cytopathic effect that occurs, even in the presence of ZVAD.fmk or Bcl-2 or B C I - X L suggest that other mechanisms are causing the change in morphology. It is well known that the viral proteases cleave host proteins as well as the viral polyprotein during the virus lifecycle, therefore, a potential mechanism of viral induction of cytopathic effect is viral protease cleavage of host structural or regulatory proteins. In the course of the viral life cycle we demonstrated that RasGAP was cleaved by viral proteases as well as caspases (Figure 44). The implications of this cleavage are not completely unknown, but may actually play a role in induction of apoptosis as will be discussed in Section 10. Preliminary studies on HeLa cells infected with CVB3 suggested that ERK 1/2 and p38 were activated late during CVB3 infection as determined by presence of phosphorylated residues using a phosotyrosine specific antibody (Figure 45). These initial studies have been elegantly followed up by H. Luo et al in the McManus laboratory and have suggested a biphasic ERK 1/2 activation, but did not notice p38 135 1.0E+12 1 1.0E+10 3 £ 1.0E+08 S 1.0E+06 ° 1.0E+04 1.0E+02 1.0E+00 z z r j r j r j D Z Z c a r j r j < D < D O O f l > < D O O O O T T O O T ~ . M X . NJ X + r- + r-O O o o c c N N § ,1 D O I I I extracellular intracellular Figure 43. Infectious virus in supernatants of ZVAD-treated and Bcl-2 and Bcl-xL transfected HeLa cells. HeLa/neo, HeLa/Bcl-2, HeLa/Bcl-xL, and HeLa/neo plus 100 uM ZVAD.fmk cell were infected with CVB3 (MOI 10), plaque assay was utilized for determination of intracellular and extracellular virus titre at 10 hours post infection. Experiment was repeated 3 times. Plaque assay was done in duplicate per experiment. 136 c L o JO "35 o hours post CVB3 infection B FAK procaspase 3 >> 3 6 8 9 10 12 14 14 § I I E ™ hours post CVB3 infection -£ o FAK < Ra sGAP Caspase 3 >- , 3hrp.i. o 1 + - + 6hrp.i. 9hrp.i. ZVADfjnk =| hours post CVB3 infection o Figure 44. Expression of RasGAP (A) and focal adhesion kinase (B) during the course of CVB3 infection. HeLa cells were infected with CVB3 or mock infected and lysed from 3 h to 9 h pi or 3 to 14 h pi. Cell lysates were subjected to Western blotting with RasGAP or FAK-specific antibodies. C) CVB3-induced RasGAP and FAK cleavage in the presence of the general caspase inhibitor ZVAD.fmk. HeLa cells were mock infected (leftmost lane) or infected with CVB3 for 3,6, and 9 h in the presence (+) or absence (-) of 100 uM ZVAD.fmk. Cell lysates were subjected to Western blotting. Caspase-3 processing was prevented by ZVAD.fmk. 137 Phospho-ERK E R K Phospho-p38 P38 >» 3 6 9 § hours post CVB3 infection Figure 45. Activation of MAPK during CVB3 infection. HeLa cells were CVB3 (Gauntt strain) infected or mock infected (leftmost lane) and lysed at 3,6, and 9 h pi and subjected to Western blot (WB) analysis with antibodies specific for the dually phosphorylated MAPK Erk-1 and Erk-2 and p38. 138 activation(449). Interestingly, the H. Luo et al study uses the Kandolf variant of CVB3 while earlier studies used the Guantt variant of the virus. We had initially demonstrated p38 activation as well as ERK 1/2 activation using the variant of CVB3 obtained from Gauntt. (Figure 45). It is not known if this difference in p38 activation status is related to the virus, the HeLa cells used, or false positive or false negative staining on behalf of either study. Regardless, this requires further examination to determine if p38 is also activated concomitantly to ERK 1/2 during CVB3 infection. In models of apoptosis, it has been suggested that focal adhesion kinase degradation by caspases may be responsible for the characteristic cell rounding that is noted (231, 745). FAK is non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). In Figure 44 we demonstrate that caspases as well as some unknown protease can cleave this protein in a similar fashion to RasGAP. Similar to as we have demonstrated with RasGAP, treatment with ZVAD.fmk does not prevent FAK cleavage, yet prevents caspase 3 processing. Based on this we suspect that one.of.the viral proteases is responsible for the degradation of FAK and further investigations are being carried out in the McManus Laboratory to identify the responsible protease. 9.4. Aim 4 Time-course of viral infection in the heart Adolescent A/J mice were infected with 1x10s pfu of myocarditic CVB3 or PBS (sham) and euthanized on days 3, 9 and 30 post-infection. To confirm infection and disease and evaluate variability amongst animals, ISH using anti-sense CVB3-specific riboprobes was performed on a myocardial sections from each mouse at each time-point. At day 3 post-infection, viral RNA was detected in the myocardium of all animals by in situ hybridization in a pattern as described in section 9.1. By day 9 post-infection, there was a significant increase in the amount of viral RNA in the myocardium. At day 30 post-infection there was no detectable viral RNA in the myocardium of infected animals (Figure 46A). 139 Figure 46A. Coxsackievirus B3 RNA localization in A/J mice. A/J mice were infected with 1x105 PFU of CVB3 and tissues were harvested at 3 (A), 9 (B) and 30 (C) days post-infection. CVB3-positive myocytes are present at days 3 and 9 post-infection. Little or no in situ hybridization positive myoyctes could be detected at day 30 post-infection. (Magnification 20X) Figure 46B. Direct viral Injury of CVB3-infected myocytes. Contiguous 4 M sections were stained by in situ hybridization for viral RNA localization (A,D), hematoxylin and eosin (B,E), and Masson's trichrome (C,F) for histologic examination. At day 3 post-infection, cells that contain viral RNA are also injured as determined by the presence of cytopathic effect (A,B,C) and coagulation necrosis (D,E,F). (Magnification 40X) 140 The characteristic localization of viral RNA within the myocardium was assessed (data not shown). During the viremic and inflammatory periods, at days 3 and 9 post-infection, respectively, there was a clear lack of preference for infection of endothelial cells, either those lining the ventricular cavities or those in small arteries, veins, or lymphatics within the tissue. As well, there did not appear to be a detectable preference for infection of interstitial cells, such as fibroblasts. Indeed, the most visibly and clearly infected cell type is the cardiac myocyte, harboring both positive strand genome and replicative negative strand intermediate. Replication diminished over time. A striking feature of the infectious process relates to geographical heterogeneity of infection in the heart, highlighted by cardiac myocytes that are quite apparently infected in small clusters or singly. There is often a clear demarcation of infected versus uninfected myocytes at the level of the intercalated discs that can be appreciated in longitudinally sectioned myocytes. Viral involvement of the ventricular septum and the left and right ventricular free-walls appeared generally similar in pattern, although the extent of right ventricular free wall involvement might have been more variable. Atrial tissue had a more limited presence of viral genome compared to ventricular cardiac myocytes. Coexistence of cardiac muscle cells that had coagulative changes, contraction bands, calcification, or numerous coalescent vacuoles indicative of cytopathic effect, often immediately adjacent to "healthy" cells, reflected the range of cellular dynamics in a particular susceptible cell type, the cardiac myocyte. Thus, certain cells with an apparently high-titer viral replication, but which had undergone cytopathic effects, might have been resisting death in a fashion distinct from neighbouring coagulated cells on the one hand, and neighbouring uninfected cells on the other. The composite in vivo host responses of a local nature in a target organ will reflect the likelihood of organ or host survival. Examination of contiguous sections stained with in situ hybridization for viral RNA, hematoxylin and eosin, and Masson's trichrome (Figure 46B) from mice at 3 days post infection (prior to the immune response) illustrates that infected myocytes are undergoing virus-induced cell death without immune involvement. Histology 141 In addition to establishing the presence and localization of viral RNA at each time-point, histologic examination of CVB3 or sham-infected murine hearts is valuable for understanding the phenotypic changes associated with viral infection of myocardium. This showed three distinct phases of the myocarditic process. As illustrated in Figure 47, as early as day 3 post-infection transversely sectioned ventricular myocardium revealed a large number of cardiac myocytes to be visibly injured in association with coxsackievirus infection. Numerous individual cardiac myocytes and groups of such showed coagulative "necrotic" changes and a small number of cells showed contraction band necrosis. In areas where numerous cardiac myocytes had coagulative changes, many cells exhibited vacuolization consistent with cytopathic effects. At this time-point no interstitial inflammatory infiltrate was seen and there were no obvious changes in extracellular matrix or in interstitial mesenchymal cells. By day 9 post-infection, roughly half of the myocytes in the ventricular walls were involved in the injury and inflammatory process (Figure 47). Regions of injury and early repair appeared to be slightly more prominent in the outer and inner thirds of the ventricular walls. The right ventricular free wall was markedly injured and granulation tissue was prominent. In about half of the hearts there was a prominent thrombus in the right ventricular cavity, with distension of the ventricle. Neither endothelial cells lining the ventricular chambers nor underlying myocytes appeared to be affected. There was no evidence of vascular or vasculocentric lesions. At day 9, numerous foci of dead myocytes with finely granular calcification, marginal zones of fibroblastic proliferation, macrophage infiltration and focal lymphocytes characterized areas of myocyte injury. Dying myocytes had a predominant coagulative phenotype, but often there were cells with cytopathic effects. Viable myocytes within the markedly injured tissue appeared to have more prominent nuclei and nucleoli compared to uninfected murine heart. Thus, the possibility of a localized host response to neighbouring infection by the remaining uninfected viable cells was quite likely. By day 30 post-infection (Figure 47), the myocardium exhibited a spectrum of healing responses, with variability between individual animals of a greater degree than seen at days 3 142 Figure 47. Phenotypic changes following CVB3 infection as determined by histology. Four uM sections were stained with Masson's trichrome. The extent and severity of virus-induced injury, inflammation, cellular remodelling, and calcification was evaluated. At day 3 post-infection coagulation necrosis and cytopahtic effect is prevalent (A,D). At day 9 post-infection cellular inflammation and cellular injury is present (B,E). At day 30 post-infection cellular remodelling and changes in extracellular matrix are apparent (C,F). (Magnification 40X) 143 and 9. Thus, certain animals, doing relatively well during the healing phase of enteroviral infection, had virtually no notable inflammation, fibroplasia, calcification, or myocyte changes such as hypertrophy, nuclear enlargement for hyperchromatism or ongoing cell death. On the other hand, certain animals, with dilated left ventricles, had prominent, multi-focal, interstitial and replacement fibrosis, areas with prominent fibroblasts, occasional clusters of macrophages, and numerous areas of calcification where prior cell death had occurred, the latter in areas adjacent to scarring. The percentage of myocardium apparently lost in these hearts ranged widely with the complex remodelling process (with both fibrosis and hypertrophy) rendering a morphological appearance that encompassed both tissue loss and gain. Cardiac myocytes had enlarged nuclei. In certain animals, there were widely dispersed, albeit infrequent, myocytes in an apparent state of degeneration or death. Microarray analysis of gene expression DNA microarrays were utilized to evaluate changes in gene expression in the myocardium at 3, 9, and 30 days post infection. Approximately 7000 heart cDNA clones, representing approximately 4200 distinct genes (at the time of analysis) were printed as high-density arrays. Arrays were probed in duplicate with fluorescently labelled probes generated from mRNA extracted from infected and control myocardial tissue at each time-point. Genes that were increased or decreased by 1.8-fold, relative to control, were considered differentially expressed. A total of 619 cDNA clones were determined to be differentially expressed in CVB3-infected myocardium. Using this spotted cDNA array approach we identified that approximately 9% of the genes were differentially expressed according to our limits (1.8 fold). It could be expected that using modern cDNA or oligonucleotide arrays, with an approximate 30,000 human genes, we might have seen at least 2500 genes differentially expressed at one or more of the time points. Not considered were cDNA clones that had no significant match, or matched only to an expressed sequence tag (EST), in GenBank ( at the time of analysis (December, 1999). We narrowed the list to 169 individual known genes (clones 144 corresponding to the same gene were enumerated as a single gene) (Figure 48). Table 2 lists the five most over- and under-expressed genes for each time-point. Figures 49-53 demonstrate the expression patterns of genes that have been arbitrarily divided into functional classes. 145 Figure 48A. Gene expression profile of 169 clones that display differential expression in murine coxsackievirus-induced myocarditis. Each row represents a different cDNA and columns pertain to data collected at 3 time points (day 3, 9 and 30 post-infection). Normalized expression values displayed in shades of red and blue color, represent elevated and repressed expression, respectively, in infected hearts as compared to sham-infected animals. Genes are divided arbitrarily into functional groups. 146 Figure 48B. Gene expression profile of 169 clones that display differential expression in murine coxsackievirus-induced myocarditis. Each row represents a different cDNA and columns pertain to data collected at 3 time points (day 3, 9 and 30 post-infection). Normalized expression values displayed in shades of red and blue color, represent elevated and repressed expression, respectively, in infected hearts as compared to sham-infected animals. Genes are divided arbitrarily into functional groups. 147 Table 2: Myocarditis top 30: Transcripts showing the greatest variability in expression at day 3, 9, and 30 post-infection. D3 up MHC class III antigen D3 down -> cardiac a-actin BTG2 Transferrin receptor 1-8U Hsp56 MHC class I 21-GARP p-2 microglobin p-globin D9 up MHC class I D9 down -> p-globin Cathepsin L HRC binding protein galectin-3 Cyt c oxidase (COX7A) P-2 microglobin Transferrin receptor MHC class III antigen Hsp56 D30 up MHC class I D30 down -> chaperonin 10 Galectin-3 p-globin p-2 microglobulin Ubiquitin conj enzyme M4 cathepsin L PEMT ANF Mito 12S and 16S rRNA 148 Cell Defense Glutathione peroxidase GPX3 1 Breast tumor antigen Complement C l q C-chain Tapasin 0 3 9 30 Thioredoxin Mctallothioncin (mt-1) Plasminogen Activator Inhibitor-1 Heat shock protein 86 Heat Shcok protein 27 Beta-2-microglobulin M H C class 1 antigen M H C class I R T l . A u heavy chain M H C class 1 RT1 (RTA) M H C class III antigen Beta-globin Heat shock protein 60 3 9 30 3 9 30 Transferrin receptor p59 immunophilin (aka HSP56 and FKBP52) I 1 3 9 30 Ca2+-ATPase Alpha-subunil of PA28 activator complex Figure 49. Gene clusters within cell defense functional group. Genes were divided into functional groups and then clustered by virtue of similarity in expression patterns. Expression curves were generated and clustered using the GExpA program (SCIOS Inc.). Differential expression values were normalized to produce a value of 1.0 or-1.0 for point of maximal elevation of repression, respectively. 149 Cell Signalling a t & 1 1 3 ^ £ 2 'E. .£ 'c C W u. u-O m It s C .5 N 1 1 I el a f 1 | J | 8 3 | J s 8 § § f & t O m- o + Figure 50. Gene clusters within Cell Signalling functional group. Genes were divided into functional groups and then clustered by virtue of similarity in expression patterns. Expression curves were generated and clustered using the GExpA program (SCIOS Inc.). Differential expression values were normalized to produce a value of 1.0 or -1.0 for point of maximal elevation of repression, respectively. 150 Cell structure Figure 51. Gene clusters within Cell Structure functional group. Genes were divided into functional groups and then clustered by virtue of similarity in expression patterns. Expression curves were generated and clustered using the GExpA program (SCIOS Inc.). Differential expression values were normalized to produce a value of 1.0 or -1.0 for point of maximal elevation of repression, respectively. 151 Gene Expression Poly-A-binding protein CRP-2 (C/EBP-related protein) Intcrlcukin-6-dcpendant binding protein BTF-2 hnRNP Transcription factor ATF4 Estrogen-responsive linger protein U7 small nuclear ribonucleoprotein (snRNP) 3 9 30 Figure 52. Gene clusters within Gene Expression functional group. Genes were divided into functional groups and then clustered by virtue of similarity in expression patterns. Expression curves were generated and clustered using the GExpA program (SCIOS Inc.). Differential expression values were normalized to produce a value of 1.0 or -1.0 for point of maximal elevation of repression, respectively. 152 Metabolism .3 If Figure 53. Gene clusters within Metabolism functional group. Genes were divided into functional groups and then clustered by virtue of similarity in expression patterns. Expression curves were generated and clustered using the GExpA program (SCIOS Inc.). Differential expression values were normalized to produce a value of 1.0 or -1.0 for point of maximal elevation of repression, respectively. 153 10 DISCUSSION 10.1. Aim 1 Molecular techniques have demonstrated the important conversion of early (acute) viral infection to viral persistence in the myocardium, and have suggested a role for chronic infection in the pathogenesis of myocarditis and cardiomyopathy (382). In myocarditis-susceptible mice viral RNA cannot only be detected early, but also during the late (chronic) stage of disease in myocardium and lymphoid organs (384). Such virus appears to be directly injurious to infected myocardial cells (28, 139, 488, 489, 498). The magnitude of this injury is most easily observed early following infection in the myocardium prior to immune cellular infiltration, after which the mechanism of cell injury remains controversial. Cardiac cytopathic lesions are present by day 2 post-infection, and cytopathic, coagulative and contraction band lesions progressively increase in number and severity days 3, 4, and 5 post-infection (489). Importantly, from work cited above (28, 139, 488, 489), the key concept that has emerged in the last six years versus the previous 20 is the attribution of the vast majority of irreversible cellular injury and death to viral mechanisms rather than immune processes. Molecular techniques can also be used to examine the tissue dissemination of virus following infection. Non-radioactive ISH with riboprobes in this study revealed wide viral dissemination in the early days following CVB3 infection (Figure 17). Heart, liver, exocrine pancreas, endocrine pancreas, splenic lymph node germinal centres, splenic marginal zone macrophages and red pulp, salivary glands, brain, thymus, kidney, testis, lung, skeletal muscle, all localized positive strand viral RNA. There are potentially more cell types that are infected during systemic dissemination as we took a widespread, random analysis of most organs, and might have missed distinct cell types. Early infection of multiple tissues and organs emphatically illustrates the multi-organ disease that occurs in mice following infection. This murine model has similarities to clinical neonatal and infant coxsackievirus infections in humans where multiple organs are involved, with high mortality (40, 137, 324, 505). 154 In consideration of this multi-organ involvement, coxsackievirus RNA is able to enter multiple cell types. The coxsackievirus and adenovirus receptor has been determined (65, 102, 698), while other proteins exist that may facilitate viral binding to cells (64, 67, 177, 632). Tomko et al. (698) have demonstrated murine expression of CAR in kidney, liver, lung, brain and heart. It is interesting that kidney and lung usually have very little viral RNA as detected by in situ hybridization even though they express relatively high levels of CAR mRNA. Spleenocytes which does not contain the CAR mRNA, localizes viral RNA to the germinal centres and marginal zone macrophages (16). Viral RNA localization in this region may be due to surface IgM on germinal centre B cells, complement component 3 (15), or phagocytosis by macrophages. Receptors are not the only potential limiting factor in tissue tropism. Host accessory proteins responsible for viral translational and transcriptional events may be equally important to receptors as limiting factors in viral replication. Numerous host proteins have been shown to be involved in picornavirus replication (75, 76, 227, 269). Key regulatory sequences in the 5' non-translated region of CVB3 (771) offer several sites for regulation by host and viral proteins that alter tropism. Murine lung and kidney cells may be deficient in critical host accessory proteins that are required for viral replication. Cheung et al (verbal communications) in the McDonald Research Laboratories at St. Paul's Hospital has examined the organ-to-organ variability of host proteins that may interact with CVB3 RNA. He has identified potential factors in the kidney that bind to the viral RNA and are not present in other organs. It is also possible that host proteins exist that may inhibit viral RNA after it has entered the cell. Therefore, it can be conceived that beyond the presence of viral receptor on the surface of a host cell, intracellular factors may determine whether or not the cell is permissive for viral replication. Not all cells express the same repertoire of proteins, so there may be proteins in some cells that are required for viral transcription or translation that are lacking, or alternatively, there might be proteins in some cells expressed that can act as inhibitors of viral replication. The pathological character of tissue damage to organs beyond the heart in the murine CVB3 enteroviral model has not received detailed attention beyond the early observations of 155 Dalldorf, Pappenheimer, Melnick, and Godman (163, 239, 498, 553). In studies of CVB4-induced pancreatitis and diabetes, the histomorphology of exocrine and endocrine cellular injury has been described (577, 721, 722). B cells, T cells and macrophages predominate in an inflammatory infiltrate that has generally been studied at late time-points during the chronic phase of infection. Diabetes classically takes 6 weeks from infection to onset of disease (113, 115). It is interesting that viral tropism has been controversial and varyed between endocrine and exocrine localization (722, 780). Obviously, different B4 strains might have different tissue tropism, but fundamental questions still remain as to in vivo localization of viral RNA and protein. Differing models have shown islet cell protein localization (780), and some have shown exocrine necrosis and inflammation with islet atrophy (577, 721, 722). While it is recognized that pancreatitis occurs with CVB3-infection (239, 245, 246, 553, 726), the exceedingly early exocrine cell death (2-4 days), intense viral replication, and relative sparing of the islets is worthy of emphasis. With prominent CVB3 RNA localization and nearly total cell death in the exocrine pancreas as demonstrated in this work, (Figure 23), it will be both interesting and important to examine the extent of exocrine cell death following CVB4 infection and whether early organ destruction and subsequently compromised blood supply to endocrine pancreas could overshadow any immunopathological phenomena as a basis for eventual diabetes mellitus. In CVB1-induced skeletal myositis there are contradictory data on the persistence of CVB1 RNA during the progression of long-term disease (680, 681, 812). The mechanisms of viral RNA entry into skeletal muscle cells deserves attention with the potential deficiency of murine coxsackievirus and adenovirus receptor (698). Despite the incompleteness of our understanding of dissemination, replication, persistence and organ injury in group B coxsackievirus infections, all members share similar patterns, which may be altered by viral genetics and host immunogenetics. As discussed in section 5, viral infection of skeletal muscle may be a site of persistence, since this type of muscle contains many of the intrinsic anti-apoptosis features found in the cardiac myocyte. 156 Selectivity of CVB3 for the serous salivary gland component (Figure 17G), with limited infection in the mixed component, and virtual absence of infection in the mucuous glands is intriguing. The salivary gland is an organ that has been shown to be a preferred site of persistence in other infectious models including members of Herpesviradae family such as cytomegalovirus (CMV) (458, 786) and Epstein-Barr virus (EBV) (215, 746), or other viruses such as human immunodeficiency virus (409, 777). These are first data of CVB3 infection, replication and potential persistence in salivary glands and suggests a potential mode of transmission of virus from one host to another. The development of hepatitis in CVB3-infected mice was another early event, with a striking predilection for hepatocytes (Figure 17). Although Kuppfer cells were also infected, their role in antigen trapping does not seem sufficient to control parenchymal infection. The liver does not appear to be a site for viral persistence, as there was rapid clearance in this organ with little evidence of massive infection remaining beyond day 5 to 6 post-infection. The clearance may be due to massive burn-out where the virus has infected and killed the susceptible cells, while non-specific and humoral immune mechanisms limit viral load in the serum, limiting further infection of hepatocytes. The brain has been known to be susceptible to CVB3-infection (239, 553), although only limited information regarding localization is available (384). The localization of positive strand viral RNA to the cerebellum and presence of negative strand viral RNA in the Purkinje cells (Figure 17D) raises a number of new questions. The basis of such highly selective cellular localization in specific sites in the brain may relate to particular expresion of receptors, viral replicative machinery, methods of transport into the CNS, as well as cyto-pathologic effects of the virus in certain cells. Murine models show agitative, withdrawn and erratic behavior following CVB infections, and dependency of this behavior on viral infection of the brain may provide important clues to mechanisms of injury. TUNEL staining provides a useful and definitive measure of tissue injury, perhaps more distinctive than other available histopathologic techniques. This method is classically employed 157 as a marker of apoptosis, but evidence suggests that this method can be applied as a marker of cell death (255). Other studies have suggested that discrimination between apoptotic and necrotic cells can be determined by patterns of TUNEL staining (189). Cells with extensive cytoplasmic staining due to destruction of the nuclear membrane are thought to have undergone necrosis. On the other hand, apoptotic cells, with their characteristic maintenance of nuclear and cytoplasmic membranes have intense nuclear stains and apoptotic bodies. This may be more difficult with coxsackievirus B3 infection since a hybrid form between apoptosis and necrosis may be occuring where caspases are activated, specific substrates are cleaved resulting in morphological and structural changes, while viral proteins such as the 2B protein may be altering membrane integrity (716) or viral release may rupture plasmalemma. Concordant localization of viral RNA with tissue injury in multiple organs as determined by TUNEL staining predates and overshadows any association of cell death with inflammation in CVB3 infection of the heart. The immune system has numerous mechanisms of killing virally infected cells involving non-specific mechanisms such as TNF-a, macrophage free radicals, complement, natural killer cells, and specific mechanisms including cytotoxic T cells (CTL's) and antibodies. CTL's are capable of killing virally infected cells by Fas and granule-mediated pathways (130, 169, 341). Fas ligand on T cells can activate the Fas receptor on Fas expressing cells, quickly resulting in caspase activation and death by apoptosis (197). This mechanism may be most important for regulation of cell survival in immune clones, while perforin and granzyme release from T cells and NK cells, including granzyme A and B, would be the major mechanism of death in viral infected cells (340). Perforin forms pores in target cells which may result in cell lysis, but probably more important, such pores serve as a pathway for the entry of granzyme B into cells, with subsequent cleavage and activation of the apoptotic caspases (168, 575). Once cysteine proteases have been activated, cells exhibit morphologic features of apoptosis including membrane and nuclear changes. What is important in this thesis is that target-organ cells were dying prior to activation either of the humoral or cytotoxic cellular responses as evident by cellular injury beginning well before activation of the acquired 158 immune response. This implies that early injury is mediated directly by the virus. Documentation that the majority of cell death occurs prior to immune infiltration and considering that during peak periods of immune response, there was comparatively less damage, raises many questions regarding the role of immune, autoimmune, and mimcry mechanisms in the pathogenesis of CVB3-induced disease in murine models. Certainly, the mechanism of cell injury following infection with a cytopathic or cytolytic virus such as coxsackievirus B3 is emerging. Cytopathic effects are classically described as morphologic changes in cells following infection (238, 592). Whether this change is attributed solely to viral proteins and the viral life cycle with events such as inhibition of host RNA and protein synthesis (194, 765, 767) or membrane lysis during viral release from cells, or requires the participation of host cell death machinery was further elucidated in Aim 2 using HeLa cells. In most cells numerous agents and stimuli can result in cell death. The division between apoptosis and necrosis has become less distinguishable with new knowledge regarding cell death. A major difference between certain cell death processes, apoptotic versus necrotic, may be the membrane changes. The major homeostatic role of apoptosis is to package cells for rapid phagocytosis without the involvement of inflammation (761). Less overt membrane changes occurring during apoptosis, accompanied by maintenance of membrane integrity and movement of phosphatidlyserine from the inner leaflet to the outer cell membrane (717), do not occur during necrosis. Thus an inflammatory response is pre-empted in the former, and promoted in the latter. Coxsackievirus B3 infection does not result in cell death that can be differentiated by standard histochemichal stains as either necrosis or apoptosis. Clearly, morphologic criteria of both processes can be seen in early death in different cell types (Figure 22 and 23). In the heart, myocytes were found showing loss of nuclear membrane integrity as well as DNA fragmentation. Apoptosis in heart muscle has been demonstrated following ischemic injury, allograft transplantation, and chronic heart failure (21, 173, 319, 433, 517, 542). Myocyte apoptosis following CVB3 infection has not been adequately determined (310). Confusing the 159 issue is that infiltrative immune cells in the myocardium during the period of a specific immune response can and do undergo apoptosis. Cells of the inflammatory infiltrate have a short lifespan and die by programmed cell death (616). the results of this thesis again support the hypothesis that the major time frame for myocyte injury and death is during the early viremic period of viral infection. In the liver there are clearly both apoptotic and necrotic hepatocytes. Certain cells exhibit only nuclear staining, while others have cytoplasmic staining. Potentially, cells may begin to undergo an apoptotic process, but proceed to necrosis with loss of membrane integrity following infection. In consideration of TUNEL staining patterns in the heart, liver, pancreas, and other organs, the prominent final common pathway in the early period of CVB3 infection is one of necrosis. In immune organs with low levels of normal adaptive apoptosis such as thymus, spleen and lymph nodes, there was enhanced apoptosis following infection (Figure 19). Whether this apoptosis was directly mediated by viral infection of lymphocytes (16, 384, 727), or indirectly due to death receptor activation (by Fas ligand, TRAIL, or TNF-a) remains to be determined. Work using Ipr and gld mice (deficient in Fas and Fas L) did not result in any reduction of apoptosis in the spleen of infected mice (data not shown), suggesting that cytokines or ligand interactions other than the Fas system may be responsible for this massive death. Apoptosis occurred extensively in the white pulp regions of the spleen and did not only correspond with viral RNA localization. The dying cells appeared as aggregates suggesting cell-to-cell interactions. It is worth noting that the extensive apoptosis related to immune activation appears to exceed the normal basal levels of apoptosis usually encountered following a viral infection, including those associated with senescence of the immune system as seen in LCMV models (582). As outlined in Section 2.1.4, it is possible that viral modulation of cell viability in the immune system is an important factor in the pathogenesis of end-organ disease, partially determining cell composition of these organs, as well as the cell populations in immune organs. Persistence of viral RNA may also be affected by death pathways. Studies have noted a lymphoid involution early following coxsackievirus B3 infection (479). Thymic involution or loss 1 6 0 of cellularity was one of the striking features first noticed by Pappenheimer et al. (553) in 1950 who noticed "acute involution," pyknosis, and fragmentation of small lymphocytes. Our examination of the thymus revealed extensive apoptosis of the cortical region where the thymocyte CD4CD8 double positive population normally resides (Section 9.1). The effect of the lymphoid involution on pathogenesis of end-organ disease remains to be determined, but there is suggestive evidence that an immunosuppressive state follows infection (59). In addition to the pronounced apoptosis in the spleen and thymus in infected mice as demonstrated in Section 9.1, we have also demonstrated a decrease in IL-2 and IL-4 protein levels in the spleen and heart of infected A/J and C57BL/6J mice (Figure 21). This observation has been recently supported by work of Hofmann et al (292) who noted impaired production of inflammatory cytokines and an increase in IL-10 expression. Comparison of "susceptible" A/J and "resistant" C57BL/6J mice revealed little difference between host strains in the systemic distribution of viral RNA early following infection. Both murine strains had apparently similar viral receptor distributions. During early viremia C57BL/6J mice had extensive immune cell and hepatic viral RNA localization. Since the myocytes in the C57BL/6J animals were far less infected as compared with the A/J mice during a heavy systemic viral burden, it can be hypothesized that the C57BL/6J myocytes have differential myocyte receptor densities or intracellular factor(s) that is required for viral replication. Following maximal viral RNA localization at day 3 and 4 post-infection the C57BL/6J mice cleared viral RNA from cells much more rapidly than did A/J mice. The latter contained virus in multiple sites for prolonged periods. C57BL/6J mice may have a modulating factor in the myocardium and a more aggressive immune response to virus. Comparison of adolescent 4 week old and adult 10 week old A/J mice revealed little difference about systemic cellular distribution, but did highlight significant changes in organ viral loads and patterns of distribution. 10 week old A/J mice had less localization within the myocardium, but did have increased loads in other organs. This is probably due to either changes in expression of viral receptors or to intracellular proteins that may aid in viral 161 replication. As mentioned before, we are actively looking for host cellular proteins that either facilitate or inhibit viral replication within a cell after the viral RNA has entered via the surface receptor. Understanding all the host protein partners that play a role in viral replication will help elucidate why certain organs are permissive to viral replication and suggest potential novel therapies (See Section 12 Future Directions). These studies indicate that coxsackievirus B3 can infect multiple tissues following infection. The viral RNA and translated proteins have the capacity to directly injure cells targeted for infection. The anti-apoptotic intrinsic nature of myocytes and other cells may allow for persistence of infectious virus for extended periods of time and thus enabling low grade tissue destruction. Further, the immune system may be dysregulated by the viral association with lymphocytes. 10.2. Aim #2 A classic feature of viruses in the family Picornaviridae (except for Hepatitis A virus) is the cellular cytopathic effect noted early following infection of most cells. Since the discovery of an extraneural cell culture technique for the multiplication of poliovirus (199), degenerative changes in cell morphology have been noted. First described by Robbins et al. (592) in 1950, these cytopathic changes include nuclear shrinkage, condensation of chromatin, cell rounding and release from the monolayer, with eventual progression to acidophilic cytoplasm, nuclear pyknosis, and fragmentation of the nuclear chromatin (karyorrhexis) (587). Understanding of cell death mechanisms in other models (TRAIL and PDT) set the stage for examination of host cell death proteins and their possible role in the cytopathic effects of CVB3 infection. Many viruses inhibit or activate cell death, strategies that convey distinctive aspects of cell injury, inflammatory responses, or viral persistence as described in detail in Section 4. As noted, previous picornavirus studies have shown the morphologic features of apoptotic cell death including cell shrinkage, DNA fragmentation, and nuclear condensation (238, 334, 587). In this aim we examined the biochemical pathways of cell death to establish whether caspases are activated, specific substrates cleaved, and whether or not this proteolytic 162 cascade could be responsible for the cytopathic effect. Caspase 3 is considered one of the key proteins involved in the execution stage of cell death. Apoptosis induced by almost all stimuli demonstrate procaspase 3 processing and subsequent activation (197, 254, 329, 359, 610). Beginning 7-8 h post-infection with CVB3 infection of HeLa cells, procaspase 3 was depleted and caspase activation assays demonstrated that this protein was cleaved into its active state (Figure 27). Several proteins can activate caspase-3, including caspase-8 via signalling through TNF or Fas receptors, granzyme B from cytotoxic lymphocytes, or caspase 9 via mitochondrial cytochrome c release and the assembly of apoptotic protease activation factors (Apafs). Once activated, caspase 3 can degrade specific substrates, which in turn results in structural alterations and loss of homeostatic regulation of cellular processes. Numerous proteins have been shown to be cleaved by activated caspases. Consistent with activation of caspase 3, both PARP and DFF were cleaved following CVB3 infection. Caspase activation and DNA fragmentation were directly linked through the cleavage of DFF (438). DFF was cleaved beginning at 9 h post-infection resulting in a 30 kDa fragment (Figure 27), which can be further processed to a 11 kDa fragment. PARP is located in the nucleus and involved in DNA repair. Cleavage of PARP began at 9 hour following infection suggesting that once caspases were activated in the cytosol they were able to access nuclear localized substrates. Importantly, caspase inhibition with the general caspase inhibitor ZVAD.fmk did not prevent the cytopathic effect induced by coxsackievirus B3 following infection. Between 6 and 7 h the cytopathic effect became apparent by phase contrast microscopy in our CVB3 infection model. The time following infection to appearance of the cytopathic effect as observed by phase contrast microscopy was not effected by ZVAD.fmk concentrations from 25 to 100 uM. In addition to not affecting the time to cytopathic effect, ZVAD.fmk treated cells had a similar morphologic appearance to untreated, infected cells (Figure 30). We used PDT treated HeLa cells as an alternative method to induce apoptosis (103). Inhibition of caspase activation with the inhibitor ZVAD.fmk prevented the apoptotic phenotype (Figure 30). From these results we 163 conclude that caspase activity and cleavage of substrates do not account for the characteristic cytopathic effect occurring following picornavirus infection, but instead are activated subsequent to the morphologic changes. The point of intersection between the viral replicative cycle and activation of the host cell death pathway was not fully established in Aim 2. Picornavirus infection results in inhibition of cellular RNA and protein synthesis soon after infection (202, 767). Early studies of relationships between picornavirus-induced metabolic alterations and virus induced cytopathic effect indicated that the inhibition of RNA and protein synthesis was not directly related to cell morphological changes (32). RNA and protein synthesis inhibitors delayed cell death but the cells displayed fewer morphological changes than those exhibited by picornavirus infected cells (33). Inhibition of protein and RNA synthesis with multiple agents including actinomycin D, puromycin, or diptheria toxin results in apoptosis (464). Early studies in poliovirus infection systems with puromycin, an inhibitor of the translation of viral as well as host proteins, delayed the cytopathic changes suggesting that certain viral proteins may be directly cytotoxic (32). It has been shown that the viral encoded 2B protein in coxsackievirus and poliovirus may associates with cellular membrane fractions, including the plasmalemma and endoplasmic reticulum, and disrupt ion movements, including the movement of Ca 2 + to the cytosol (8, 716). Ca 2 + influx occurs in apoptosis (72, 546), but it is not clear if the influx occurs prior to or following caspase activation. Examination of ionic requirements of caspases, it has been determined that calcium ion concentration has little effect on caspase activity (657). An early calcium influx following coxsackievirus infection could result from the influence of the 2B protein on membrane permeability, and the large late calcium influx noted-(>6 h) (716) could be a downstream effect of caspase activation. During the early phases of infection it would be advantageous for the virus to inhibit host cell death thereby allowing for maximal replication of viral progeny virus. At late stages of the viral life cycle it would also be beneficial to the virus to induce apoptosis rather than necrosis. Such a mechanism of death is a potential means to allow viral release to surrounding tissue 164 while evading the immune system is evaded. Apoptosis is characterized by the rapid phagocytosis of affected cells without the release of pro-inflammatory cytokines (624). Aim 2 demonstrated that caspase 3 activation follows rather than precedes coxsackievirus B3-induced degenerative morphological changes in infected HeLa cells (Figure 26 and 27). Activated caspases process specific substrates including PARP and DFF. However, inhibition of caspase activity did not eliminate the morphologic changes induced in virus-infected cells as determined by phase contrast microscopy. On the other hand, caspase processing and cleavage of substrates may be important in the ultimate alteration of normal homeostatic processes in infected cells and may be facilitate final clearance of virus-infected cells. The viral proteases 2A, 3C, and 3CD may cleave specific structural proteins, resulting in morphologic alterations consistent with the cytopathic effect in a fashion analogous to the action of caspases which cleave separate substrates to achieve a distinct apoptotic phenotype. 10.3. Aim #3 Viral regulation of host cell viability is becoming an ever-more important area of investigation as discussed in Section 4. Certain viruses try to prevent cell death by expression of viral proteins that can inhibit or regulate important host proteins involved in initiation or execution of cell death (253, 685). Still other viruses do not contain gene products that can inhibit death, but instead have relatively small genomes and a short lifecycle that can be efficiently replicated prior to host cell death. A third scenario may be that of a virus that is benefits from the initiation of apoptosis within a cell. It is becoming evident that apoptosis in picornavirus-infected cells may not be a host response to limit viral replication and viral dissemination to other organs and cells, but rather a process triggered or accelerated by the virus to facilitate viral release. Once the virus has translated all the necessary non-structural and structural proteins and the genome has been replicated, there is no need to maintain the viability of the host cell. Yet, there are host cells that express a repertoire of regulatory proteins that make them more resistant to apoptosis, such as the myocyte, and may create an environment where the virus can persist for extended periods of time. More recently, it has also 165 been demonstrated that CVB3 replicates more slowly in cells that are quiescent such as the myocyte (209), which may additionally enhance persistence. Picornaviruses have a short replication lifecycle. Almost immediately following viral RNA internalization the viruses are capable of parasitizing the host and controlling normal cellular functions. Host transcription and translation are inhibited to allow for maximal use of host accessory factors for viral RNA transcription and RNA translation. Within h of infection, infectious progeny virus can be detected in the cytosol of the host cell. This differs (although Feuer et al recently suggest that it may be similar for picornaviruses (209)) from many DNA viruses, as discussed in Section 4.3.1, that require cells to be undergoing active DNA proliferation to allow for viral DNA replication, and therefore have longer replicative cycles. Viral release from picornavirus infected cells occurs by unknown mechanisms. These may include cell ionic gradients disruption leading to cell bursting, or the virus may encode proteins such as 2B which increase membrane permeability (716) as mentioned in Section 1.3.3. The cytoskeleton and plasmalemma maintain the progeny virus within the cell. Caspases can cleave multiple substrates to regulate translation, transcription, DNA synthesis and repair, stress signaling, and to disrupt the cellular cytoskeleton by degradation of important structural proteins. Data presented in Aim 3 showing that inhibition of caspase retained cellular virus suggests that caspase activation in a picornavirus-infected cell would enhance viral release from cells. Such a consequence would facilitate the virus movement to new host cells and possibly to increase the ability of the virus to circumvent host mobilization of a non-specific immune response. Further, caspase activation would decrease inflammation, since substrates expressed on apoptotic cells are known to induce rapid phagocytosis with limitation of inflammation. Activation of apoptotic machinery can be demonstrated in cell cultures following picornavirus infection as demonstrated in Aim #2 (104). In Aim 3, we further demonstrated that in addition to caspase 3 activation and PARP and lamin A/C cleavage, that multiple members of the caspase family are activated (Figure 31). Apoptosis-related caspases -2, -3, -6, -7, -8, and -9 were all activated or degraded into their characteristic large and small subunit components 166 during CVB3 infection. Effector caspases -3, -6, and -7 can efficiently cleave host proteins including PARP, a nuclear protein involved in DNA repair, and lamin A/C, a structural protein. Initiator caspases-2, -8, and -9 were also activated during picornavirus infection (Figure 31). Caspase-8 and -3 can degrade Bid (80, 427) and caspase-9 can degrade and thus activate caspase -3, -6, or -7 (549). Inflammation-related caspase-1 and -4 were also processed, probably as a result of profound proteolytic events that are occur during CVB3 infection. Of three primary mechanisms of caspase activation, death receptor ligation, mitochondrial release of apoptogenic factors, or granzyme B entry into cell cytosol, mitochondria appear to have key involvement in caspase activation during picornavirus infection. We compared 3 models of apoptosis in this Aim to further identify the mechanisms of apoptosis inductions. HeLa cells treated with recombinant TRAIL showed apoptosis within 2 to 3 h, a process that is clearly initiated by caspase 8 processing and susbsequent Bid degradation and cytochrome c release (Figure 32). HeLa cells treated with PDT demonstrated rapid apoptosis that was initiated at the mitocondrial membrane, as one of the first features noted subsequent to light treatment was the movement of cytochrome c from the intermitochondrial membrane to the cytosol. In the CBV3 infection model, at about the same time that caspase or substrate proteolysis was noted, cyt c was released from the inter-mitochondrial membrane space to the cytosol. This is at the same time as compared to the other models wherein TRAIL results in caspase 8 processing prior to cytochrome c release and BPD and light results in cytochrome c release preceding any caspase or substrate proteolytic events. In all three models, once released, cytoplasmic cyt c can associate with Apaf-1 in the presence of dATP and trigger caspase-9 autocatalysis. Evidence for the importance of the mitochondrial events such as cyt c release in CVB3 infection was also obtained based upon the appearance of the the mitochondrial epitope 7A6. In the CVB3 model, 7A6 expression occurred despite ZVAD.fmk treatment, eliminating the possibility that cyt c release was related to Bid processing by caspase-8 or -3. The release of cyt c during ZVAD.fmk treatment also suggests that there are no pathways in CVB3 infected 167 cells whereby a viral protease either to directly or indirectly activates a caspase-dependent proteolytic cascade leading to cyt c release. There are published reports that expression of picornaviral proteases in cells induces apoptosis. The human glioblastoma SF268 cell line was used to investigate the induction of apoptosis by the 3C protease of enterovirus 71 (EV71) (428), and resulted in morphologic alterations consistent with apoptosis. When two residues in the viral protease catalytic site were mutated, this protein lost its ability to induce apoptosis, suggesting that viral cleavage of host proteins was sufficient to induce apoptosis. HeLa cells expressing an inducible poliovirus 3C protease underwent apoptosis upon protein expression, in a process that was inhibited by ZVAD.fmk (44). Similarly, when inducible expression of picornaviral 2A occurred in a cell line, inhibition of protein synthesis was noted followed by induction of apoptosis (241). In TRAIL-treated HeLa cells, treatment with ZVAD.fmk inhibited the expression of the 7A6 antigen on the outer surface of the mitochondrial membrane confirming that its expression was dependent on Bid degradation by caspase 8. In the PDT model, there was immediate expression of 7A6 on the surface of the mitochondrion following exposure to light at the same time as cytochrome c release, suggesting that this is a reliable marker for cytochrome c release. Other mitochondrial alterations induced during picornavirus infection may relate to apoptotic signalling; previous studies have demonstrated the generation of reactive oxygen species (288), downregulation of various mitochondrial transcripts (684) as observed in aim 4 (Figure 48), and a decrease in ATP levels (731). The Bcl-2 family of proteins includes family members that contain Bcl-2 homology (BH) domains as described in Section 3.3. Over-expression of anti-apoptosic members, Bcl-2 and Bcl-xL, can inhibit or delay multiple mitochondrial alterations including cyt c release, the permeability transition, and generation of reactive oxygen species. There is also evidence that these proteins can depress or inhibit caspase activation once cyt c has been released into the cytosol (103). Over-expression of Bcl-2 and Bcl-xL inhibited cyt c release from the mitochondria to the cytosol for at least the first 10 h following CVB3 infection of HeLa cells (Figure 34). This 168 correlated with both a delay in the onset and a in the magnitude of DEVDase-like cleavage activity. PARP substrate processing is then limited. Similar to ZVAD.fmk treatment of HeLa cells during infection as demonstrated in Aim 2, CPE was not inhibited when apoptosis was inhibited by overexpression of Bcl-2 or Bcl-xL in CVB3 infected cells (Figure 39). This further confirms that CVB3 CPE as determined by light microscopy is a process independent of caspase processing. Viral mechanisms likely contribute to CPE. It has recently been shown that picornaviral proteases cleave structural proteins early during infection (34, 628). Protease 2A cleavage of dystrophin disconnects the cytoskeleton from the extracellular matrix anchor, completely disrupting the cytoskeletal architecture (34, 36). Potentially, many different structural substrates of viral proteases exist that may result in cytopathic effect. We identified focal adhesion kinase as a protein that can be degraded by caspases and potentially viral proteases (Figure 44). Cleavage of this protein by caspase 3 during apoptosis is believed to play a role in degenerative morphological changes (745) in many models of infection. Cleavage of this protein during CVB3 infection, at a site that is distinct from caspase cleavage sites, suggests that this protein may be one of the structural proteins that might play a role in viral protease induced cytopathic effect. It is important to note that this does not necessarily mean that all morphological changes seen during picornavirus infection are independent of caspases. Many structural changes noted during electron microscopic evaluation of picornavirus infected cells are characteristic of apoptosis (489). Examination of the viability of infected HeLa/neo, HeLa/Bcl-2, and HeLa/Bcl-xL cells with or without ZVAD.fmk treatment at 18 h post-infection demonstrated that caspases played a role in the loss of cell viability. Although the Bcl-2 and Bcl-xL overexpressing cells exhibited cytopathology (cell rounding) (Figure 39), mitochondrial function as determined by MTT assay was preserved relative to control and this was shown to be statistically significant (Figure 41). Plaque overlay assay of the supernatants of cell cultures and freeze thawed cell pellets suggested that when caspases were inhibited and cells appeared to be 'viable', they had 169 retained a greater proportion of the infectious progeny virus within the cytoplasm (Figure 43). Taken together, these data suggest that caspases, although not directly responsible for the cytopathic effect noticed during CVB3 infection, do play a major role in the eventual demise of the cell including loss of viability and release of infectious virus particles from the cytoplasm. In conclusion, Aim 3 suggests that the loss of viability of picornavirus infected cells may be attributed to several potential events including: cell bursting during viral release, inhibition of host transcription and translation, and loss of cellular homeostasis due to direct viral protease cleavage of structural proteins. We present evidence that caspases play a role in loss of function and viability of cells during picornavirus infection, and thus consequences are partially prevented by overexpression of Bcl-2 and Bcl-xL or treatment with the pan-caspase inhibitor ZVAD.fmk. 10.4. Aim 4 Using a well-established model of viral infection and microarray technology, we examined temporal changes in gene expression in a single target organ system. The corresponding phenotypic changes we're monitored and characterized as described in Section 9.4. This study was carried out with the intent of extending significantly previous observations in our Laboratory (772) and to identify genes critical to the molecular mechanisms of initiation and progression of viral myocarditis. As noted, coxsackievirus B3 infection in mice with consequent myocyte cell death, myocarditis, and reparative heart disease provides an excellent model in which to examine differential gene expression within a diseased organ. Three distinct phases were captured at different time points following infection. At day 3 post-infection, changes in gene expression would be hypothesized to occur in acutely infected myocytes including initiation of cell death signals, mobilization of other adverse signaling, stress responses, or first-line innate immune responses. At this time, the virus would be "hijacking" host cellular machinery for its own propagation and evading the host cellular apoptosis and anti-viral immune responses. Similarly, neighboring myocytes that are not infected will be bombarded with the interferons and distress 170 signals and will also contribute to the overall gene expression patterns within the organ. By day 9 post-infection there is still on going infection of the myocytes with concomitant cellular response of the immune system. Host responses would be hypothesized to entail anti-viral, antigen specific immune responses, genes associated with myocyte injury and survival, and early reparative processes. Differential gene expression would be expected to reflect the infiltration of immune cells and the organ's increasing extracellular matrix synthesis in the wake of myocyte death and tissue collapse. Day 30 post-infection would occasion prominent extracellular matrix gene expression and genes associated with tissue remodeling and possibly ventricular decompensation as the number of healthy myocytes remaining in the myocardium will be fewer. Virus-mediated changes Early events in CVB3 infection of susceptible mice have been postulated to include gene regulation directly related to viral replication in the host cell. In this study, the up-regulation of poly(A)-binding protein (PABP), ubiquitin-specific protease UBP41, and inorganic pyrophosphatase genes appear to support this concept (Figure 48). All such genes may be up-regulated in response to an environment created by viral infection of the host cell. PABP transcript up-regulation is most likely compensatory for viral degradation of the PABP protein, which is cleaved by the CVB3 viral genome encoded protease 2Ap r 0 (367) and as described in Section 1.3.3. Surviving cells may require an elevated level of PABP in order to increase translation of other cellular proteins that facilitate myocyte repair and restoration of normal cardiac muscle cell activity and integrity. In addition to PABP, the gene of another protein involved in ribonucleoprotein complexes, heterogeneous nuclear ribonucleoprotein (hnRNP), was found to be up-regulated at days 3 and 9 this model of CVB3 myocarditis. hnRNP may significantly impact gene expression by modulating the accessibility and/or interactions of trans-acting factors with particular DNA/RNA sequence elements and has been implicated in the translational inhibition of certain viral mRNA's (405). 171 The expression of other host genes modulated either through viral action or by host reaction involves a "struggle" between the expression of pro-apoptotic and anti-apoptotic molecules. Pro-apoptotic transcription events included the over-expression of the insulin-like growth factor binding protein-3 (IGFBP-3) gene, the protein of which is known to induce apoptosis via an insulin-like growth factor-independent pathway and the over-expression of the BTF-2 gene, encoding a pro-apoptotic transcription factor known to interact with anti-apoptotic family members Bcl-2 and Bcl-xL (357). In addition, other regulatory events contributing towards anti-apoptotic processes included over-expression of the ATF-4 gene. ATF-4 encoded a member of the activating transcription factor/cAMP-responsive element binding protein (ATF/CREB) family of transcription factors, which mediates apoptosis through binding to the cell-death inducer ZIP kinase (361) and down-regulation of gene expression in the anti-apoptotic adhesion related kinase UFO (Ark) involved in growth arrest and induction of apoptosis (10). In contrast, several transcripts with proposed anti-apoptotic protein functions were also over-expressed (Figure 48). One example was the small GTPase RaplB, which may antagonize mitogenic and transforming activity of Ras through the formation of non-productive complexes with critical Ras effector targets (13). Other examples included l-kappa B, an inhibitor of the potent anti-apoptotic transcription factor NF-kappa B (354) and clusterin, a serum protein thought to inhibit the membrane attack complex cytolytic activity of complement (724) and found to be associated with areas of tissue remodeling (6). BTG2, econdes a protein whose expression is induced through a p53-dependent mechanism and with functions relevant to cell cycle control and cellular responses to DNA damage (193, 598) was also induced in response to CVB3. Several other genes encoding signaling molecules may also play a role in either pro- or anti-apoptotic pathways. These include up-regulated H(+)-transporting ATPase, galectin-3, epithelial membrane protein 3 (EMP-3), five-lipoxygenase activating protein (FLAP), PTP-S protein-tyrosine phosphatase, cystatin-beta, and transforming growth factor beta-2 (TGF beta-2). Firm conclusions cannot be made regarding the impact of transcriptional regulation of most 172 signaling molecules since all of these factors may also be post-transcriptionally regulated and have diverse and unknown functions beyond these briefly denoted here. There is normally a balance between apoptotic and anti-apoptotic signals and cell death occurs in response to a persistent shift in this balance. The present profile of gene expression certainly suggests that viral infection invokes countervailing transcriptional regulation that must contribute to the cellular and organ-based disease phenotype. In addition to the above anti-apoptotic gene products, up-regulation of the peripheral-type benzodiazepine receptor (PBR) gene on days 3 and 9 may contribute to cell survival as well. The PBR localizes to the mitochondrial membrane. While its precise physiological role is still unclear, it has been found physically associated with a voltage-dependent anion channel (VDAC) and is a component of the mitochondrial permeability transition pore complex (PT) formed at the contact site between the mitochondrial inner and outer membranes (78). Since the PT is known to be involved in apoptosis signaling (87) and opening of the PT and VDAC pores are regulated by Bcl-2 and Bcl-xL (640), it is likely that PBR is a critical component in an anti-apoptotic process. This is supported by the wealth of PBR agonists that are potent anti-apoptotic compounds (78). Thus, up-regulation of the PBR transcript at days 3 and 9 may represent a host stress response aimed at cell survival. Host defense response Other host cell responses to viral infection are reflected in the differential regulation of inflammatory response genes, stress response genes, and genes pertinent to generation of reactive oxygen species. Concomitant to the inflammatory response, typical of myocarditic disease, mRNA modulation of immune responses genes was a prominent feature by day 9 post-infection (Figure 48 and 49). Correspondingly, transcripts of complement C1q and several genes involved in stimulation of the innate and antigen-specific immune responses were up-regulated. These included: CCAAT/enhancer-binding protein (C/EBP) related protein CRP-2 gene, the interleukin-6-dependent binding protein gene, and the interferon-inducible 1-8U gene. CRP-2 is a transcription factor involved in the regulation of inflammatory cytokines (569) 173 analogous to other members of the C/EBP family of transcription factors whose expression is stimulated by IL-6. These factors are known to mediate expression of several acute-phase genes, and are thought to act as pivotal regulators of cellular differentiation, effector function and the response to an inflammatory insult (569) . One of the positively induced targets of C/EBP transcription factors is p8, a gene whose expression is known to be induced by injury and has a probable role in promoting cellular growth (719). Not unexpectedly, the up-regulated expression of the CRP-2 gene in our model coincides with up-regulated expression of the p8 gene. The CRP-2 gene may also be linked to the heart's hypertrophic response to injury, since it may be involved in the regulation of monocyte chemotactic protein-1 (MCP-1) (303, 304) whose over-expression in the myocardium of transgenic mice elicited cardiac hypertrophy and dilation (391). Other strong regulatory responses by the host to CVB3 viral infection were seen with the up-regulation of host genes relating to processing and presentation of antigen to the immune system in the context of MHC (Figure 48). These host responses were reflected in the up-regulation of the MHC class I structural genes, including MHC class I heavy chain, MHC class I antigen and beta-2 microglobulin, and other proteins involved in the processing and presentation of the viral antigen in the MHOantigen complex. These latter genes included tapasin and cathepsin L (19, 548, 713). Stress response genes are activated in response to a variety cellular insults and are thought to enhance both cell survival and the immune response. Heat shock proteins (HSP) are well-known stress response molecules induced during viral infection. These molecules function as critical modulators of three-dimensional folding of nascent proteins within the cell, assist in the repair of denatured proteins, or promote their degradation after stress or injury. In addition, these stress-activated proteins themselves, in the context of MHC, are recognized and elicit an immune system (434). On days 3 and 9 in our model, hsp86 and hsp27 genes were found to be up-regulated and hsp60 (on day 9 only) and hsp56 were found to be down-regulated (Figure 48). Hsp60 is a potent antigen recognized by the innate immune system and is proposed to 174 have an important role in chronic tissue inflammation. It has been found to trigger the release of tumor necrosis factor alpha (TNF-alpha) and production of nitric oxide (123). Down-regulation of the hsp60 gene may be a cell survival strategy invoked by the host or virus to evade immune-mediated damage of myocytes or immune clearance of infectious virus. Concordant with this possibility, the T-Complex Polypeptide-1 (TCP-1) gene was down-regulated on days 3 and 9. This gene encodes a molecular chaperone thought to belong to the same family as HSP60 (403). Similarly, there was down-regulation, at all three time-points, of the transferrin receptor gene whose protein is usually expressed on activated T lymphocytes and is a target for immunosuppressive therapy (758). We also observed up-regulation at days 3 and 9 of the thymic shared antigen-1 (TSA-1) gene, whose protein inhibits T cell receptor-mediated T cell activation and apoptosis (602). Hsp 27 up-regulation may play a role in resistance to apoptosis. As discussed in Section 3.4, Hsp 27 can bind and sequester cytochrome c in the cytosol, thus preventing its interaction with Apaf-1 and activation of caspase 9. Reactive oxygen species are believed to be important in myocardial injury occurring with certain pathophysiological conditions (425). There is increasing evidence that viral infection triggers production of reactive oxygen species (625) and that oxygen free radical scavengers protect against virus-induced myocarditis (53). In this context, it is of interest that host responses to CVB3 included increased expression of oxygen free radical scavenger genes such as glutathione peroxidase (GPX3), metallothionein (mt-1) and thioredoxin. All of these proteins would convey protection to cardiac myocytes from reactive oxygen species (53, 325, 352) . Of particular note, expression of the phosphotidylethanolamine N-methyltransferase (PEMT) gene, whose protein activity is necessary for maintenance of membrane permeability integrity, and whose concentrations in cardiac sub-cellular fractions is decreased by excess oxygen free radicals, was also found to be decreased at days 3, 9, and 30 in our analysis (Figure 48). This finding is consistent with reports that phosphotidylethanolamine N-methylation activity is decreased in diseased hearts wherein oxygen free radicals are suspected to play a role in pathogenesis of cardiac dysfunction (351). Gene expression of another enzyme involved in the 175 recycling of phopholipids (420), lysophospholipase, was also found to be down-regulated. These findings are intriguing because mitochondrial membrane phospholipid composition may impact electron transport chain enzyme composition and cellular respiration (554). Such changes are supported by our model and are described in other models of cardiac dysfunction (185). Changes in host metabolism and contractility Typically, a switch from the chief myocardial energy source of fatty acid B-oxidation to glycolysis is observed both in hypertrophy and in the failing heart. These changes were highlighted by human patients with dilated cardiomyopathy wherein enzymes in the mitochondrial fatty acid oxidation cycle are downregulated (601). Furthermore, downregulation of the expression of the medium-chain acyl-coenzyme A dehydrogenase gene, a key fatty acid B-oxidation enzyme, has been demonstrated during the progression from cardiac hypertrophy to ventricular dysfunction (601). In our model, the observed downregulation of genes (Figure 48) encoding enzymes apparently involved in the B-oxidation of saturated fatty acids and metabolic mitochondrial enzymes involved in the citric acid cycle and electron transport chain agrees with other models of cardiac dysfunction. In addition, transcripts encoded by the mitochondrial genome, namely cytochrome b, the mutants of which have been found to include cardiac conduction block phenotypes, (790) and 12S and 16S rRNA genes, the mutants of which have been reported with hypertrophic cardiomyopathy, (187) were found to be downregulated on days 3, 9, and 30, respectively. Because mitochondrial mutations have been implicated in both hypertrophic and dilated cardiomyopathies, (187) it is tempting to speculate that downregulation of these and other mitochondrial transcripts may be responsible for the loss of function/cardiomyopathy observed in our model. In parallel to myocardial energy source switching from B-oxidation of fatty acids to glycolysis, a reversion to the fetal energy substrate preference pattern and the regulation of other "hypertrophy" genes in the myocarditis model may be important. Myocardial hypertrophy is often an early hallmark of heart failure, occurring in response to a variety of stimuli, including 176 pathologic factors. The hypertrophic response occurs as the heart attempts to adapt to increased demands on individual myocytes for cardiac work. Our model would satisfy such a setting, as noninjured myocytes compensate for injured and dead myocytes. In most forms of cardiac hypertrophy, there is an increase in the expression of embryonic genes, including the genes for natriuretic peptides and fetal contractile proteins (315). This re-expression of fetal isoforms mimics the mitogenic response of many terminally differentiated cell types, such as cardiomyocytes. Such responses were seen in our model with the upregulation of genes encoding B-type natriuretic peptide, atrial natriuretic factor, B-actin, and -actin (Figure 48). Changes in extracellular matrix genes Studies using genetically modified mice have also implicated extracellular matrix (ECM) proteins in the pathogenesis of heart disease. Both disruption of desmin, a component of muscle adherens junctions in mice, and an ECM protein defect in syrian hamsters lead to cardiomyopathy, implicating a link between myocardial ECM and the pathogenesis of cardiomyopathy (315). In our model, a large number of genes encoding ECM proteins and ECM/cytoskeletal linker proteins were up-regulated during the course of disease, beginning as early as day 3. These genes included structural protein genes actinin-associated LIM protein (ALP), moesin, desmin, alpha-1 type IV collagen, fibrillin-1, vimentin, decorin, fibronectin, beta-galactoside-binding lectin, and enigma, in addition to genes encoding the regulatory protein such as actin-related protein Arp2/3 complex subunits and cofilin, (involved in organizing and controlling actin polymerization), and lysyl oxidase and TIMP-3 (involved in maintenance and stabilization of the ECM). Overexpression of these genes not only corresponds to an observed increase in ECM seen in the day 30 post-infection myocarditic phenotype, but also suggests an active reparative and remodeling effort in response to myocardial damage and cell loss. This initial portrait of an in vivo model of human disease is considered a first step, inasmuch as microarray results and trends were limited to a portion of the total regulatory picture in cellular processes. In addition, the demonstration and exploration of transcriptionally regulated events using expression microarray technologies, while qualitative, is only quantitative 177 in a relative fashion (192). Furthermore, the process involves a mixed, interactive cell population for origin of the probe transcripts in our model tissue. The mixed population of cells used for probe construction not only contained infected myocytes, endothelial cells, fibroblasts, smooth muscle cells and, especially by day 9, innate and specific immune cells, but also non-infected myocytes with infected neighbors in close proximity, releasing stimuli or signaling factors (autocrine or paracrine). Thus, gene regulation observed in these whole-heart gene expression assays was due to a composite portrait rather than sole regulation of myocyte gene expression in response to viral infection. Although not unicellular, our approach does capture the entire pathophysiological microenvironment associated with the myocarditic disease process and as such may provide directions of greater pertinence to the pathogenesis and ultimate treatment of viral myocarditis. Our observations do not exclude the possible influence of systemic factors, originating from other organs and tissues, on the gene expression profile of the myocardium. 178 11 SUMMARY AND MAJOR CONCLUSIONS 11.1. Aim#1 In this Aim we examined the dissemination of coxsackievirus B3 in multiple organs and tissues and the occurrence of cellular injury in myocarditis-susceptible A/J and myocarditis-resistant C57BL/6J mice during the early period of infection. Viral RNA and cellular injury were localized by non-radioactive in situ hybridization (ISH) and terminal deoxynucleotidyl transferase (TdT) -mediated dUTP-digoxigenin nick end-labelling (TUNEL), respectively. In both A/J and C57BL/6J mice, viral dissemination was rapid and widespread following infection, with viral RNA detected systemically within 24 h. Maximal detection of positive and negative strand viral RNA occurred during peak viremia on days 3 and 4 post-infection. Heart, liver, spleen, lymph nodes, thymus, salivary glands, exocrine and endocrine pancreas, skeletal muscle, central nervous system, kidney, testis, lung and visceral fat all contained virus-positive cells. Multiple cells positive for viral RNA were TUNEL positive, reflecting cellular injury during this early acute period. Differential patterns of TUNEL positivity in injured cells, nuclear only, and nuclear plus cytoplasmic, could be visualized, perhaps reflecting different mechanisms of cell death in different organs and tissues, apoptotic versus necrotic/cytopathic effect. Caspase activation assays confirmed that effector caspases such as caspase 3 and 7 were activated in organs with TUNEL positivity. At day 7, after peak viremia, viral RNA could be localized to heart, salivary glands, pancreas, central nervous system, and lymphoid organs. The patterns of infection indicated the particular in vivo susceptibility of certain cell types. 11.2. Aim #2 In this Aim we moved to an in vitro model of CVB3 infection to evaluate the characteristic cytopathic changes noted in multiple susceptible organs and tissues (as in Aim 1) in vivo and in vitro. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the caspase 3 was cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirmed that cleaved caspase 3 was proteolytically active. Caspase 3 substrates were degraded at 9 h following infection, yielding 179 their characteristic cleavage fragments. Inhibition of caspase activation by ZVAD.fmk did not inhibit the virus induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzophorphyrin derivative monacid ring A (BPD-MA) and visible light inhibited the apoptotic phenotype. This aim importantly suggested that caspase activation and cleavage of substrates is not responsible for the characteristic cytopathic effect produced by picornavirus infection, yet may be related to late stage alterations of cellular homeostatic processes and structural integrity. 11.3. Aim #3 This Aim extended the work established in Aim 2 where caspase-3 was activated and caspase-specific substrates were cleaved, but where these events did not appear to be required for the morphologic changes noted following infection. In aim 3 we demonstrate cytochrome c release from the inter-mitochondrial membrane space to the cytosol and caspases-1, -2, -3, -4, -6, -7, -8, and -9 processing. Bcl-2 and Bcl-xL markedly reduced early release of cytochrome c and depressed subsequent activation of caspases following infection. The mitochondrial epitope 7A6 was recognized by the monoclonal antibody Apo2.7 in HeLa cells infected with CVB3 or exposed to TRAIL, but not in infected HeLa/Bcl-2 or Bcl-xL cells. 7A6 appearance was not inhibited by ZVAD.fmk treatment at concentrations that prevented caspase activation and substrate processing of infected HeLa cells. In contrast, ZVAD.fmk did prevent expression in cells exposed to TRAIL, a positive receptor-mediated apoptosis control. Enforced Bcl-2 or Bcl-xL expression, or treatment with ZVAD.fmk, delayed the loss of host cell viability following CVB3 infection although it did not prevent cytopathic effects. Overexpression of Bcl-2 and Bcl-xL or treatment with ZVAD.fmk also decreased the amount of progeny virus released into the supernatant, while allowing cellular virus titres to be sustained at control levels. Focal adhesion kinase was cleaved by a non-caspase protease, and is likely to be a viral protease, and may play a role in the cytopathic effect noted following infection. RasGAP was also cleaved at late time points following infection, another degradation that was shown to be independent of the caspases. The ERK 1/2 and p38 MAPK were activated at late timepoints following infection as 180 determined by use of phospho-specific antibodies. RasGAP degradation by viral proteases may be a mechanism that facilitates ERK 1/2 activation. Subsequent work by H. Luo et al have demonstrated that ERK plays a role in loss of cell viability as inhibition extends cell viability, depresses apoptosis, and decreases viral progeny production (449). Although the inciting incident that induces the release of cytochrome c release from the mitochondria is not yet established, these data suggest that mitochondrial release of cytochrome c is the primary mechanism of caspase activation in CVB3 infection, and, as such, is important in the loss of host-cell viability and release of progeny virus. 11.4. Aim #4 Much has been learned about the pathogenesis of coxsackievirus-induced diseases by study of murine models. In adolescent mice, at least three distinct phases of systemic disease are evident, several days of viral injury, followed by host immune responses, and subsequent tissue repair. We selected days 3, 9, and 30 as timepoints that in our experience reflect these different phases of disease. Gene expression profiling was performed using cDNA microarrays to establish the relative change in mRNA species in infected myocardium at days 3, 9, and 30 post-infection in myocarditis-susceptible, male adolescent A/J mice as compared to sham-infected animals. Each expression profile represents the average of two separate experiments for all genes studied. Of 7000 clones initially screened, 169 known genes had a level of expression significantly different at one or more postinfection time points as compared with baseline. Expression patterns were interpreted in light of known contributors to the myocarditic process. Genes were sorted according to their functional groups including those involved in structural and contractile, energy production, immune responses, signalling related to cell death and cell survival, and unclassified (or unknown). A classical marker of heart failure, ANF, is upregulated at all three time periods. General trends include upregulation of genes involved in antigen processing, immune and stress responses, mixed-regulation of genes involved in myocyte contractility and myocardial structure, and downreregulation of genes associated with energy utilization. 181 12 FUTURE DIRECTIONS The completion of the human genome project and the many technological advances that have occurred in the 1990's will enable dramatic extensions of the data that has been described in this thesis. What took years of research to give us a limited understanding into the pathogenesis of CVB3 induced disease is about to dramatically change. Rather than studying one viral protein or one host cellular response pathway at a time, researchers will be able to screen all the viral and host genes at a single time, expression levels and protein partners (protein-protein interactions), along multiple timepoints during disease progression. The cDNA arrays described in this thesis are now outdated and have already been significantly improved upon by companies like Affymetrix, Inc. (Santa Clara CA) and their rapidly evolving GeneChips™. Rather than use full length cDNA's spotted to glass slides, the GeneChip™ consist of short oligomers that are synthesized on silicone wafers. Each 'gene' consists of 10 individual 'match' and 10 individual 'mismatch' probes. The latest release, at the time of writing this Thesis, was their HG-U133 set of GeneChips™, where almost all genes present in the human genome can be assayed on two chips. This is a powerful tool that will give great insight into the early events within an infected cell and within an infected organ. Ideally, one would want to array a variety of different cell types and coxsackievirus variants at various time points following infection. Such an experiment might consist of cells such as HeLa cells, Vero cells, transformed human cardiac myocytes, isolated primary murine cardiac myocytes, cells with functional and truncated CAR receptors. One would want to infect with the Gauntt and Kandolf strains of CVB3, as well as UV inactivated virus and other coxackievirus serotypes or mutants. It would also be of interest to collect cultures at regular intervals between zero and 18 h following infection to determine the temporal change in expression profiles. In addition to experiments using isolated cells, comparing such results in a variety of organs from infected mice (susceptible and resistant to myocarditis, immune-competent and immune-deficient) at regular intervals following infection should be informative. The animal 182 studies would be a direct extension of the initial work demonstrated in this Thesis. The whole organ studies would compare infected as well as neighboring and uninfected cells and mobilized cells of the immune system. A great number of time points would allow for better dissection of the early direct viral events, the immune response, and late stage remodeling and cardiac dysfunction. These whole organ results could be directly compared to the arrays that were undertaken within the in vitro models of replication. Additional studies to extend this thesis would be to identify all the host protein partners of each of the CVB3 proteins in different permissive and non-permissive cell types. Each of the viral structural and non-stuctural genes can be cloned into expression vectors that produce epitope-tagged versions of the viral protein in transiently-transfected or stably cell lines. The expressed proteins would interact with a variety of host protein partners in a number of different cells. The protein complexes would be purified with the use of epitope tags and the composition of the complexes determined using mass spectrometry. It would be predicted that numerous interactions between host and viral proteins would be observed, some of which might offer unique points for therapeutic intervention. Work in this thesis has focussed on a small subset of the human genes that were annotated at the time of the research and will likely lead to numerous extensions based on discovery of new interaction networks. A fundamental problem in high throughput research has been the gap between the abilty to work with most genes in the genome, and the relatively little amount of annotation available for each gene. The names, the function, the tissues and organs or natural expression are all areas that will require extensive research over the near term. Logical direct extensions of this thesis are to further clarify the intersection between cell death and cell survival pathways in various cell types following CVB3 infection. Prior to 8 h post-infection when cytochrome c is released into the cytosol and caspase 9 is activated, there appears to be multiple signaling pathways that are activated. In addition to the ERK activation, it must be determined if p38, JNK, PI3K/Akt, N F K B , or other kinase and signaling cascades are activated. In addition to determination of activitation status, these pathways can be modulated 183 prior to and during infection by small molecule inhibitors or growth factors and other agonists. As was demonstrated by ERK1/2 inhibition (450), these pathways may play a direct role in either creating an enviroment that is suitable for viral replication or may influence host survival or death. As work in this thesis utilized HeLa cells, a transformed human cell type that is commonly used to propogate enteroviruses, further work must be done to dissect the life and death signaling pathways in a variety of cells. With regards to ERK1/2 activation, it will be interesting to see if this non-protective role is common amongst cell types. As mentioned in Section 5, ERK 1/2 signalling appears to be an antiapoptotic response in stressed myocardium. This potentially may be one method that the virus has adapted to facilitate its replication within this organ. Primary murine myocytes are being optimized for infection in the Cardiovascular Research Laboratory, but there are some hurdles that need to be overcome. First, the timeframe for induction of apoptosis may be longer since the myocytes may have intrinsic anti-apoptotic factors (as discussed in Section 5) or the variety of growth factors used to maintain viability may influence the viral replicative cycle or inhibit apoptosis. Maintaining these cells for long periods of time to support productive infection may be a challenge. In addition to cardiac myocytes, one would want to assay a variety of cell lines for CAR and DAF expression and then determine the viral replicative and host signaling responses in the different combinations of these cells where there was productive and nonproductive infection. It is assumed that there will be cell types that express the appropriate viral receptors but do not support viral replication. It will be interesting to see if the host signaling responses are activated. The principal event leading to cytochrome c release, whether directly downstream of a viral protease cleavage event (degradation or activation of apoptosis regulatory proteins) or a host signaling event such as p53 activation, or a MAPK signaling event needs to be further identified. The use of recombinant, biologically active 2A and 3C viral protease and cell free systems of apoptosis could be used to determine if these two proteins degrade a host protein(s) that results either directly or indirectly in apoptosis. Caspase activation assays and immunoblot analysis can be done on these cell free systems as an indicator that the appropriate proteolytic environment 184 exists. Further, protein-protein interaction studies between the 2A and 3C and host cellular proteins may identify interacting partners and give further insights into potential intersection been host and viral proteins that are responsible for apoptosis induction. These studies may also give further insight into potential host proteins that are a degraded by viral proteases and may be responsible for the CPE such as additional structural proteins. The work presented in thesis has been subsequently fortified by additional studies within the McDonald Research Laboratories, St. Paul's Hospital. Of note, the group has focused on three primary areas; the mechanism of apoptosis induction in infected cells during CVB3 infection, the role of intracellular kinase signaling pathways on cell viability and viral replicative events, and the interaction between viral and host proteins during the virus life cycle. Recent studies by H Luo et al have examined the role of the ERK 1/2 activation during CVB3 infection of HeLa cells (450). I am eagerly awaiting further discoveries and extensions of this work to eminate from the rich research environment of the McDonald Research Wing. I hope this work stimulates others within the Laboratory to continue where I have left off. 185 13 MATERIAL AND METHODS 13.1. Coxsackievirus B3 Stock CVB3 was generously provided by Charles Gauntt, PhD, and stored at -80°C. Virus was grown in HeLa cells and routinely re-titered at the beginning of all individual experiments. 13.1.1. Propogation and handling of virus Virus propagation was performed in HeLa cell, which were 95% confluent, at an moi 0.1. The supernatant and cells were harvested when virus infected HeLa cells demonstrated 100% cytopathic effects. The supernatant and cells were frozen. Thawing once liberated cellular virus which was centrifuged (500 X g, 10 minutes) to pellet cellular debris pipetted into 1 ml aliquots, and frozen at -80°C. Supernatant virus-stocks were consistently titered at 5 X 107 to 108 pfu/ml. Working virus was prepared by diluting stock virus in Dulbecco's phosphate buffered saline without magnesium (DPBS) as defined by each experimental protocol. 13.2. HeLa Cell Culture HeLa cells (Cat # CCL-2, ATCC) were grown in minimal essential media (MEM) with penicillin/streptomycin (10,000 units/ml), L-glutamine (200 mM), HEPES buffer (10 mM, pH=7.35), and 10% fetal bovine serum (FBS) at 37°C. Cells were incubated until 100% confluent, harvested with 1% trypsin/EDTA, centrifuged (500 X g, 5 minutes), resuspended in media, counted using a hemocytometer and seeded into a new flask(s). Cell viability was consistently 95-99%. 25 cm2 flasks were seeded with 106 cells in 10 ml of media, and 75 cm2 flasks were seeded with 3 X 106 cells in 20 ml of media and cultured until confluent (within 2 days). Each culture series was subcultured 10-13 times and discarded; 107 fresh cells (frozen in 50% MEM, 30% FBS and 20% dimethylsulfoxide) were removed from liquid nitrogen long term storage, thawed, washed with media and seeded into a 75 cm2 flask. Frozen stocks of HeLa cells are negative for Mycoplasma infection as determined by Hoechst 33258 staining (273). 13.3. Generation and culture of stable-transfected cell lines HeLa cells overexpressing Bcl-2 and Bcl-xL were generated as previously described (718). Briefly, Bcl-2 and Bcl-xL inserts were cloned into EcoRI site of pSFFV-Neo vector (217). Vectors 186 containing inserts, or no insert (neo), were transfected into HeLa cells by electroporation. After selection with 1 ng/ml G418 (Gibco/BRL Life Technologies), cells were cloned by limiting dilution and transfectants screened for Bcl-xL or Bcl-2 by Western blot. Transfected cell lines were maintained in complete DMEM with 1 jig/ml G418. 13.4. Collection of total cell lysates Cells were washed twice in cold PBS and suspended in 1 ml of cold lysis buffer (20mM Tris pH 8, 137mM NaCI, 10% glycerol, 1% Noniodet P-40, 1mM phenylmethylsulfonyl fluoride, 10 ug/ml aprotinin) per 75cm2 culture area. After 20 minutes on ice, supernatant was collected following centrifugation at 10,000x g. Cell lysate protein concentration was determined by the BCA method (Pierce Chemical Company, Rockford, IL) 13.5. Collection of cytosolic extracts Cells were washed twice in cold PBS and resuspended in 1 ml of ice cold buffer (250 mM sucrose, 20 mM Hepes pH 7.4, 10 mM KCI, 1.5 mM MgCI2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, supplemented with 1 mM PMSF and 10 ug/ml aprotinin) per 75cm2 culture area. Cells were disrupted by 20 strokes with the B pestle of a Kontes™ dounce homogenizer. The supernatant was collected following centrifugation at 10,000x g and further centrifuged at 100,000 x g for 1 hour at 4°C in a Beckman Optima™ ultracentrifuge using a TL-100 rotor. Cell lysate protein concentration was determined by the BCA method (Pierce Chemical Company, Rockford, IL). 13.6. Caspase activation assays To evaluate DEVDase-like (caspase-3 and -7) cleavage activity following CVB3 infection, total HeLa cell lysates were incubated with a caspase-specific fluorescent substrate as previously described (23). Briefly, lysates were incubated with reaction buffer (20mM Tris pH 7.5, 137mM NaCI, 1% NP-40, 10% glycerol) containing 100 |aM of the caspase substrate DEVD-AMC (Calbiochem, Cambridge, MA). The reaction mixture was incubated at 37°C for two h and fluorescence excitation at 380/400 nm was measured at 460/505nm (AMC/AFC) using a CytoFluor™ 2350 from Perseptive Biosystems (Norwalk, CT). 187 13.7. Western blotting Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred to nitrocellulose (Hybond ECL, Amersham Pharmacia Biotech, Uppsala, Sweden). Following blocking, and incubation with primary and secondary antibodies, horseradish peroxidase-conjugated secondary immunoglobulins were detected using the enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech, Uppsala, Sweden) and exposure to Hyperfilm™ (Amersham Pharmacia Biotech, Uppsala, Sweden). 13.8. MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay To determine cell viability, cells were cultured in Falcon 96-well microtitre plates and the MTT assay was performed as previously described (508) with modifications. Briefly, following CVB3 infection culture medium was decanted and replaced with complete DMEM containing 10% FBS. At 10 h following CVB3 infection or 24 h following TRAIL (150 ng/ml) treatment, culture medium was aspirated and 50 ng of MTT in 100 u.l of PBS was added to each well. Cells were incubated for one hour and the reaction was stopped by the addition of 150 jal of acidified isopropanol. Color development correlating to cell viability was measured at 590 nm with a Dynatech Laboratories microtitre plate reader (Chantilly, VA). 13.9. Morphology Differential interference contrast (DIC) morphological analysis for degree of CPE was assessed using images taken with a Nikon Eclipse TE300 inverted scope (Nikon, Tokyo, Japan) and images were collected using SPOT imaging software (Diagnostic Instruments, Inc.; Sterling Heights, Ml). 13.10. Flow Cytometry At 10 h following CVB3 infection or 2 h following TRAIL (150 ng/ml) treatment, HeLa cells were washed 2X in cold PBS and dispersed into suspension by gentle agitation with 2 mM EDTA-PBS. Cells were permeabilized with 0.1% digitonin in PBS and exposed to phycoerythrin (PE) conjugated mouse lgG1 monoclonal antibody AP02.7 (Beckman-Coulter, Fullerton, CA) in a solution of PBS containing 2% FCS for 30 min on ice. Control cells were treated with a PE-188 conjugated isotype-matched antibody. Cells were washed twice with the labeling solution and read immediately on a Coulter XL flow cytometer (Coulter Electronics, Inc., Hialeah, FL). Mean channel fluorescence intensity (MCFI) values were determined (5000 cells analyzed per sample). For isolation of thymocytes, single cell suspensions of thymocytes were prepared by delicately homogenizing the spleen in a Wheaton™ glass tissue homogenizer and transferred to a 15 ml conical tube. Delicate homogenization and treatment of cells allowed collection and analysis of splenocytes from uninfected and infected animals, since infected virus exposed lymphocytes appear to be extremely friable. Tissue debris was discarded, the cell suspension was centrifuged (500 X g, 5 min), and the supernatants archived for plaque assay. Cell counts were determined using a hemocytometer or Coulter™ counter. Single cell suspensions of lymphocytes were adjusted to 2x107 cells/ml in DPBS, and stained for surface antigens CD4, CD8, with antibodies conjugated to FITC or R-PE (Gibco/BRL), at a dilution of 1:200. Immune cells were washed three times with DPBS then fixed with 10% buffered formalin (overnight [O/N], 4°C). After washing three times with TSK-BT (100 mM Tris-base, 550 mM NaCI, 10 mM KCI, 2% bovine serum albumin, 0.1% Triton X-100™), immune cells were then analyzed on a Coulter™ flow cytometer. Experimental results from each sample reflect percentages determined from more than 1000 fluorescent events. 13.11. Animal handling and sacrafice, and tissue distribution and evaluation A/J (H-23), and C57BL/6J (H-2") mice (Jackson Laboratories, Bar Harbor, Maine) were 4 weeks of age when received at St. Paul's Hospital Animal Care Facility, University of British Columbia, and 5 weeks of age at the onset of each experiment. Mice were euthanized by C0 2 narcosis. 13.12. Tissue histopathology Sections from the heart, liver, spleen, lymph nodes, pancreas, salivary glands, testis, thymus, lung, kidney, brain, and skeletal muscle were fixed in fresh DPBS-buffered 4% paraformaldehyde overnight at 4°C. Fixed tissue was dehydrated in graded alcohols, cleared in xylene, embedded in paraffin, and sectioned for in situ hybridization and nick-end labelling. Serial sections were also stained with hematoxylin and eosin and Masson's trichrome. The extent and severity of virus-189 induced injury including coagulation necrosis, contraction band necrosis, and cytopathic effects were evaluated and scored as previously described (140) 13.13. Virus content of tissues and cells Ttiters of CVB3 in tissues, cells or serum were determined on monolayers of HeLa cells by agar overlay plaque assay in duplicate, with modifications (16). HeLa cell cultures were periodically tested for Mycoplasma infection utilizing Hoechst 33258 stain (273). Fresh frozen tissues or cells were homogenized with an OMNI 2000™ homogenizer equipped with a 5 mm generator. Multiple samples were diluted in a serial 10-fold fashion in 96-well plates with a multi-well pipetter, and 200 pi of each dilution series sample was overlaid on 95-100% confluent monolayers of HeLa cells in 6-well plates and incubated (5% C0 2, 1 hour , 37°C). Virus was removed by pipetting, and warm media (MEM plus penicillin/streptomycin [10,000 units/ml], L-glutamine [200 mM], HEPES buffer [238.3 g/l], 10% FBS [heat-inactivated], and 0.75% agar) was overlaid in each well. Plates were incubated (2-3 days) and fixed with Carnoy's fixative (30 min). The solidified MEM/agar overlay was removed, the monolayers were stained with 1% crystal violet, plaques were counted, and viral concentration was calculated as plaque forming units (pfu) per mg wet tissue, cell number, or ml. 13.14. Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end-labelling (TUNEL) TUNEL was carried out on tissue sections using ApopTag in situ apoptosis detection kits (Oncor, Gaithersburg, MD) following manufacturer's instructions. Briefly, slides were deparaffinized, proteinase K digested (15 minutes at 37°C), and endogenous peroxidase activity was quenched (3%H 20 2 for 5 minutes). TdT enzyme and digoxigenin-labelled dUTP in reaction buffer were added to slides and incubated for 1 hour at 37°C. Slides were washed and incubated with peroxidase-conjugated anti-digoxigenin antibody for 30 minutes at room temperature and developed using diaminobenzidine as a substrate and copper solution as an enhancer. Slides were counterstained with dilute carmalum and examined for reaction product by light microscopy. The specificity of TUNEL signals was confirmed by omission of either TdT enzyme or digoxigenin-190 dUTP. Slides for uninfected and infected animals were assayed concurrently. Positive control slides included testis and thymic tissue, both of which undergo spontaneous apoptosis. 13.15. In Situ hybridization In situ hybridization was carried out as previously described (16, 293). Briefly, tissue sections were incubated overnight in hybridization mixture containing digoxigenin-labelled, CVB3 strand-specific riboprobes. Post-hybridization washing was followed by blocking with 2% normal lamb serum. A sheep anti-digoxigenin polyclonal antibody conjugated to alkaline phosphatase (Boehringer Mannheim PQ, Laval, Canada) was developed in Sigma-Fast nitroblue tetrazolium-BCIP [5-bromo-4-chloro-3-indolylphosphate tuluidinium] (Sigma Chemical Co.). The slides were counterstained in fresh carmalum and examined for reaction product by light microscopy. 13.16. RNA isolation/cDNA probes Mouse hearts isolated for each time-point were flash-frozen in liquid nitrogen and stored at -70°C. Hearts from each experimental group were pooled, whole tissue mRNA was extracted and twice selected by oligo dT chromatography. Fluorescently labelled cDNA probes were then generated by reverse transcription of poly(A)+ mRNA in the presence of Cy3 or Cy5 dCTP (Amersham). 13.17. DNA microarray. Microarrays were fabricated and contained ~ 7000 cDNA clones randomly collected from a normalized cDNA library prepared hearts of 3 male Wistar rats (656). Microarray fabrication and hybridization were performed at Incyte Pharmaceuticals (Palo Alto, CA) as described (326, 619). Each pair (CVB3-infected and sham-infected) of fluorescently labeled cDNA sample probes was applied to the microarray and allowed to hybridize competitively to each of the 7000 elements. Degree of hybridization was quantitated by sequential excitation of the two fluorophores with a scanning laser. Differential expression values were represented as a ratio of intensities. 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