Role of the Ubiquitin/Proteasome System in Coxsackievirus Induced-Myocarditis by Guang Gao A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2010 © Guang Gao, 2010 ii Abstract Viral myocarditis, an inflammatory disease of the myocardium, can lead to the development of dilated cardiomyopathy (DCM), a common cause of heart failure. Coxsackievirus B3 (CVB3) in the family of Picornaviridae is one of the primary causative agents of viral myocarditis. The ubiquitin/proteasome system (UPS), a primary intracellular protein degradation system in eukaryotic cells, has emerged as a key modulator in viral infectivity and virus-mediated pathogenesis. Our laboratory has previously demonstrated a potential role of the UPS in CVB3 infection. However, the effect of proteasome inhibition on CVB3-induced myocarditis in vivo has not been assessed and the underlying mechanism by which the UPS regulates CVB3 replication remains unclear. In this dissertation, my hypothesis is that the UPS plays a critical role in the pathogenesis of CVB3-induced myocarditis through promoting CVB3 replication and by regulating host protein degradation. To test this hypothesis, I proposed three aims. In aim 1, using a myocarditis-susceptible mouse model, I demonstrated that treatment with a proteasome inhibitor MLN353 significantly attenuates CVB3-induced myocardial damage, suggesting that proteasome inhibition may provide a therapeutic means for viral myocarditis. During this study, however, the potential toxicity of general inhibition of proteasome was recognized, which prompted me to search for the specific targets within the UPS utilized by CVB3. In aim 2, collaborating with others, I showed that protein ubiquitination is enhanced and CVB3 protein 3D is ubiquitinated during viral infection. Gene-silencing of ubiquitin significantly reduces viral titers. However, this reduction is not as potent as by iii proteasome inhibition, suggesting that ubiquitin-independent proteasomal degradation may also play a role during CVB3 infection. In aim 3, I showed that REGγ, which mediates ubiquitin-independent protein degradation, enhances CVB3 replication via facilitating p53 degradation. During CVB3 infection, REGγ is sumoylated and translocated. Taken together, the results suggest a therapeutic value of proteasome inhibition in the treatment of viral myocarditis. The data also demonstrate important roles of both the ubiquitin-dependent and -independent pathways in the regulation of CVB3 infection. Identification of the specific substrates within the UPS during CVB3 infection and the potential mechanisms involved allows for more precise targeting in drug therapy. iv Preface This study was carried out in strict according with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Canadian Council on Animal Care. The protocol was approved by the Committee on Animal Care of the University of British Columbia (A03-0144). All efforts were made to minimize animal suffering. v Table of Contents Abstract ............................................................................................................................... ii Preface................................................................................................................................ iv Table of Contents ................................................................................................................v List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix List of Acronyms ............................................................................................................... xi Acknowledgements .......................................................................................................... xiv Chapter 1: Background .......................................................................................1 1.1 Overview of myocarditis................................................................................................1 1.2 Viral myocarditis ...........................................................................................................4 1.3 Coxsackievirus ...............................................................................................................7 1.3.1 CVB3 genome .................................................................................................7 1.3.2 CVB3 life cycle and encoded viral proteins ...................................................8 1.3.3 CVB3 receptors .............................................................................................12 1.4 Interaction between CVB3 infection and the host cells ...............................................15 1.4.1 Gene profile after CVB3 infection ................................................................15 1.4.2 CVB3-induced myocarditis: a tri-phasic disease ..........................................16 1.5 The ubiquitin/proteasome system (UPS) .....................................................................21 1.5.1 Ubiquitin and ubiquitination .........................................................................21 1.5.2 20S proteasome .............................................................................................24 1.5.3 Proteasome activators ...................................................................................25 1.5.4 Deubiquitination ...........................................................................................28 vi 1.5.5 Functions of the UPS ....................................................................................29 1.6 The UPS and virus infection ........................................................................................31 1.6.1 The UPS and viral immune evasion..............................................................34 1.6.2 The UPS and viral progeny release and (or) budding ...................................35 1.6.3 The UPS and transcriptional regulation of virus ...........................................36 1.6.4 The UPS and apoptosis suppression .............................................................37 1.6.5 Deubiquitination in viral infection ................................................................38 Chapter 2: Rationale, Hypothesis, and Specific Aims ................................40 2.1 Rationale ......................................................................................................................40 2.2 Overarching hypothesis ...............................................................................................42 2.3 Specific aims ................................................................................................................42 Chapter 3: Proteasome Inhibition Attenuates CVB3-Induced Myocardial Damage in Mice ..................................................43 3.1 Introduction ..................................................................................................................43 3.2 Experimental design.....................................................................................................44 3.3 Results ..........................................................................................................................46 3.4 Discussion ....................................................................................................................64 Chapter 4: Ubiquitination is Required for Effective Replication of CVB3 ..........................................................................................69 4.1 Introduction ..................................................................................................................69 4.2 Experimental design.....................................................................................................70 4.3 Results ..........................................................................................................................71 4.4 Discussion ....................................................................................................................83 vii Chapter 5: Proteasome Activator REGγ Enhances CVB3 Infection via Facilitating p53 Degradation .......................87 5.1 Introduction ..................................................................................................................87 5.2 Experimental design.....................................................................................................88 5.3 Results ..........................................................................................................................90 5.4 Discussion ..................................................................................................................115 Chapter 6: Conclusions and Future Directions ..........................................121 6.1 Conclusions ................................................................................................................121 6.2 Future directions ........................................................................................................124 Chapter 7: Materials and Methods .................................................................127 Bibliography ............................................................................................................136 Appendix 1: List of publications, abstracts and presentations .............155 viii List of Tables Table 1. Function of CVB3 encoded viral proteins ...........................................................13 Table 2. Examples of viruses or viral proteins interacting with the UPS ..........................33 ix List of Figures Figure 1. Myocarditis is an inflammation of the myocardium ..........................................2 Figure 2. CVB3 genome and viral proteins .......................................................................9 Figure 3. Proteolytic cleavage of CVB3 viral polyprotein ..............................................10 Figure 4. Pathogenesis of viral myocarditis: a tri-phasic disease ....................................18 Figure 5. Protein degradation by UPS .............................................................................22 Figure 6. Proteasome activators and ubiquitin-dependent and –independent Degradation ......................................................................................................26 Figure 7. CVB3 infection leads to an accumulation of protein-ubiquitin conjugates in mouse heart ................................................................................48 Figure 8. Expression of ubiquitinating enzymes is upregulated in CVB3-infected mouse heart .............................................................................50 Figure 9. Expression of deubiquitinating enzyme is increased in CVB3-infected mouse heart .............................................................................53 Figure 10. Proteasome inhibitor MLN353 inhibits CVB3 replication in mouse Cardiomyocytes ....................................................................................56 Figure 11. MLN353 treatment reduces proteasome activity in mouse heart .....................59 Figure 12. Effect of proteasome inhibition on CVB3 viral titer in mice ...........................61 Figure 13. MLN353 treatment attenuates CVB3-induced myocardial injury in mice ..................................................................................................62 Figure 14. Proteasome inhibitors decrease CVB3 RNA expression, viral protein synthesis and viral progeny release in HeLa cells ..........................................73 Figure 15. Knockdown of ubiquitin expression by siRNA reduces CVB3 replication .....76 x Figure 16. CVB3 infection results in increased protein polyubiquitination and decreased free ubiquitin ..................................................................................79 Figure 17. CVB3 RNA-dependent RNA polymerase 3D is ubiquitinated .......................82 Figure 18. A proposed model for UPS regulation of CVB3 replication ...........................86 Figure 19. Knockdown of REGγ reduces CVB3 progeny titers .......................................93 Figure 20. Overexpression of REGγ promotes CVB3 replication ....................................95 Figure 21. Overexpression of REGγ decreases p21 and p53 levels .................................98 Figure 22. Blockage of p21 and p53 degradation by proteasome inhibitor lactacystin is associated with decreased virus protein expression ................101 Figure 23. Overexpression of p53 inhibits CVB3 infection ...........................................103 Figure 24. Overexpression of p53 attenuates the effect of REGγ on promoting CVB3 replication.........................................................................106 Figure 25. CVB3 infection leads to redistribution of REGγ ...........................................109 Figure 26. CVB3 infection promotes REGγ sumoylation ..............................................113 Figure 27. In vivo sumoylation assay by ELISA kit .......................................................119 Figure 28. A proposed mechanism by which REGγ enhances CVB3 Infectivity ......................................................................................................120 xi List of Acronyms The following is a list of abbreviations used in this dissertation in alphabetical order: CAR Coxsackievirus and adenovirus receptor CEP Carboxyl extension proteins CTL Cytotoxic lymphocytes CVB3 Coxsackievirus B3 DAF Decay-accelerating factor DAPI 4,6 diamidino-2-phenylindole DCM Dilated cardiomyopathy DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide DTT Dithiothreitol DUB Deubiquitinating enzyme EBNA Epstein-Barr virus encoded nuclear antigen EBV Epstein-Barr virus eIF4G Eukaryotic translation initiation factor-4G ELISA Enzyme-linked immunosorbent assay GAPDH Glyceraldehyde-3-phosphate dehydrogenase GPI Glycosyl-phosphatidylinositol H&E Hematoxylin and eosin HCMV Human cytomegalovirus HECT Homologous to E6-AP carboxyl terminus HHV Human herpesvirus xii HIV Human immunodeficiency virus HPV Human papillomavirus HSV Herpes simplex virus HTLV Human T-cell leukemia virus IFN Interferon IL Interleukin IRES Internal ribosome entry site ISH in situ hybridization IκB Inhibitor of nuclear factor κappa B JAK Janus kinase JNK c-Jun N-terminal kinase MAPK Mitogen-activated protein kinase MEK Mitogen-activated protein kinase kinase MHC Major histocompatibility complex MOI Multiplicity of infection NCS Newborn calf serum NFκB Nuclear factor κappa B PABP Poly (A) binding protein PCR Polymerase chain reaction PDTC Pyrrolidine dithiocarbamate PFU Plaque forming unit PHD Plant homeodomain PI3K Phosphatidylinositol-3 kinase xiii REG Also known as proteasome activator 28 protein RING Really interesting new gene ROS Reactive oxygen species RSV Rous sarcoma virus RT Reverse-transciption SAPK Stress-activated protein kinase STAT Signal transducers and activators of transcription SUMO Small ubiquitin-related modifier SV Simian virus TNF-α Tumor necrosis factor-α UBP Ubiquitin-processing protease UCH Carboxyl-terminal hydrolase UCHL1/3 Ubiquitin c-terminal hydrolase L1/3 UPS Ubiquitin/proteasome system USP7 Ubiquitin-specific protease 7 UTR Untranslated region VP1 Viral protein 1 zVAD.fmk Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone xiv Acknowledgements This dissertation would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, I offer my sincerest gratitude to my supervisor, Dr. Honglin Luo, who has supported me throughout my thesis study with her patience and knowledge whilst allowing me the room to work in my own way. I attribute the level of my Ph.D. degree to her encouragement and effort and without her this thesis would not have been completed or written. Deepest gratitude is also due to my co-supervisor, Dr. Bruce McManus, for his patience and steadfast encouragement to complete this study. One simply could not wish for better or friendlier supervisors than them. Special thanks to my supervisory committee members, Drs. Decheng Yang, Weihong Song, and Richard Hegele, as well as my Committee Chair, Dr. Haydn Pritchard, for their precious guidance, advice and time. This dissertation bears on imprint of many people. I sincerely thank my friends and colleagues, Dr. Xiaoning Si, Jingchun Zhang, Jerry Wong, Mary Zhang, and Zongshu Luo, as well as many colleagues and staff around in my daily work, for their technical expertise and great support. I gratefully acknowledge financial support from the Michael Smith Foundation for Health Research and the Heart and Stroke Foundation of BC and Yukon. Finally availing myself of this opportunity, I wish to express my love and gratitude to my beloved families, my wife and son, for their understanding and endless love, xv through the duration of my study; to my beloved parents for their moral and spiritual support. 1 Chapter 1: Background The overall objective of this dissertation is to better understand the role of the ubiquitin/proteasome system (UPS) in coxsackievirus B3 (CVB3)-induced viral myocarditis. In this section, I will first introduce the background knowledge about CVB3, viral myocarditis, and the UPS, and then I will review recent findings on the interactions between host UPS and viral infection. 1.1 Overview of myocarditis According to the Dallas criteria (1987), myocarditis is clinically defined as an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes in the absence of ischemic event [1, 2] (Figure 1). Myocarditis can be caused by various etiologies including toxic substances, allergic and hypersensitive responses, immune diseases such as rheumatic fever and systemic lupus erythematosus, cardiac transplant rejection, and infectious agents such as bacteria, viruses, protozoa, and fungi [3-5]. In Central and South America, myocarditis is highly associated with Chagas disease caused by Trypanosoma cruzi, whereas viruses are the prevalent infective agents in North America and developed countries [6-8]. Myocarditis is a major cause of sudden unexpected death in young adults less than 40 years old, and contributes significantly to the incidence of heart failure . It can result in rapidly progressive heart failure and cardiac arrhythmia in a healthy person; while in some cases, the myocardial inflammatory may persist chronically and progress to dilated cardiomyopathy (DCM), a common cause of heart failure . It is estimated 2 Figure 1. Myocarditis is an inflammation of the myocardium. Acute viral myocarditis is induced most often by Coxsackie B virus and echovisuses. Myocardial interstitium presents an abundant edema and inflammatory infiltrate, mainly with lymphocytes and macrophages. Focal destruction of myocytes may be present, generating loss of contractile function of the myocardium. (H&E, ob. x10). This figure is from Atlas of Pathology (2nd Edition, www.pathologyatlas.ro). 3 that about 10-20% of patients with histological evidence of myocarditis will develop chronic disease, eventually progression to DCM . Early postmortem studies on autopsies from victims of sudden death has revealed a prevalence of more than 1% for myocarditis . However, the real incidence of myocarditis is still not clear, largely due to asymptomatic, subclinical, and misdiagnosed cases. In late 1970s, introduction of endomyocardial biopsy provided the clinicians and scientists with a useful diagnostic tool to assess the potential myocarditis in living patients . However, examination of endomyocardial biopsy samples form patients with new-onset congestive heart failure has reported a wide-ranging incidence of 0% to 67% for myocarditis [11-24]. The inconsistencies are most likely due to differences in host ethnic and genetic backgrounds, susceptibility to infection, pathogen epidemiology, sampling errors, diagnostic application of Dallas histological criteria, and the lack of unanimity among pathologists as to what constitutes myocarditis [25, 26]. Despite the low sensitivity, ventricular endomyocardial biopsy still represents the gold standard for diagnosis of myocarditis [4, 27]. The clinical presentation of myocarditis is often associated with diverse, nonspecific signs such as shortness of breath and chest pain. Only about 10% of cases are with typical symptoms including palpitation, ventricular dysfunction, and acute heart failure. While the histological analysis according to the Dallas criteria is often inadequate in the diagnosis of myocarditis, additional methods, such as histoimmunological, serological, electrophysiological (electrocardiogram and echocardiography), and genetic analyses, as well as a detailed history and careful physical examination, may provide a more definitive diagnosis [28-31]. 4 So far, no specific and effective therapy is available for myocarditis. Current therapeutic strategies for myocarditis are primarily supportive (e.g. avoidance of exercise, fever reduction, control of arrhythmias, and treatment of heart failure) depending on the etiology and presenting symptoms. For clinical symptoms of heart failure or arrhythmias, basic medications such as angiotensin-converting enzyme inhibitors or angiotensin- receptor blocking agents, diuretics, β-blockers, and anti-arrhythmic drugs should be administered. When a patient presents with symptoms of end-stage DCM, heart transplantation still represents the only definitive therapeutic option . The 5-year survival in biopsy-proven myocarditis/DCM is approximately 50% . 1.2 Viral myocarditis Numerous viruses have been associated with viral myocarditis and DCM in humans, including adenoviruses, enteroviruses, parvovirus, influenza viruses, cytomegalovirus, herpesviruses, hepatitis C virus, and human immunodeficiency viruse-1 (HIV-1) [6, 33-37]. However, coxsackieviruses are considered to be one of the prominent causes of viral myocarditis, particularly in neonates and young children [38-41]. It has been reported that 5-15% of patients of myocarditis have a viral infection some time during the course of their illness [42-44]. Endomyocardial biopsy examination demonstrates that up to 60% of patients with myocarditis and DCM are virus-positive in the heart [45-47]. Many factors have been reported to contribute to the susceptibility to viral myocarditis, which include age, gender, nutrition, and pregnancy [6, 48-51]. Young children, particularly infants under six months of age, are extremely sensitive to 5 enteroviral infections [6, 52-54]. In North America, viral myocardits is the cause of about 20% of sudden unexpected death among children and youth [55-60]. Using molecular diagnostic techniques, the enteroviral sequences were isolated from 17-21% of heart biopsies taken from children with acute myocarditis [61-63] Clinical presentations of viral myocarditis vary from flu-like symptom and/or gastrointestinal illness to cardiac dysfunction and congestive heart failure with a poor prognosis [4, 6]. Patients usually present with shortness of breath, chest pain, and ventricular arrhythmias. About one third of patients with myocarditis develop a chronic form of disease at times associated with viral persistence, progressing to end-stage DCM and heart failure [4, 6]. In recent years, the advanced molecular techniques, such as reverse-transciptional PCR (RT-PCR), in situ hybridization (ISH), biomarker assessment (troponin I and T), and noninvasive imaging techniques have been combined with traditional methods (serology) to enhance and increases certainty of the histology-based diagnosis of viral myocarditis [6, 29, 32]. Like for other myocarditis, treatment of viral myocarditis is mostly surpportive. For early onset of this disease, antiviral agents eliminating the invading virus may help control the disease progression. Currently, specific antiviral drugs for viral myocarditis are under development and evaluation. Antiviral drugs such as Isoxazoles (WIN54954) and Pleconaril (VP63843) are viral capsid function inhibitors that block enterovirus attachment and entry, and have been shown to have some protective effects against enterovirus infection [64-68]. Several clinical trails using Pleconaril in children and 6 adults have reported a decrease in severity and duration of symptoms in patients with enteroviral meningitis and upper respiratory tract infections [69, 70]. Immunomodulatory therapy such as immunosuppression, interferon (IFN) treatment, and immunoglobulin administration has originally considered being beneficial to patients with viral myocarditis. However, administration of immunosuppressive agents such as prednisolone, FK-506, cyclophosphamide, or azathioprin in animal model or clinical trials has shown few success in ameliorating myocarditis [71, 72]. Targeted immunomodulation using interferon and immunoglobulin has been reported to have some benefits in treating myocarditis. Studies have shown that the interferon anti-viral system can significantly decreases viral replication and dissemination in vitro and in vivo. IFN-α was successfully used to treat a group of patients with acute enterovirus-induced myocarditis , and both of IFN-α and IFN-β have been shown to improve the prognosis for patients with viral myocarditis or DCM [74, 75]. High-dose of intravenous immunoglobulin therapy has been shown to be effective in managing myocarditis in animal models and achieved some improvement of cardiac function in several clinical trails [76-80] Its protective mechanisms may include direct inhibition of virus replication  and reduce production of pro-inflammatory tumor necrosis factor-α (TNF-α) coupled with increased anti-inflammatory interleukins . Despite the great efforts in the last two decades to develop a therapeutic means for viral myocarditis, there is currently no vaccine against coxsackievirus infections and no effective treatment for viral myocarditis and DCM. Therefore, new preventive or therapeutic treatments are greatly needed. 7 1.3 Coxsackievirus Coxsackievirus is originally found as a filterable infectious agent associated with a small outbreak of paralytic disease in children in Coxsackie, New York, and was initially isolated from new born mice in early 1948 . There are two subgroups, A and B, with 23 known Coxsackie A serotypes causing mainly enteric diseases, and 6 Coxsackie B (CVB) serotypes associated with severe diseases in the heart, pancreas, and central nervous system . For CVB, by definition, immunity against 1 serotype does not confer immunity against any of the other 5 serotypes. Among the CVBs, CVB3 has been considered as one of the primary causative agents of viral myocarditis. In recent years, the development of experimental in vivo and in vitro model of CVB3 infection has provides scientist with the opportunity to explore various genetic, cellular, and molecular aspects of CVB3 pathogenesis. To gain a better understanding of the pathology of CVB3 infection, it is crucial to comprehend important characteristics of viral structure and life cycle. 1.3.1 CVB3 genome CVB3 is a nonenveloped, cytolytic single-stranded RNA virus, belonging to the family of Picornaviridae. The mature virions of CVB3 are approximately 30nm in diameter with hexagonal external structure and the interior of the capsid shell is packed with the viral RNA. The CVB3 genome is a positive sense RNA with ~7400 nucleotides in length. Similar to other enteroviruses, the genome contains a single open reading frame flanked by the 5’ and 3’ untranslated region (UTRs). Instead of a cap structure 7-methyl guanosine triphosphate group, the 5’ end is linked covalently to the viral protein, Vpg 8 (3B). The 5’UTR contains 741 nucleotides representing 10% of the viral genome and forms a highly ordered secondary structure, functioning in both viral protein translation and viral genome replication. The highly structured internal ribosome entry site (IRES) in the 5’UTR directs viral translation initiation. The 5’UTR has been identified as a major determinant of the cardiovirulence phenotype from two clinical CVB3 isolates. The 3’ UTR contains three stem-loops followed by a poly(A) tail sequence which has been suggested to participate in the regulation of viral replication through interactions with cellular proteins [84, 85]. 1.3.2 CVB3 life cycle and encoded viral proteins A rapid replication cycle of virus takes place in the cytoplasm following the viral attachment, uncoating and release of viral RNA into cytoplasm. The CVB3 genome encodes eleven proteins within a polyprotein of 2,185 amino acids, including four capsid proteins VP1-VP4 and seven nonstructural proteins (Figure 2). Viral genomic RNA can serve directly as the mRNA template for translation of a single large polyprotein of about 250 kDa via a cap-independent mechanism utilizing the IRES at the 5’ end. The newly synthesized polyprotein is subsequently cleaved into three primary precursor molecules P1, P2 and P3 by virus-encoded proteases 2A and 3C. The P1 portion will be further cleaved by 3C to generate the structure capsid proteins VP1, VP2, VP3, and VP4, while the P2 and P3 segments will produce non-structural viral proteases and polymerases (2A, 2B, 2C, 3A, 3B/VPg, 3C, 3D)[86-89] (Figure 3). 9 Figure 2. CVB3 genome and viral proteins. CVB3 RNA encompasses a single and large open-reading frame flanked on both 3’ and 5’ termini by untranslated regions (UTR). Upon entry, the RNA encodes four structural proteins (capsid proteins) VP1-VP4 and seven non-structural proteins. VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D5’UTR 3’UTR Structural protein Non-structural proteins Protease RNA-dependent RNA polymerase CVB3 genome and viral proteins Capsid 10 Figure 3. Proteolytic cleavage of CVB3 polyprotein. The viral polyprotein is cleaved into three precursors, P1, P2, and P3 by 2A and 3C proteases. Autocatalytic cleavage by 2A of the polyprotein at the P1-P2 junction separates the structural proteins from the non- structural proteins. Subsequently 3C cleavages at multiple sites to release the rest of the capsid/proteases proteins. Polyprotein Precusor polyproteins Individual proteins 2Apro 3C pro 3C pro P1 P2 P3 VP1VP2 VP3VP4 2A 2B 2C 3A VPg 3C 3D 11 Viral protein 2A has various functions. It cleaves viral polyprotein at VP1-2A junction [90, 91], stimulates translation initiation on the cognate viral IRES [92, 93], and cleaves a number of host cell proteins including the 220 KDa component of the eukaryotic initiation factor-4G (eIF4G) and the poly(A)-binding protein (PABP), two crucial components of host cell mRNA translation and protein synthesis machinery [94- 96]. 2A further contributes to virus-induced myocardial injury through cleavage and disruption of cytoskeletal proteins dystrophin and dystrophin-associated glycoproteins α- sarcoglycan and β-dystroglycan in both in vitro and in vivo models [97-99]. In endomyocardial biopsy specimens, it was found that the presence of focal dystrophin and β-dystroglycan disruption in human myocarditis . Viral protease 3C cleaves and inactivates cAMP response element binding protein (CREB/ATF), octamer-binding transcription factor (Oct-1), cellular TATA-binding protein, and the histone protein H3, leading to profound shutdown of host cell transcriptional and translational machinery [101-104]. Following the early translational event, viral 3D, a RNA-dependent RNA polymerase, uses the positive sense RNA as a template to construct negative strand RNA intermediates that are subsequently used for synthesis of multiple copies of positive RNA strands . 3D initiates RNA synthesis by generating the protein primer VPg-pU-pU (uridylylation), which is required for the initiation of RNA synthesis at the 3’ poly (A) region of viral genome [106-108]. A single negative RNA can produce several copies of positive strands. Hence, at any given time, the ratio of positive to negative RNA strands is between 30:1 and 50:1 [109, 110]. The importance of viral protein 3D in CVB3 infection and replication is also recognized recently. Two siRNAs directed against the 3D 12 RNA-dependent RNA polymerase were found to inhibit virus propagation by 80-90%. The protective effect of the efficient siRNAs lasted for several days . The positive RNA are then packed and eventually released as progeny viruses to initiate new rounds of infection. The actual mechanism underlying virus release is not well understood. However, there is some evidence demonstrating that viral protein 2B increases the permeability of the plasma membrane and facilitates the release of progeny viruses [112, 113]. In addition, polioviruses carrying a 2B mutation show defects in RNA amplification and viral release in cell culture . Table 1 summarizes the role of individual CVB3-encoded viral proteins during the replicatin process. 1.3.3 CVB3 receptors An early and important biological stage of the virus infection cycle is host cell receptor attachment. To initiate a productive infectious cycle, CVB3 binds to the decay- accelerating factor (DAF) as a primary attachment protein (co-receptor) and the coxsackievirus and adenovirus receptor (CAR) as an internalization receptor. DAF (CD55), a 70 kDa membrane glycosyl-phosphatidylinositol (GPI)-anchored protein, is widely expressed in human tissues; and normally protects cells from complement- mediated lysis by preventing the formation of C3 convertase . DAF contains four contiguous short consensus repeats linked to GPI-anchored C-terminal domain. It is suggested that binding to DAF protein facilitates the access of virus particles to CAR without causing any conformational changes in viral capsid proteins [116, 117]. Through the GPI anchor, DAF also interacts with the Src-family protein tyrosine kinase, p56lck, 13 Table 1. Function of CVB3 encoded viral proteins Viral Protein Proposed Function VPg (3B) Priming RNA synthesis VP1-VP4 Viral capsid proteins 2A Host protein shutoff, cleavage of cellular dystrophin 2B Increased plasma membrane permeability and viral release, inhibition of cellular secretion 3A Inhibition of cellular secretion 3C Cleavage of cellular transcription factors 3D RNA-dependent RNA polymerization, VPg uridylylation 14 which reportedly plays a critical role in CVB3 infection in T lymphocytes and virus- induced myocardial injury [118-120]. CAR protein (46 kDa) was cloned and characterized by three independent groups using immuno-affinity purification , SDS-electrophoresis with 35S-labeled CVB3 , and expression cloning techniques . CAR is a trans-membrane component associated with tight junctions in human epithelial cells; and has been known to play a role as an adhesion molecule involved in neuro-network formation in developing mouse brain [124-126]. CAR protein comprises an extracellular domain composed of two immunoglobulin-like motifs D1 and D2, a trans-membrane helical domain, and a highly conserved cytoplasmic tail. The cytoplasmic domain contains a potential tyrosine phosphorylation as well as a palmitylation domain . The cytoplasmic and trans- membrane domains of CAR are not required for virus binding, while the extracellular domain is necessary and sufficient for a productive coxsackievirus and adenovirus infection . Recently, it is reported that CVB3 enters HeLa cell via CAR-mediated internalization and clathrin-dependent endocytosis, and that intracellular trafficking of CVB3 is highly dependent on intact endosomal function . It has been found that CAR mRNA expresses in the heart, brain, pancreas, prostate, and testis, with lower expression level in liver, lung, and intestine, which is consistent with the pattern of tissue susceptibility and clinical presentations in CVB3 myocarditis [123, 130]. In addition, a significant decrease of CAR expression has been reported in mouse heart and brain with increasing age, indicating an age-related pattern of tissue tropism and host susceptibility . 15 1.4 Interaction between CVB3 infection and the host cells After CVB3 infection, there are interactions between viruses and host cells. The interactions are dynamic, multifaceted and temporal in nature [99, 120, 131-135]. It is commonly accepted that the balance of host anti-viral responses and virus-mediated pro- viral mechanisms determines the outcome of infection . Thus a comprehensive analysis of them may provide us better understanding of the pathogenesis of CVB3- induced myocarditis. 1.4.1 Gene profile after CVB3 infection Because gene is involved in metabolism, cell cycle, cell defense and chemokine expression, as well as in an immediate early response, gene profile study may provide us an answer on how to comprehensively and integratively control the interaction between microbial pathogens and their hosts. The understanding of the pathophysiology and mechanisms contributing to the progression of human viral myocarditis is based on the studies in experimental murine models. Because of various characteristics such as the genetic similarity to human, easy and cost-efficient handling/breeding, availability of transgenic strains, and sensitivity to cardiotropic viruses, the mouse has become an excellent and well-defined model for CVB3-induced myocarditis. In suckling and weanling mice, CVB3 replication happens in the heart, pancreas, spleen, and brain causing clinical symptoms that resemble human diseases [137-142]. In murine model, the host genetic elements that are responsible for the changes observed in the different time points of post-infection have been determined . In this study, cDNA microarray was utilized to profile multiple genes’ expression 16 after CVB3 infection, instead of traditional molecular approaches that only monitor one gene at a time. 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. Among those genes, 85 known gene sequences found to be differentially expressed in the context of their expression trends at each of three postinfection time points. For example, poly (A) binding protein (PABP) gene was found to be upregulated in this study, and seemingly compensating viral degradation of the PABP, which is cleaved by CVB3 viral protease 2A . While the gene profile in the CVB3-infected mouse heart provides us with valuable insight into global transcriptional alterations within the heart, gene profile in vitro model of CVB3 infection would dissect out the transcriptional changes in CVB3-infected host cells in the absence of immunological processes such as inflammatory infiltration and fibrosis. The in vitro model of CVB3 infection aimed for gene profiling was successfully established and showed a subset of genes related to cytokine induction and stress signaling responses were significantly upregulated following virus infection in HeLa cells. 1.4.2 CVB3-induced myocarditis: a tri-phasic disease CVB3 infection can cause the myocardial injury. Evidences suggest that both direct virus-mediated injury and subsequent inflammatory immune response contribute to the damage of cardiomyocytes [144-146]. Histological analysis of infected murine hearts showed that death and destruction of cardiomyocytes in early viral infection are common before the infiltration of immune cells [144, 146, 147]. However, the host inflammatory 17 response, as well as autoimmune response, may further cause myocardial injuries [145, 148]. CVB3-induced myocarditis is a complicated disease because it progresses through stages with distinctly mechanisms and manifectations. Recent studies of available data have suggested that the disease can be characterized in the three distinct stages: (1) acute viral infection (2) inflammatory cell infiltration and (3) myocardial remodeling ( Figure 4 ). In acute viremic stage, early cardiomyocyte damage occurs. The damage is usually associated with prominent viral replication in the absence of significant host immune responses. It has been demonstrated that virus can directly injure the infected cardiomyocytes and contribute significantly to the pathogenesis of viral myocarditis [43, 136], which is different to pancreatitis induced by CVB4 infection, in which the tissure damage of pancreas is considered to be caused by inflammatory response rather than direct viral infection . In immune competent mice model (such as CD-1 mice) infected with CVB3, there were cytopathic lesions in ventricular myocytes, starting in the cell cytoplasm featuring by single or multicell vacuolar changes early on 2 days postinfection. By day 3, 4, and 5 postinfection, mice had increasingly numerous and widespread islands of myocytes with cytopathic changes and accompanying areas of contraction band and coagulation necrosis, and early, punctuate single-cell calcification. By day 6, 7, and 8 postinfection, the extent of necrosis and cytopathic injury was further advanced [144, 146]. A co-localization of viral genome with damaged cardiomyocytes has been shown by In situ hybridization [136, 147]. Recently, viral proteases are considered as an important pathogenic mechanism. Cardiac-restricted overexpression of 18 Figure 4. Pathogenesis of viral myocarditis: a tri-phasic disease. It is generally accepted that viral myocarditis is a triphasic disease that occurs in three distinct stages: acute viral infection, inflammatory cell infiltration, and myocardial remodeling. This figure is modified from  Acute Myocarditis Subacute Myocarditis Chronic Myocarditis 0 4 days 14 days 90 days Viremia Absence of Virus Viral Clearing • Early cardiomyocyte damage • Viral replication • No visible host immune responses, but early antibody responses • Inflammatory cell Infiltration • Further damage to myocardium • NK, macrophages TNF-α,IL-1,IL-2, IFN-α • T-lymphocytes, B-cells • Cardiac remodelling Fibrotic reparation Cardiac dilatation Three Stages of CVB3 Viral Myocarditis 19 enteroviral 2A can induce cardiomyopathy, probably through the cleavage of dystrophin, causing the detachment of the cardiomyocyte cytoskeleton from the external basement membrane and subsequently disrupt myocyteintegrity which not only reduces myocyte contractility, but also induces cell death [99, 136, 150]. The direct injury induced by CVB3 infection has also been observed in cultured cells. Viral infection was shown to induce a direct cytopathic effect and cell apoptosis in HeLa cells . The stage of inflammatory cell infiltration is characterized by inflammatory cell infiltration that results in further damage to the myocardium. After viral infection, the initial host immune reaction is first evoked by nature killer cells and macrophages, causing inflammatory cell infiltration of myocardium and profound cytokine production, including tumor necrosis factor-α, interleukin-1, interleukin-2, and IFN-γ, [43, 136]. These nature killer cells and macrophages aid in clearance of the virus in infected cells and mediate cytolysis of infected cells. The secondary immune response is executed by the antigen-specific T-lymphocytes and antibody-producing B-cells . The T- lymphocytes are activated to produce CD8+ or cytotoxic lymphocytes (CTL) and CD4+ helper cells willing to eliminate infected cells and produce anti-virus antibodies respectively. The host immune response plays a critical role in host defense mechanism by eliminating viral particles and infected cardiomyocytes, however, persistent immune response by ongoing production of cytokines and chemokines such as TNF-α, IL-1β, IFN-γ, and IL-6, can cause further damage to the heart. The first evidence of such inappropriate and destructive responses was provided by Woodruff et al. , who demonstrated that depletion of T lymphocytes using irradiation or anti-thymocyte serum significantly decreased immune infiltrates and mortality rate in a CVB3 experimental 20 model. In another study, the absence of both CD4+ and CD8+ T-cell subpopulation in A/J mice has been shown to markedly reduce inflammatory infiltrates and mortality . In addition, autoimmune response, which is elicited by exposure to cardiac- antigens released from damaged cardiomyocytes, such as cardiac myosin and troponin-I that are usually secluded from the reactive immune system, may also induce the further damage of myocardium [136, 153]. Early evidence for such deleterious effect was provided by the detection of auto-antibodies against cardiac myosin heavy chain, in the sera of patients with myocarditis and DCM . There is also evidence to support the hypothesis of “antigenic mimicry” in viral myocarditis . Antigenic mimicry between epitopes on cardiac muscle cells and virus particles may induce a strong immune response form sensitized B and T lymphocytes. Antibodies against cardiac α-myosin and the cardiomyocyte sarcolemma have been detected in the heart and sera of myocarditis patients, and have been shown to cross-react with Coxsackie B viruses . The idea of the involvement of the immune system and auto-immunity in viral myocarditis is a continuing paradigm. Unquestionably, the observation that a reactive immune system may be involved in the severity of viral myocarditis highlights the importance of the balance between the protective and deleterious effects of the immune response. Myocardial remodeling means the molecular and cellular restructuring of the myocardium and interstitium. During this stage, the heart processes fibrotic reparation and cardiac dilatation in absence or the presence of low-level persistent viral genomes. Due to the extensive myocyte loss, progressive replacement fibrosis occurs within the myocyte dropout regions, which is featured by abundant collagen accumulation. 21 Meanwhile, interstitial or reactive fibrosis extends to areas of normal viable tissue. The extensive fibrosis may lead to ineffective contraction and improper electrical signal conduction, while degradation of the interstitial collagen network can result in the loss of structural support and lead to wall thinning and left ventricle dilatation. Thus, reparative fibrosis and cellular alterations in this process may ultimately lead to DCM . The exact mechanism of this reparation and resolution process is still elusive. However, several factors, including the degree of injury or amount of remaining viable tissue, persistence of virus and inflammation, and the balance of matrix regulators, influence this remodeling process [158, 159]. 1.5 The ubiquitin/proteasome system (UPS) The ubiquitin/proteasome system (UPS), a major intracellular system for extralysosomal protein degradation, plays an important role in a wide variety of cellular functions, including antigen presentation, cell cycle regulation, apoptosis, signaling transduction, transcriptional regulation, and DNA repair [160, 161]. The significance of the discovery of UPS has been recognized by the award of 2004 Noble Prize in Chemistry to three distinct scientists. There are two successive steps involved in protein degradation by UPS: (1) ubiquitination, covalent attachment of ubiquitin to the target protein substrate, and (2) proteasome-mediated degradation, degradation of the ubiquitinated protein by the 26S proteasome with the release of recycled ubiquitin [160, 161] (Figure 5). 1.5.1 Ubiquitin and ubiquitination 22 Figure 5. Protein degradation by the UPS. (1) Covalent attachment of ubiquitin (Ub) to the target protein substrate through three enzymatic reactions of E1, E2 and E3. (2) 26S proteasome degradation of the Ub-tagged protein with release of recycled Ub by deubiquitinating enzyme (DUB). This figure is from . E1Ub + ATP AMP+PPi Ub E1 E1 E2 E2 Ub protein E3 E3 E2 protein Ub protein UbUb UbUbUbUb α β β α 19S 20S 19S DUBs p rotein Ub Ub Ub Ub Ub Ub R ec yc le d U b Peptides (2) (1) ATP ADP+Pi 23 Ubiquitin is a small protein composed of 76 amino acids and highly conserved during evolution. The best known function of ubiquitin in proteolysis is to serve as a tag on the target protein that is recognized and further degraded by the proteasome. The ubiquitination step involves three sequential enzymatic reactions. First, ubiquitin is activated in an ATP-dependent manner by the ubiquitin-activating enzyme (E1) to form a thiol ester bond between its C-terminal glycine residue and the active cysteine residue of E1. The activated ubiquitin is then transferred to an ubiquitin-conjugating enzyme (E2) through an additional thiol ester linkage. Finally, ubiquitin-protein ligase (E3) transfers the ubiquitin to the target protein by the formation of the covalent isopeptide bond between ubiquitin and the ε-amino group of lysine residues in the substrate protein. After several rounds of ubiquitination, multiple ubiquitin molecules are attached to the substrate. Once a polyubiquitin chain composed of at least four ubiquitins is formed, the substrate is quickly recognized and subsequently degraded by the 26S proteasome, and ubiquitin is recycled via the actions of deubiquitinating enzyme (DUBs) [160, 163, 164]. The protein degradation process is highly specific and regulated. The specificity of proteolysis appears to be achieved primarily at the step of ubiquitination. The structure of the ubiquitin conjugation system is hierarchical: there is only one E1 enzyme in humans, which activates ubiquitin for all conjugation reactions. Dozens of E2 enzymes have been identified, each of which interacts with one or several E3 enzymes. The number of known E3 ligases is growing rapidly. Each E3 targets one or a few substrates. Therefore, the substrate specificity of the ubiquitin conjugation system is conferred by the E3s [161, 163, 164]. E3s associate with both E2 and the target protein to facilitate the transfer of ubiquitin from E2 to the substrate or to the last ubiquitin of a polyubiquitin chain through 24 an isopeptide bond between the ε-amino groups of lysine residues on adjacent ubiquitin molecules. E3s can be divided into three groups according to the proteins with which they interact: the single-subunit really interesting new gene (RING)-finger subfamily (both substrate recognition site and RING domain reside in the same protein); the multi- subunit RING-finger subfamily (protein recognition and RING domain are found in different proteins), and the E6 associated protein carboxyl terminus (HECT)-domain subfamily E3s. The importance of E3 in viral replication has been demonstrated. It was reported that, in most cases, viruses modulate the ubiquitin/proteasome function at the levels of the ubiquitin-protein ligase, either by encoding an E3 functional domain or by redirecting a pre-existing host E3 ligase to a new target [165, 166]. 1.5.2 20S Proteasome The proteolysis of protein through the UPS is carried out by proteasome system, usually formed by 20S proteasome and its regulators, such as 19S and 11S regulators (also called as activators). The 20S catalytic core is a hollow, barrel-shaped cylinder composed of four stacked rings. The α-rings make up the two outer rings each consists of seven identical α-subunits, whereas the β-rings, each consisting of seven β-subunits, make up the two inner rings. The two outer rings, which are catalytically in active, modulate the entrance of protein substrates to the inner chamber. The proteolytic activity of the 20S proteasome locates in the two inter rings, which contain the proteolytic sites formed by N-terminal threonine residues facing the central chamber of the 20S complex. Three distinct proteolytic activities of the 20S proteasome have been reported: trypsin- like, chemotrypsin-like, and caspase-like activities. 25 The 20S proteasome is a large multicatalytic protease. In cells, it is usually latent and requires activation for its proteolytic function by binding of proteasome activators to the α-subunits, at either one end or both ends of core. Without proteasomal activators, protein substrates are barred from entering into the 20S, subsequently making the 20S latent. Tertiary structural analysis showed that in the absence of proteasomal activators, the α rings of the 20S are normally closed and blocked by peptides from the amino termini of the α ring subunits . However, when proteasomal activators bind to the α rings, the occlusion of the amino termini is removed and 13 Å pores become available for protein substrates to enter into the 20S proteasome . 1.5.3 Proteasome activators To date, at least two classes of proteasome activators have been identified to bind to the 20S proteasome and enhance its catalytic function : 19S and 11S proteasome activator (Figure 6). The 19S proteasomal activator (or PA700) is a well-studied proteasomal activator that binds to outer α-rings of the 20S, forming the 26S proteasome, which executes proteolysis of proteins in the ubiquitin- and ATP- dependent manner . Most of the cellular proteins are degraded through the 26S proteasome after ubiquitination. The 19S proteasome is involved in ubiquitin recognition, deubiquitination, substrate unwinding, and substrate translocation into the 20S catalytic core. Six different ATPase subunits are found in the 19S proteasome, and most likely produce ATP to unfold and deubiquitinate protein substrate before transferring them into the lumen of the 20S . 26 Figure 6. Proteasome activators and ubiquitin-dependent and –independent degradation The 20S proteasome is latent in cells and can be activated by either 19S activator to form 26 proteasome and perform ATP- and ubiquitin-dependent protein degradation, or 11S activator (REGγ) to form REGγ-20S- REGγ complex and involves in ATP- and ubiquitin-independent protein degradation. This figure is modified from . 20S proteasome α1 α2 α3 α4 α6 α7 α5 β1 β2 β3 β4 β7’ β6’ β5’ α7’ α6’ α5’ α4’ 19S activator α1 α2 α3 α4 α6 α7 α5 β1 β2 β3 β4 β7’ β6’ β5’ α7’ α6’ α5’ α4’ 26S proteasome (19S-20S-19S) 11S activator (REGγ) REGγ-20S-REGγ proteasome Ub-dependent pathway Ub-independent pathway α6 α7 α5 α1 α2 α3 α4 β1 β2 β3 β4 β7’ β6’ β5’ α7’ α6’ α5’ α4’ α6 α7 α5 Ub ATP protein β8’ β8’ β8’ 27 11S proteasome activator is also known as REG. It does not contain any ATPase activity and can mediate proteasomal degradation independent of ATP and ubiquitin. Thus 11S proteasome activator plays an important role in the ubiquitin- and ATP- independent protein degradation process. There REG family members have been found so far. Among them, REGα and REGβ share approximately 50% amino acid indentity, while REGγ shares only about 25% amino acid identity with REGα and REGβ . Although REGα and REGβ are primarily found in the cytosol and together form heteroheptamer caps, REGγ mainly locates in the nucleus and forms homoheptemer caps. REGα and REGβ can be induced by interferon-γ and play an important role in MHC class I antigen presentation [174, 175]. REGγ is not responsive to IFN-γ and does not appear to be heavily involved in the immune system. For example, mice deficient in REGγ do not show significant abnormalities in their immune system . REGγ has been shown to increase the proteasome activity and alter the cleavage pattern and substrate-specificity of the proteasome . REGγ was originally thought to degrade short peptide only . However, recent evidence demonstrates that intact intracellular proteins can also be targets of REGγ [179- 181]. But how intact proteins are unfolded and translocated into the 20S proteasome in an ATP-independent manner is poorly understood. The biological functions of REGγ have not been fully characterized. Recent studies support a role of REGγ in the regulation of apoptosis, cell cycle progression, and viral pathogenesis . Although the identified intracellular protein substrates for REGγ is limited to steroid receptor coactivator-3 (SRC-3) , cyclin-dependent kinase inhibitors p21, p16 and p19 [179, 181], as well as the tumor suppressor p53 , so far, 28 the importance of REGγ-mediated ATP- and ubiquitin-independent protein degradation in the fundamental cellular process has been recognized. 1.5.4 Deubiquitination Recent studies strongly suggest that protein ubiquitination is controlled by both specific processes of ubiquitination and deubiquitination. Deubiquitinating activity was first found to cleave ubiquitin from histone H2A in 1981 . After that, more than 90 deubiquitinating enzymes (DUBs) have been identified [184, 185]. DUBs hydrolyze the isopeptide bonds between two adjacent ubiquitins or between ubiquitin and the substrate protein. Base on their structure and function, DUBs can be classified into at least four distinct families. Ubiquitin-processing proteases (UBP) and ubiquitin carboxyl-terminal hydrolases (UCH) are two well-characterized classes [184-186]. The Jab1/MPN domain- associated metalloisopeptidase (JAMM) group of hydrolases [187, 188] and a family of cysteine proteases that contains an ovarian tumor domain (OTU) [189, 190] are recently described. The function of these enzymes is involved in processing ubiquitin gene products, negatively regulating the process of ubiquitination, and recycling free ubiquitin after protein recognition by the proteasome . Recent studies also suggest a key role of the DUBs in the regulation of numerous pivotal cellular functions, such as cell growth, cell differentiation, endocytosis, oncogenesis, DNA replication, and gene silencing [184, 185]. 29 1.5.5 Functions of the UPS In addition to the recycling of damaged, misfolded, or un-needed proteins, accumulated evidence suggests that the UPS is responsible for the modulation of many critical regulatory proteins which control a wide variety of cellular functions, including cell cycle regulation, apoptosis, antigen processing, signal transduction, transcriptional regulation, DNA repair, and receptor regulation . For example, several cell cycle proteins, including cyclins, cyclin-dependent kinase inhibitors (p21, p27), and tumor suppressors (p53) are all substrates of the UPS . The NFκB signaling pathway is also a critical target for this system . The NFκB p50/p65 heterodimer is retained inactive in the cytoplasm through binding to a specific inhibitor protein, IκB. Upon activation, IκB is rapidly and sequentially phosphorylated, ubiquitinated, and degraded by the proteasome. NFκB is released and translocated to the nucleus to participate in transcriptional regulation of multiple genes involved in inflammation. Increasing data have suggested that abnormalities of the ubiquitin/proteasome system contribute to the pathogenesis of many human diseases, including cancer, inflammation, cardiovascular, and neurodegenerative diseases [193, 194]. Proteasome inhibition has now been recognized as a promising therapeutic option in the treatment of several diseases [195- 197]. Apart from the best known role of polyubiquitination in protein degradation, ubiquitination has been reported to be involved in regulating protein functions. Monoubiquitination of some proteins, such as histones, calmodulin, actin, and some transmembrane proteins, serves as a signal for endocytosis and histone-mediated transcriptional regulation, without targeting for degradation [198, 199]. Finley and co- 30 workers  showed that covalent attachment of ubiquitin to ribosomal proteins is required for efficient ribosome biogenesis. In addition, ubiquitin has been suggested to be a key regulator of eukaryotic messenger RNA synthesis by regulating RNA polymerase II and related transcription factors . Cytosolic protein degradation proceeds mainly via 26S proteasome, which degrades ubiquitinated proteins in an ATP-dependent manner. As a successive processing of relatively small products of 26S proteasome, tripeptidyl peptidase II (TPPII) is also involved in protein degradation. TPPII is an aminopeptidase of the subtilision-type of serine proteases, which cleaves tripeptides from proteasome-generated peptides . In recent years, TPPII has attracted particular attention because of adaptive response of the TPPII proteolytic pathway in the presence of proteasome inhibitors and its compensatory function to impaired proteasome in mammalian cells . Inhibition of proteasome activity provides a potential therapeutic option in the treatment of several diseases through the prevention of cell proliferation and inflammatory response and the promotion of apoptosis . Of the most promise is the application of proteasome inhibitors in cancer therapy. Indeed, proteasome inhibitor bortezomib has been approved by FDA for the treatment of multiple myeloma . In addition to cancer therapy, inhibition of proteasome activity may also represent a novel approach to the treatment of cardiovascular diseases. In animal myocardial ischemia- reperfusion injury model, treatment with proteasome inhibitors reduces inflammatory infiltration, decreases myocardial infarct size, and maintains heart function . It was also found that proteasome inhibition can repress allograft rejection in mice without apparent side effects at effective doses . 31 In contrast to proteasome inhibition, stimulating proteasome function may also be a potential therapeutic means for diseases induced by damaged proteasome function, such as neurodegenerative diseases, in which the aggregation of misfolded proteins is the cause of the disease. Enhanced UPS function by proteasome stimulating may provide a strategy to prevent the accumulation of misfolded proteins in such diseases . Protein degradation inadequacy has also been suggested to play a role in human congestive heart failure. Recently, enhancement of proteasome function by REGα overexpression has been reported to protect against oxidative stress. Although REGα and REGβ are primarily involved in antigen processing, overexpression of REGα is sufficient to up-regulate 11S proteasome, enhance proteasome-mediated removal of misfolded and oxidized proteins, and protect against oxidative stress in cardiomyocytes. . 1.6 The UPS and viral infection Since the first discovery that human papillomavirus E6 protein targets the cellular tumor suppressor protein p53 for the ubiquitin/proteasome-mediated degradation , studies from many research groups, including from our laboratory, have showed that various viruses have evolved sophisticated mechanisms to utilize or manipulate the host UPS for their own needs (Table 2). For example, the UPS has been suggested to be required for avoidance of host immune surveillance, for viral maturation and viral progeny release, for efficient viral replication and for reactivation of virus from latency. Table 2 summarizes the interaction between viruses and the UPS. Although most proteasomal substrates must be ubiquitinated before being degraded, there are some exceptions to this general rule, especially when the proteasome plays a 32 normal role in the post-translational processing of the protein. Recently, the ubiquitin-and ATP-independent protein degradation has been demonstrated. Li et al  revealed that the proteasomal activator REGγ directs degradation of the steroid receptor coactivator SRC-3 by the 20S proteasome in an ATP- and ubiquitin-independent manner. Importantly, this form of protein degradation, as well as REGγ, has been reported to be involved in the viral replication process. Moriishi et al  showed that REGγ binds to and regulates the stability and nuclear retention of hepatitis C core protein, contributing to hepatitis C core protein-induced insulin resistance and hepatocarcinoma. This result suggests the potential role of REGγ and REGγ-mediated ubiquitin-independent protein degradation in viral infection and replication. 33 Table 2. Examples of viruses or viral proteins interacting with the UPS Note: HPV, human papillomavirus; EBV, Epstein-Barr virus; HCMV, human cytomegalovirus; HTLV-1, human T-cell leukemia virus 1; SV, simian virus; HSV, herpes simplex virus; HIV, human immunodeficiency virus; RSV, Rous sarcoma virus; HHV, human herpesvirus; RSV, human respiratory syncytial virus. This table is from  p53 inactivation Apoptosis suppression Viral immune evasion Viral budding Viral transcriptional regulation Others HCM US2, US11 EBV EBNA1 HHV-8 K3, K5 (PHD-E3) MHV-68 MK3 (PHD-E3) Myxomavirus M153R HIV-1 Vpu Mumps virus V protein SV V protein MHC-1 EBNA MHC-1 B7, ICAM-1 MHC-1 CD4 CD4 STAT1, STAT2 STAT1 HIV-1, HIV-2 Gag RSV Gag - - HIV-1 Tat HTLV-1 Tax CVB3 unknown HPV E6 HTLV-1 E1B 55k E4orf6 HSV ICP0 - - Cyclin D1, p53, β-catenin p53 p53 p53 Virus Viral proteins Target proteins Actions of the UPP Functional effects References Degradation Degradation Degradation Degradation Degradation Degradation Degradation Degradation Monoubiquitination Monoubiquitination Degradation Degradation Deubiquitination Monoubiquitination Monoubiquitination Degradation Adenovirus L3 23K (DUBs) Coronavirus PLpro (DUBs) Deubiquitination Deubiquitination Unknown Unknown Shamu et al. 2001 Levitskaya et al. 1997 Coscoy et al. 2001 Boname and Steveson 2001 Mansouri et al. 2003 Schubert et al. 1998 Gotoh et al. 2002 Gotoh et al. 2002 Schubert et al. 2000 Strack et al. 2000 Bres et al. 2003 Peloponese et al. 2004 Luo et al. 2003b Yuan et al. 2005 Barry et al. 1998 Yew et al. 1992 Hagglund and Roizman 2004 Balakirev et al. 2002 Sulea et al. 2005 34 1.6.1 The UPS and viral immune evasion Viruses can persevere inside cells to cause latent or chronic infection of the host, even though the host has developed a sophisticated immune system for eliminating the virus-infected cells. Down-regulation of major histocompatibility complex (MHC) class I molecules to reduce antigen presentation becomes a common mechanism used by the virus to avoid immune surveillance . Viruses have developed different strategies to escape the host immune responses by inhibiting antigen presentation from MHC molecules. US2 and US11, encoded by human cytomegalovirus, induce dislocation of MHC class I from the endoplasmic reticulum to cytoplasm where MHC molecules are polyubiquitinated and rapidly degraded by the proteasome. The Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) contains Gly-Ala repeats that interfere with antigen processing and MHC class I – restricted responses by preventing viral protein degradation through proteasome [213, 214]. Viral proteins containing ubiquitin ligase RING-finger domain trigger the cytosolic or lysosomal degradation of MHC class I products. The PHD (plant homeodomain) motif, closely related to RING-finger domaim, is found in many viral proteins, including the human herpesvirus-8 proteins K3 (also known as modulator of immune recognition 1, MIR1) and K5 (MIR2) and the murine γ-herpesvirus-68 MK3 protein. Studies have implicated a critical role of these viral proteins in the destruction of MHC class I molecules [215, 216]. It was shown that K3 and K5 proteins, acting as E3 ligases, specifically target MHC class I at the host plasma membrane for ubiquitination leading to its endolysosomal degradation . MK3 protein also down-regulates the MHC class I molecules in a similar PHD domain-dependent manner .The PHD-containing viral 35 E3 ligases, particularly K5, also induce down-regulation of other cell surface molecules other than MHC class I, such as B7 and ICAM-1 which are important for the co- stimulation of T cells . It has been reported that M153R, another PHD domain protein of myxomavirus, induces degradation of CD4 in T cells  other than MHC class I . Interestingly, the endogenous cellular E3 ligases can also be used by viral protein to induce CD4 down-regulation. Human immunodeficiency virus type-1 (HIV-1) expresses the Vpu membrane protein, which induces the down-regulation of CD4 in transfected cells. This down-regulation depends on an intact ubiquitin conjugation system and C-terminal lysine residues in CD4 . Another mechanism of viral evasion in host cells is to interfere with interferon signaling, which plays a central role in host defense against infected virus. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is required for the expression of interferon response genes . V protein of mumps virus and simian virus stimulates proteasomal degradation of the cellular STAT protein to inhibit JAK/STAT signaling pathway, escaping interferon-initiated antiviral responses. 1.6.2 The UPS and viral progeny release and (or) budding The process of viral membrane envelopment is called budding, which is a critical step in the virus lifecycle. It has been reported that the cellular ubiquitination machinery is required for viral budding of some enveloped RNA viruses, such as retroviruses. Studies by three independent groups have shown that proteasome inhibition interferes with the processing of viral Gag polyproteins and reduces viral progeny release and viral 36 infectivity. They further demonstrated that proteasome inhibition reduces the level of free ubiquitin in retrovirus infected cells and prevents monoubiquitination of p6Gag [224-226]. The Gag protein has been considered to be the essential component of virus for viral budding. The central parts of Gag are assembly domains which include a small sequence called the late domain (L domain). This L domain carries a proline-rich motif PPXY, which can interact with the host ubiquitinating enzymes [198, 227]. The WW-domain HECT E3 ligases such as Nedd4 or Nedd4 like proteins have been recently reported to interact with PPXY motif of L-domain and these functional E3 ligases are required for viral budding [228, 229]. In addition to Nedd4 protein, Tsg101, tumor susceptibility gene 101, is another binding partner of the Gag protein. Tsg101 is a protein normally involved in vacuolar protein sorting and multivesicular body biogenesis by binding to monoubiquitinated proteins . Recent studies on various viruses have suggested an important role of Tsg101 in viral budding and release. Depletion of Tsg101 by using small interfering RNAs significantly reduces viral budding from infected cells . Overexpression of the N-terminal UEV domain of Tsg101 inhibits viral progeny release . 1.6.3 The UPS and transcriptional regulation of virus The association between the protein degradation and viral transcriptional regulation has been implicated in recent studies. For example, Zhu et al.  show that the ubiquitin-mediated proteolysis is required for the transcriptional activation of synthetic herpes simplex viral VP16 transcription activators. Blockage of VP16 degradation severely impairs their transcriptional activities. Furthermore, overexpression of a subunit 37 of the proteasome reduces both the degradation and transcription activation of VP16. However, increasing evidence suggests that ubiquitination process may directly regulate viral transcriptional activities, independent of proteasome-mediated degradation [201, 234]. The HIV-1 encodes a potent transactivator, Tat, which activates the HIV-1 long terminal repeat by adapting co-activators complexes to the promoter. A recent study demonstrated that ubiquitination of Tat protein positively regulates its transcriptional properties without targeting it for proteasomal degradation . Another interesting observation was made in the research of human T-cell leukemia virus type 1. This human retrovirus encodes a 40 kDa Tax transcriptional activator which modulates expression of the viral long terminal repeat and transcription of many cellular genes. A recent report revealed that Tax is predominantly monoubiquitinated and its transcriptional function is downregulated through a proteasome-independent mechanism in mammalian cells . 1.6.4 The UPS and apoptosis suppression Apoptosis or cell death at late viral infection may facilitate virus progeny release. However, premature cell death will decrease the ability of host cells to foster virus replication. Thus, various viruses have evolved different strategies to suppress or delay apoptosis during early viral infection to provide sufficient time for the production of high yields of progeny viruses. The tumor suppressor protein p53 is an important transcription factor which plays a key role in the growth control by modulating processes leading to apoptosis and to DNA replication . Additionally, p53 has been reported to interfere with the replication of several viruses, such as human immunodeficiency virus type-1, 38 simian virus 40, hepatitis B virus and herpesvirus [238-240]. Thus, p53 appears to be a common barrier to the replication of many different viruses. The human papillomavirus E6 protein interacts with the cellular ubiquitin ligase E6-associated protein to form a complex, targeting p53 for polyubiquitination and degradation by the proteasome . In adenovirus-infected cells, the level of p53 is markedly reduced. Two adenovirus gene products, E1B 55K and E4orf6, have been shown to regulate the function of p53 by directing it for degradation. In the absence of either of these proteins, a dramatic increase in cellular p53 levels is observed, suggesting the assembly of E1B and E4orf6 complex is required for E1B 55K/E4orf6-dependent p53 ubiquitination and proteasomal degradation . Herpes simplex virus infected cell protein 0 has also been shown to inactivate p53 by a mechanism described below. Recently, we have shown that expression of p53 was markedly reduced following coxsackievirus infection and this reduction is abrogated when specific inhibitors of the UPS are used , suggesting that coxsackievirus may have developed certain mechanisms, for example the UPS, to inhibit apoptosis for viral replication by inactivating the p53 pathway. 1.6.5 Deubiquitination in viral infection Although the detailed mechanisms remain to be elucidated, the importance of the deubiquitinating enzymes in the regulation of protein degradation and several fundamental cellular functions has now been appreciated. Recent studies suggest that the deubiquitination process, similar to its counterpart ubiquitination, can be used or modified by viruses to support their infection. The number of known viruses directly 39 targeting on the deubiquitination process is increasing rapidly. The herpesvirus- associated ubiquitin-specific protease (HAUSP, also known as ubiquitin-specific protease 7 (USP7)) can suppress cell growth by removing ubiquitin from polyubiquitinated p53 to prevent it from degradation . Herpes simplex virus infected cell protein 0 binds HAUSP to inactivate p53 . Epstein-Barr virus nuclear antigen 1 is also reported to interact with HAUSP to regulate its transcriptional activities . Using a chemistry- based functional proteomics approach, it was shown that Epstein-Barr virus infection of B cells leads to the activation of a group of DUBs, which include UCH-L1, HAUSP/USP7, UCH-L5, USP15, and USP9X . Virus proteins can encode not only their own ubiquitinating enzymes but also deubiquitinating enzymes. Balakirev et al  have found that adenovirus infection is accompanied by an increased deubiquitinating activity in infected cells. They further provide evidence that adenovirus protease L3 23K, which is responsible for the cleavage of viral precursor polyproteins, may function as a deubiquitinating enzyme. By function motif search, a most recent publication also revealed that papain-like protease (PLpro), a viral cysteine protease of severe acute respiratory syndrome coronavirus, has a very similar structure to HAUSP, implicating that PLpro might have deubiquitinating activities in addition to their function in processing viral polyprotein . Evidence above reviewed the ways by which viruses interact with the host UPS. This interaction is endless, dynamic, and complicated. A better understanding of the interaction will provide us not only the insight look of the UPS in CVB3 replication and the pathogenesis of CVB3-induced myocarditis, but also the potential therapeutic strategy and drug development for viral myocarditis. 40 Chapter 2: Rationale, Hypothesis, and Specific Aims 2.1 Rationale CVB3, an enterovirus of the family Picornaviridae, is the common human pathogen that has been associated with the pathogenesis of myocarditis and idiopathic dilated cardiomyopathy. Myocarditis is a common heart disease with inflammatory injury of myocardium. Induced by virus infection, viral myocarditis is the major cause of sudden unexpected death in children and youths, and contributes significantly to the incidence of heart failure in North America. Pathology of viral myocarditis can be described as early myocyte injury or death associated with viral infection, with or without inflammation, and ultimate myocardial remodeling leading to ventricular dilation and heart failure. Numerous viruses have been associated with myocarditis and DCM, such as enteroviruses, adenoviruses, and so on. Among them, CVB3 is the most prevalent and extensively studied enteroviruses for viral myocarditis . At the onset and during the progression of viral myocarditis, there is a constant interplay between the virus and the host. Cardiomyocytes and the host immune system attempt to limit viral replication or to induce apoptosis to clear the invaded pathogen, whereas virus wants to inhibit anti-viral host mechanisms or even usurp the host intracellular machinery, such as the UPS, to facilitate viral replication and promote host cell survival . Indeed, destabilization of host intracellular proteins that perturb efficient virus infection is an important mechanism evolved by viruses to optimize progression through the viral lifecycle . 41 The UPS is the major protein degradation system in cells and it performs the rapid degradation of abnormal and short-lived regulatory proteins controlling a variety of fundamental cellular processes . Impairment of this system has been shown in the pathogenesis of various diseases, including cancer, inflammatory, neurodegenerative, and cardiovascular diseases [250, 251]. Recently, the functional significance of the UPS in the regulation of viral infectivity, inflammation and immunity, and viral pathogenicity has been increasingly recognized . Emerging evidence suggests that the replication of virus requires the function of host protein degradation systems, especially the UPS. Table 2 summarizes the viruses that utilize the UPS for their own replication in cells. As shown in Table 2, by different strategies, viruses can either induce the degradation of host anti-viral proteins or promote viral protein ubiquitination which is required for viral budding and transcriptional activity. Moreover, studies have suggested that degradation of excess viral proteins may be required by some viruses to achieve optimal replication efficiency . For better understanding the pathogenesis of CVB3-induced myocarditis, our laboratory performed studies and experiments to investigate the role of the UPS in CVB3 replication. Cell culture experiments using murine cardiomyocytes showed that inhibition of the UPS using inhibitor markedly decreases CVB3 viral RNA and protein levels, and inhibited CVB3 progeny release without direct inhibition of viral protease proteolytic activities . In addition, there is a reduction of expression level on several host proteins during CVB3 infection, including cyclin D1, p53, and β-catenin. This reduction is abrogated when specific proteasome inhibitors are used [132, 254, 255]. Interestingly, 42 pyrrolidine dithiocarbamate, an oxidant, potentially reduces CVB3 replication, likely through the inhibition of the UPS . Even though it has been demonstrated that proteasome inhibition decreases CVB3 replication in cells, the potential value of proteasome inhibition in the treatment of viral myocarditis in an animal model has not been tested. The details about the role of the UPS in CVB3 replication, in term of potential mechanism involved, also needs to be clarified further. 2.2 Overarching hypothesis The overarching hypothesis of this thesis study is that the ubiquitin/proteasome system plays a critical role in the pathogenesis of CVB3-induced myocarditis through promoting coxsackieviral replication and by regulating host protein degradation. 2.3 Specific aims Three specific aims are proposed to test the above hypothesis: 1) To determine the impact of proteasome inhibition in coxsackieviral myocarditis in mice; 2) To determine the interplay between ubiquitin-dependent proteasomal degradation and CVB3 infection; 3) To delineate the role of ubiquitin-independent proteasomal degradation in the regulation of CVB3 infection. 43 Chapter 3: Proteasome Inhibition Attenuates CVB3-induced Myocardial Damage in Mice In this chapter, I will introduce the rationale, experimental design, and present the results and discussion of the in vivo study for Aim 1. 3.1 Introduction Recent studies have revealed a pivotal role of the UPS in viral infectivity. As alluded to earlier , the UPS can be utilized or manipulated by various viruses, including CVB3, to achieve successful viral infection [84, 132, 162, 166, 253, 255, 256]. CVB3 infection facilitates ubiquitin-dependent proteolysis of cyclin D1 that is linked to CVB3- induced cell growth arrest , and stimulates glycogen synthesis kinase 3β’s activity, which contributes to virus-induced cytopathic effect and apoptosis through ubiquitin- mediated degradation of β-catenin . Importantly, proteasome inhibitor markedly reduces CVB3 replication through suppression of viral RNA transcription and protein synthesis . Moreover, pyrrolidine dithiocarbamate and curcumin can potently inhibit CVB3 replication, likely through selective inhibition of host protein degradation [255, 257]. Despite the importance of the UPS in the lifecycle of CVB3 replication is recognized, the expression and regulation of the UPS in viral myocarditis, and the direct role of proteasome dysregulation in viral myocarditis have not been determined. Thus, here, I propose to use in vivo mouse model to determine the impact of proteasome inhibition on coxsackieviral myocarditis. 44 3.2 Experimental Design In this study, a new proteasome inhibitor MLN353 (obtained from Millennium Pharmaceuticals) was first applied in the myocarditis-susceptible A/J mouse model. Comparing to the proteasome inhibitors we used in cell model (e.g.MG132 and lactacystin), MLN353 is more suitable for administration in vivo and relatively stable under physiological conditions. To verify our previous finding that proteasome inhibition blocks CVB3 replication, we first tested the effect of MLN353 on CVB3 protein expression in mouse cardiomyocytes. HL-1 cells were preincubated with various concentrations (0, 0.1, 0.5, 1, and 5 µM) of MLN353 for 30 min and then infected with CVB3 (MOI=100) for 1h. 7 hours post-infection, cell lysates were collected and immunoblotted with anti-VP1 and anti-β-actin antibodies. At 18 hours post-infection, medium was collected from CVB3- infected cells and virus titer was determined by plaque assay. To determine whether the UPS affects the pathogenesis of CVB3-induced myocarditis, we examined the effect of proteasome inhibition on viral replication, on host protein degradation and on virus-mediated myocardial damage in mice. MLN353 was applied to A/J mice, which were at age of 4-5 weeks and obtained from Jackson Laboratories. To choose a dose of MLN353 for the in vivo experiment, we performed a pilot toxicity study to test four selected doses (0.02 mg/kg, 0.06 mg/kg, 0.3 mg/kg, and 1 mg/kg) in non-infected mice. MLN353 was given at three difference times, which is at 1, 4, and 7 days. Body weight was recorded daily after first injection for 10 days. In this experiment, vehicle (PBS)-treated, age-matched control mice were used as control group. 45 In CVB3 infection study, a total of 60 male A/J mice at age 4-5 weeks were randomized to four groups: sham+vehicle (n=10), sham+MLN353 (n=10), virus+vehicle (n=20) and virus+MLN353 (n=20). Mice were either infected intraperitoneally with 105 plaque forming units (PFU) of CVB3 or sham infected with PBS. Virus- or sham-infected mice were administered the proteasome inhibitor MLN353 subcutaneously (0.02 mg/kg, once a day for 3 days, i.e. one day prior to virus infection, 3 and 6 days post-infection) or vehicle (PBS). Mice were sacrificed on day 9 post-infection and infected heart was harvested for further analysis, e.g. Western blot, plaque assay, immunostaining, histological grading, and proteasome activity assay. In Western blot, expression level of ubiquitin, as well as several key enzymes of the UPS such as E1, E2, E3 and DUBs were detected in mice heart, to examine the effect of CVB3 infection on the UPS. Meanwhile, immunohistochemical staining was performed to determine the location where the changes of expression on the UPS key enzymes occur. Proteasome activity assay was also performed using the synthetic fluorogenic substrate of proteasome, Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin, from Calbiochem. For testing the potential protective effect of proteasome inhibitor on viral infected mice, the survival rate of vehicle- and MLN353-treated mice at 9 days post-infection was calculated and analyzed by Kaplan-Meier plot. Then a plaque assay was performed for viral titers after CVB3 infection and MLN353 treatment. Finally, the histological examinations on the infected or/and MLN353-treatment mice heart tissue, including H&E staining and histological grading, were performed. Mice heart sections were graded blindly by an experienced pathologist for the severity of myocarditis based on the 46 myocardial lesion area, cellular vacuolization, calcification, necrosis, and inflammatory infiltration as previously described [258, 259], with the following scales: 0, no or questionable presence; 1, limited focal distribution; 2-3, intermediate severity; and 4-5, coalescent and extensive foci over the entirety of the transversely sectioned ventricular tissue. 3.3 Results CVB3 infection results in an increased accumulation of protein-ubiquitin conjugates in mouse heart Previously, we have shown that the UPS plays an important role in coxsackievirus infection using cultured cells . In the present study, I extend my interest using an in vivo animal model to further characterize the function of the UPS in CVB3-induced myocarditis. I first examined the protein ubiquitination after CVB3 infection of myocarditis-susceptible A/J mice and found that protein-ubiquitin conjugates were markedly increased at 9 days post-infection, comparing to the sham-infected control (Figure 7A). Effects of CVB3 infection on protein expression of key enzymes involved in the UPS It is commonly accepted that the molecular mechanism underlying regulation of the UPS can occur at two levels: (1) the protein ubiquitination which is regulated by E1, E2 and E3 ubiquitin enzymes as well as deubiquitinating enzymes; and (2) the proteasome-mediated protein degradation [161, 260]. To explore the underlying 47 mechanisms of the UPS dysregulation in the mouse heart after CVB3 infection, I examined the 20S proteasome activities in mouse heart at 9 days post-infection. The proteolytic activities of 20S proteasome were unchanged between CVB3-infected mouse heart and control heart (Figure 7B), which was consistent with our previous results using cultured cells [132, 253]. This observation suggests that the increased accumulation of ubiquitin conjugates is unlikely a result of decreased proteasome activity, prompting us to investigate whether there is difference in the process of protein ubiquitination. The protein expression of several key enzymes involved in ubiquitination, including ubiquitin-activating enzyme E1A/E1B and ubiquitin-conjugating enzyme UBCH7, were then examined. Protein levels of both E1A/E1B and UbCH7 were significantly increased in CVB3-infected hearts as compared to control hearts, suggesting that the upregulation of protein-ubiquitin conjugates was related to increased level of these key enzymes (Figure 8A). Immunohistochemical staining further demonstrated that increased expression of E1A/E1B was mainly localized to the vacuolated cells. Cellular vacuolization has been considered as a common feature of CVB3-induced myocarditic lesion (Figure 8B). These results indicate that CVB3 manipulates the UPS likely through upregulation of UPS-related enzymes. 48 64.2 181.8 115.5 82.2 MW (kDa) Sham CVB3 (9d pi) (Ub)n Figure 7. GAPDH 0 0.5 1 1.5 2 2.5 3 3.5 4 * Pr o te in - u bi qu iti n Co n jug at es (N o rm al iz ed to G A PD H ) A. B. 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 R el at iv e Fl u o re sc en ce In te n si ty Sham CVB3 (9d, pi) Sham CVB3 (9d, pi) NS 49 Figure 7. CVB3 infection leads to an accumulation of protein-ubiquitin conjugates in mouse heart. A/J mice were infected with CVB3 (105 PFU of Nancy stain) or PBS (sham infection). At 9 days post-infection mice were sacrificed and heart tissue was harvested. (A) Western blot was performed to detect the ubiquitinated proteins using an anti-ubiquitin antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed as a protein loading control. Protein levels of protein-ubiquitin conjugates (molecular weight starting from 82.2 kDa to approximately 230 kDa) were quantitated by densitometric analysis using NIH ImageJ program, and normalized to GAPDH expression. The data shown are mean ± standard error (SE) (sham group: n=3; CVB3 group: n=3) and significance was determined by Student’s t-test. *P<0.05 as compared to sham infection. (B) Heart homogenates were prepared and the proteasome activity was measured using the fluorogenic substrate SLLVY-AMC. Results are expressed as the amount of AMC formed by the enzymatic cleavage of substrate (mean ± SE of three independent measurements from each animal; sham group (n=8) and CVB3 group (n=9)). NS: no significant difference as compared to sham infection. 50 Figure 8. Ubc H7 E1A/E1B E6-AP GAPDH Sham CVB3 (9d pi) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 * * E1A/E1B Ubc H7 E6-AP R el at iv e Pr o te in Ex pr es si o n (N o rm al iz ed to G A PD H ) A. B. E1A/E1B Sham CVB3 NS 51 Figure 8. Expression of ubiquitinating enzymes is upregulated in CVB3-infected mouse heart. A/J mice were infected with CVB3 or sham-infected with PBS as described above. Nine days post-infection mouse heart was collected. (A) Western blot was performed using anti-E1A/E1B, anti-ubcH7, anti-E6-AP, and anti-GAPDH (loading control) antibodies, respectively. Levels of expression were quantitated by densitometric analysis using National Institutes of Health ImageJ 1.37 program, and normalized to GAPDH expression. The data shown are mean ± SE (sham group (n=7); CVB3 group (n=6)). *P < 0.05 or NS (no significant difference) as compared to sham infection. (B) Immunohistochemical staining for ubiquitin-activating enzyme E1A/E1B (red) was carried out as described in “Material and Methods”. The nuclei were counterstained with hematoxylin (blue). Scale bar = 50 µM. 52 I also examined the expression of p53-related ubiquitin-protein ligase E6-AP (human papillomavirus E6-associated protein). However, no noticeable differences between sham- and virus-infected hearts were observed (Figure 8A). Since E6-AP is only one of numerous E3 ligases in the eukaryotic cell, future investigation is needed to determine whether other E3 ubiquitin ligases were dysregulated during coxsackievirus infection. Protein ubiquitination can also be regulated by DUBs that specifically cleave ubiquitin from ubiquitin-conjugated protein substrates [184, 185]. I therefore examined the protein expression of ubiquitin C-terminal hydrolase L1 (UCHL1), one of the DUBs primarily localized in neurons but lately found to be significantly upregulated in cardiomyocytes of dilated cardiomyopathy . The expression level of deubiquitinating enzyme UCHL1 was significantly increased in virus-infected hearts (Figure 9). Together, these results suggest that CVB3 infection promote the process of protein ubiquitination through upregulating ubiquitin-activating enzyme and ubiquitin- conjugating enzyme and via increasing the expression of deubiquitinating enzyme. My studies also implicate that enhanced protein turn over as a consequence of increased protein ubiquitination may play an important role in the pathogenesis of viral myocarditis. 53 Figure 9. UCH L1 GAPDH Sham CVB3 (9d pi) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 * R el at iv e Pr o te in Ex pr es si o in (N o rm al iz ed to G A PD H ) Sham CVB3 (9d, pi) 54 Figure 9. Expression of deubiquitinating enzyme is increased in CVB3-infected mouse heart. A/J mice were infected and mouse heart was collected as described above. Anti-UCHL1 antibody was used for immunoblotting of deubiquitinating enzyme UCHL1. Protein expression was quantitated and analyzed as described in Figure 14. The data shown are mean ± SE (sham group (n=7); CVB3 group (n=6)). *P < 0.05 as compared to sham infection. 55 MLN353 inhibits CVB3 viral protein expression in murine cardiomyocytes Proteasome inhibitor MLN353, suitable for administration in vivo and relatively stable under physiological conditions, was obtained from Millennium Pharmaceuticals, Inc. To verify the previous finding that proteasome inhibition blocks CVB3 replication, I first tested the effect of MLN353 on CVB3 protein expression in mouse cardiomyocytes. As shown in Figure 10A, MLN353 inhibited CVB3 capsid protein VP1 expression in a dose-dependent manner. In addition, the titer of released virus in the supernatant was also reduced dose-dependently (Figure 10B). The results suggest that, like other proteasome inhibitors (MG132 and lactacystin), MLN353 also potently inhibits CVB3 replication in cardiomyocytes. Toxicity and survival rates after proteasome inhibitor treatment of CVB3-infected mouse To choose a dose of proteasome inhibitor for the in vivo experiment, a pilot toxicity study to test four selected doses of MLN353 (0.02 mg/kg, 0.06 mg/kg, 0.3 mg/kg, and 1 mg/kg) in non-infected mice was performed. MLN353 was given at three difference times as described. Body weight was recorded daily after first injection for 10 days. It was found that administration of MLN353 at the doses of 0.06 mg/kg, 0.3 mg/kg, or 1 mg/kg produced toxicity after either one, two or three injections, as judged by weight loss or lethality. However, as compared to vehicle-treated, age-matched control mice, the body weight of mice treated with 0.02 mg/kg of MLN353 was no significant difference. In both groups, mouse body weight was gradually increased (10.31% versus 9.23% 56 β-actin VP1 Sh a m 0 0.1 0.5 1 5 MLN353 (µM) CVB3 (7h pi) Figure 10. MLN353 (µM) 0.5 5 Vehicle H L- 1 Ce ll Vi ra l T itr e (10 8 pf u /m l) CVB3 (18h pi) 0 10 20 30 40 50 60 70 80 * * A. B. 57 Figure 10. Proteasome inhibitor MLN353 inhibits CVB3 replication in mouse cardiomyocytes. HL-1 cells were preincubated with various concentrations of MLN353 for 30 minutes and then infected with CVB3 (MOI = 100) for 1 hour. (A) Seven hours post-infection, cell lysates were collected and immunoblotted with anti-VP1 and anti-β-actin (as loading control) antibodies. The data are representative of three independent experiments. (B) Eighteen hours post-infection, medium was collected from CVB3-infected cells and virus titer was determined by plaque assay. The values are mean ± SE of three independent experiments. *P < 0.001 as compared to vehicle-treated cells. 58 increases at day 10 after vehicle or MLN353 treatment) and there was no mortality. Thus, the dose of 0.02 mg/kg was chosen for the subsequent mouse experiment. In CVB3 infection studies, it was further demonstrated that treatment with this dose of MLN353 significantly reduced the 20S proteasome activities in mouse heart (Figure 11B). The survival curves over the whole time period were presented in Figure 11A, showing that the survival rates were 50% for vehicle group and 75% for MLN353 group at 9 days post-infection, respectively. There was no statistical difference between two groups (P>0.05). MLN353 protects CVB3 induced murine myocarditis Adolescent A/J mice were infected with CVB3 in the presence or absence of MLN353. Nine days after viral inoculation, mice were sacrificed and heart tissues were harvested. To determine whether in vivo application of MLN353 can reduce CVB3 replication in heart, plaque assay for viral titers after CVB3 infection and MLN353 treatment was performed. As showed in Figure 12, the reduction of CVB3 viral titer after MLN353 treatment is not significant comparing to the vehicle control. Finally, the histological changes after MLN353 treatment were examined. H&E staining (Figure 13 upper panel) showed that the extent of myocarditis, especially inflammation, induced by CVB3 infection was significantly decreased by the treatment of MLN353, comparing to the vehicle controls. The corresponding histological grades of the extent of myocarditis were shown in Figure 13 lower panel. These results strongly suggest that MLN353 protects myocarditis induced by CVB3 in mice. This is the first in vivo evidence demonstrating the effect of the proteasome inhibitor in CVB3 induced myocarditis. 59 Figure 11. 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Vehicle MLN353 CVB3 (9 d pi) * R el at iv e Fl u o re sc en ce In te n si ty A. B. Su rv iv al (% ) Days after CVB3 infection 1 2 3 4 5 6 7 8 9 Vehicle (11/20) MLN353 (15/20) 0 20 40 60 80 100 120 60 Figure 11. MLN353 treatment reduces proteasome activity in mouse heart. (A) Kaplan-Meier plot of survival curves of vehicle- and MLN353-treated mice 9 days after CVB3 infection. The animal numbers shown at 9 days post-infection are the numbers of surviving mice over the numbers of total experimental mice. P > 0.05 as compared to vehicle-treated mice by the log-rank test. (B) A/J mice were sham infected with PBS or CVB3 infected in the treatment of vehicle or MLN353. At 9 days post- infection, heart homogenates were prepared and the proteasome activity was measured as described in Figure 1. Results are mean ± SE of three independent measurements from each animal (vehicle group (n=9); MLN353 group (n=11)). *P<0.05 as compared to vehicle-treated mice at 9 day post-infection. 61 Figure 12. Effect of proteasome inhibition on CVB3 viral titer in mice. A/J mice were CVB3 infected with vehicle or MLN353 treatment. Heart tissues were collected at 9 days post-infection and heart homogenates were used for plaque assay (mean ± SE; vehicle group (n=8) and MLN353 group (n=11)). NS: no significant difference as compared to sham infection, P>0.05. Figure 12. CV B 3 Ti tr e (10 4 pf u /m l) 0 5 10 15 20 25 30 Vehicle MLN 353 CVB3 (9d pi) NS 62 Vehicle MLN 353 Vehicle MLN 353 H is to lo gi ca l G ra de (0- 5+ ) Figure 13. CVB3 (9d pi) 0 0.5 1 1.5 2 2.5 3 * 63 Figure 13. MLN353 treatment attenuates CVB3-induced myocardial injury in mice. A/J mice were CVB3 infected in the presence of vehicle or MLN353. At 9 days post- infection, heart tissue from both vehicle and MLN353 groups were collected and H&E stained (A) and the extent of myocarditis was histologically graded (B) based on the intensity and character of injury and inflammatory infiltration as described in “Material and Method”. The results shown are mean ± SE (vehicle group (n=11); MLN353 group (n=15)). * P<0.05 as compared to vehicle-treated mice at 9 day post-infection. 64 3.4 Discussion The severity of viral myocarditis is determined by virus infectivity and host responses: CVB3 directly injures the infected myocardium and the extent of such injury determines the severity of late stage organ dysfunction . Host-responses to viral infection may reflect a host defense mechanism. However, improper host-protein regulation, such as aberrant host protein degradation, may cause further tissue damage . In the present study, I examined the expression and regulation of the UPS in CVB3-induced murine hearts, and explored the direct role of proteasome inhibition in the pathogenesis of coxsackieviral myocarditis. It was shown that protein-ubiquitin conjugates are abnormally accumulated in CVB3-infected hearts. I further demonstrated that increased accumulation of ubiquitin conjugates is attributed to augmented protein ubiquitination, but not to decreased proteasome proteolytic activites. Finally, I showed that application of a proteasome inhibitor attenuates CVB3-induced myocardial injury. The study reveals a novel mechanism of coxsackievirus infection, and suggests that the ubiquitin-proteasome system may be an attractive therapeutic target against coxsackievirus-induced myocarditis. Indeed, the level of protein-ubiquitin conjugates is determined by a balance between the rates of protein conjugation and degradation [161, 260]. A breakdown in this balance will lead to abnormal expression of ubiquitin conjugates. The finding in this study that proteolytic activities of proteasome are unchanged after CVB3 infection of mouse heart is consistent with our previous observation in cultured cells , indicating that the proteasome is unlikely a direct target of the virus. These results also exclude a possible contribution of proteasome dysfunction in the pathogenesis of viral myocarditis. 65 As alluded to earlier, three classes of enzymes, known as E1, E2, and E3, catalyze the conjugation reaction of ubiquitin to the protein substrates . In the present study, it was found that CVB3 infection induces the expression of ubiquitin enzymes E1A/E1B and UbcH7, suggesting that increased protein ubiquitination may be a factor resulting in the aberrant accumulation of ubiquitin conjugates. Although protein level of p53-related E3 ligase E6-AP is not different after virus infection, we cannot rule out the influence of CVB3 infection on other E3 ligases since more than 500 E3s are predicted based on the human genome . It is known that protein ubiquitination can also be regulated by deubiquitinating enzymes . In eukaryotes, ubiquitin is generated in the cells only by proteolysis of polyubiquitin chains or ubiquitin fused to carboxyl extension proteins . Protein deubiquitination has been reported to play a critical role in the supply of free ubiquitin to the cells for protein ubiquitination. In this study, it was demonstrated that CVB3 infection increases UCHL1 expression, suggesting that enhanced protein deubiquitination appears also a cause of the increased accumulation of protein conjugates, as UCHL1 may increase protein ubiquitination by providing the available pool of free ubiquitin. The mechanisms leading to the increased expression of E1A/E1B, UbcH7 and UCHL1 are unclear. Experimental and clinical studies have indicated a pivotal role of myocardial inflammation in the development and progression of viral myocarditis. Increased release of pro-inflammatory cytokines has been implicated as contributing to the pathogenesis of this disease . Recent studies have suggested that cytokines are important modulators for protein degradation through the regulation of protein ubiquitination and degradation/deconjugation [264-266]. Thus, we speculate that CVB3 66 infection leads to increased inflammatory cytokine release, which stimulates protein ubiquitination by upregulation of ubiquitin enzymes. Moreover, the fact shown in this study that increased expression of E1A/E1B appears to localize to virus damaged, non- inflammatory cells, together with our previous observation that CVB3 infection of cultured murine cardiomyocytes is unable to induce E1A/E1B expression (data not shown), suggest that this enzyme may be influenced by inflammatory cytokines in a paracrine or autocrine mechanism. Proteasome inhibitors with low molecular weight have been widely used in basic research and in the clinical trials of many diseases based on the observation that 26S proteasome is a primary component of the protein degradation system of the cell. Bortezomib (also known as Velcade or PS-341), developed by Millennium Pharmaceuticals, is a dipeptidyl boronic acid that potently inhibits 26S proteasome activity in a specific and reversible manner. This chemical has been shown to have significant antitumor activity as a single agent and in combination with other cytotoxic drugs [267, 268]. Other proteasome inhibitors, such as MG132 and lactacystin, have been frequently used in basic research. We previously reported that these two agents can reduce CVB3 replication in murine cardiomyocytes . In the present study, we used a new proteasome inhibitor, MLN353, which has a similar structure to Bortezomib and is more soluble and suitable for animal study than MG132 and lactacystin, to investigate the effect of proteasome inhibitor on CVB3-induced mycoarditis. Proteasome inhibitors have been shown to dramatically reduce the production of multiple inflammatory mediators and leukocyte adhesion molecules through their ability to block the activation of NFκB, which play a crucial role in many diseases . Viral 67 myocarditis is an inflammatory disease of the heart. It was classically considered that infiltrating immune cells play a critical role in the host defense mechanism by clearing the invaded viruses. However, accumulating evidence suggests that an inappropriate immune response may also lead to tissue damage . It has been shown that depletion of T lymphocytes results in a reduction in mortality and a decrease in the inflammatory infiltrate following CVB3 infection . Transfer of mononuclear cells from mice infected with CVB3 or from patients with myocarditis into genetically identical or immunodeficient mice, respectively, exacerbates myocardial damage [271, 272]. In this study, it was shown that treatment with MLN353 attenuates the severity of myocarditis, especially inflammation, suggesting that the UPS play an important role in the pathogenesis of viral myocarditis. Although it was demonstrated in vitro that MLN353 treatment reduces viral replication in cardiomyocytes, animal study showed no direct anti-viral effect of this inhibitor. As stated above, early host immune response plays a critical role in clearance of virus. It is speculated that, after treatment with proteasome inhibitor, a balance between a direct inhibition of viral replication and a suppression of host immune response determines the final virus load inside the heart. It is believed that an appropriately regulated immune system is crucial in virus clearance and in the regulation of myocardial damages. But we still cannot rule out the possibility that the observation of no effect on virus titers may be also due to a suboptimal efficacy of the dose of MLN373 administered and/or the experimental model and timing employed under current study. In summary, I demonstrate that protein-ubiquitin conjuates are aberrantly accumulated in CVB3-infected hearts, which is attributed to increased protein 68 ubiquitination/deubiquitination. I further demonstrate that proteasome inhibition attenuates myocardial injury induced by coxsackievirus infection. Our data suggest that the UPS may be an attractive therapeutic target for virus-induced myocarditis. 69 Chapter 4: Ubiquitination Is Required for Effective Replication of CVB3 In this chapter, the background, experimental design, results and discussion for the specific Aim 2, which is the in vitro study of the UPS on CVB3 replication, will be presented. The content of this chapter has been published in 2008 . I am the second author of this paper. 4.1 Introduction As reviewed earlier, in addition to the degradation of damaged and misfolded proteins, the UPS is also responsible for the modulation of many regulatory proteins such as cyclins , inhibitors of cyclin-dependent kinases p21 and p27 , tumor suppressors p53 , and inhibitor of NFκB (IκB) . These proteins are essential for a variety of fundamental cellular functions, including cell-cycle regulation, apoptosis and host immune responses . Thus it is expected that general inhibiton of the proteasome function will lead to the interference of normal cellular functions by which the UPS is involved in. Additionally, during the in vivo study presented in the previous chapter, I also recognized the toxicity of systematically applying proteasome inhibitor as a therapeutic means. Thus, we need to further identify specific targets utilized by CVB3 within this pathway. In this Aim, we focus on studying the contribution of ubiquitin- dependent proteasomal degradation in CVB3 infection. 70 4.2 Experimental design For this Aim, HeLa cell, a well-established cell model for CVB3 research, was used to explore the molecular mechanism by which the UPS regulates CVB3 infection. HeLa cells were grown in complete medium to 70 -80% confluence, and then infected at a multiplicity of infection (MOI) of 10 with CVB3 of Kandolf strain or sham infection with PBS for 1h, washed with PBS, and then incubated with DMEM without /or containing various concentration of proteasome inhibitor (MG132: 0, 1, 5, 10, and 20µM; lactacystin: 0, 1, 2.5, 5, 10, 20µM). Seven hours post-infection, positive sense of CVB3 viral RNA was determined by in situ hybridization using riboprobes for CVB3; cell lysates were collected and immunobloted with anti-VP1 and anti-β-actin (as loading control) antibodies in Western blot. At 16 hours post-infection, cell medium were collected from CVB3-infected cells and virus titer was determine by plaque assays; cell viability assay was performed by the MTS assay which measures mitochondrial function. The siRNA of ubiquitin was also used for role of the UPS in CVB3 replication. HeLa cells were grown to 50% confluency and then transiently transfected with ubiquitin-specific siRNA (200nM) using oligofectamine according to the manufacturer’s suggestion. A scramble siRNA (200nM) was used for control. After 24 hours of transfection, cells were infected with CVB3 as above described. The silencing efficiency was detected by immunoblot ananlysis using the anti-ubiquitin antibody. To demonstrate the interaction between CVB3 and the UPS during viral infection, we first examined the changes of the UPS after CVB3 infection. Western blots were performed using anti-ubiquitin antibody and antibodies against several key enzymes of the UPS, such as ubiquitin-activating enzyme E1A/E1B, ubiquitin-conjugating enzyme 71 Ubc H7, ubiquitin C-terminal hydrolase and two p53-related E3 ligases, on CVB3 infected cells. Next, we wanted to know if CVB3 viral proteins can be ubiquitinated during the viral replication cycle. Immunoprecipitation experiment was performed to test the ubiquitination of viral protein 3D. Cells were lysed using lysis buffer with freshly added 20 mM iodoacetamide. A total of 500µg of cell lysates were incubated with a monoclonal anti-ubiquitin antibody (1:100) at 4°C overnight, followed by 2 h incubation with protein G-agorose beads (Amersham). Immunocomplexes were washed five times with the lysis buffer containing 20mM iodoacetamide, and then boiled for 5 min in the 2 × non- reducing sample buffer which lacks both β-mercaptoethanol and DTT, but with addition of 20mM iodoacetamide. After centrifugation, the precipitated proteins were separated by SDS-PAGE. Ubiquitin conjugates were analyzed by immunoblot using polyclonal anti- 3D antibody. 4.3 Results Proteasome inhibition reduces CVB3 infection in HeLa cells To uncover the underlying mechanisms of the antiviral activities of proteasome inhibitors, we chose to use the well-characterized HeLa cells to further our study. We first examined the role of proteasome inhibition in CVB3 replication. It was found that proteasome inhibitor, MG132, significantly reduced CVB3 viral RNA synthesis (Figure 14A). Both proteasome inhibitors used in the study, MG132 and lactacystin, decreased the synthesis of CVB3 capsid protein, VP1, in a dose-dependent manner (Figure 14B). In 72 addition, two inhibitors inhibited CVB3 viral titers by up to fifteen folds (Figure 14C). Although MG132 and lactacystin significantly inhibited cellular 20S proteasome activities, we have previously demonstrated there was no apparent difference in proteasome activities between CVB3-infected and sham-infected HeLa cells . Together, these results suggest that efficient replication of CVB3 requires the intact UPS function rather than the core proteasome activity alone. Cell viability assay and morphological examination were also performed to determine whether inhibiting viral replication by proteasome inhibitors is due to the toxicity. We found that there was no measurable cell death throughout the incubation period for all doses of proteasome inhibitors used in this study (Figure 14D). On the contrary, virus- induced cell death was markedly inhibited after the treatment of proteasome inhibitors as a result of decreased viral replication (Figure 14D). 73 A Figure 14. CVB3 +DMSO CVB3 +MG132(20µM)CVB3 +MG132(5µM) B VP1 0 1 2.5 5 10 20 lactacystin (µM) CVB3 Sh am VP1 0 1 5 10 20 MG132 (µM) CVB3 Sh am β-actin β-actin C 0 20 40 60 80 100 MG132 (5µM) Lactacystin (5µm)DMSO CV B3 Ti te r (10 8 pf u/ m l) # # D 0 20 40 60 80 100 120 0 10 0 2.5 5 10 MG132(µM) Sham CVB3 Ce ll Vi ab ilit y (% co nt ro l) # # # 74 Figure 14. Proteasome inhibitors decrease coxsackieviral RNA expression, viral protein synthesis and viral progeny release in HeLa cells. HeLa cells were sham- infected with PBS or infected with CVB3 in the presence or absence of MG132 or lactacystin. (A). Seven hours post-infection (pi), positive-stranded viral RNA was determined by in situ hybridization using anti-sense riboprobes for CVB3 (red). Cell nuclei were counterstained with hematoxylin (blue). (B). Cell lysates were collected at 7 h pi and immunoblotted with anti-VP1 and anti-β-actin (loading control) antibodies. (C). Medium was collected from CVB3-infected cells at 16 h pi and virus titer was determined by plaque assays. The data shown are mean ± SE (standard errors) from three independent experiments. # p<0.001 as compared to CVB3 infection without treatment. (D). Cell viability assay was performed at 16 h pi by the MTS assay which measures mitochondrial function (mean±SE, n=3). One hundred percent survival was defined as the level of MTS in sham-infected cells in the absence of MG132. # p<0.001 as compared to CVB3 infection only without MG132 treatment. 75 Knockdown of ubiquitin by siRNA reduces CVB3 infection In addition to blocking proteasome proteolytic activities, proteasome inhibitors are known to reduce free ubiquitin levels in treated cells . It has been suggested that proteasome inhibition negatively affects the budding of retroviruses through reducing free ubiquitin level and subsequently interfering with ubiquitination of viral Gag proteins [224-226]. Ubiquitin is generated in the cell by proteolysis of polyubiquitinated proteins or ubiquitin fused to carboxyl extension proteins (CEPs) . To investigate whether protein ubiquitination is beneficial to CVB3 replication in HeLa cells, we used the ubiquitin-specific siRNA to gene-silence the expression of human ubiquitin-CEP Uba80, which codes for ubiquitin fused to ribosomal protein S27a . As shown in Figure 15A, both ubiquitin conjugates and free ubiquitin levels were markedly knocked down after the treatment of ubiquitin siRNA. We further showed that viral titers were significantly reduced in the ubiquitin siRNA-transfected cells as compared to scramble siRNA control (Figure 15B), suggesting that protein ubiquitination is a critical process adopted by coxsackievirus for the successful completion of its lifecycle. 76 Figure 15. A B 0 4 8 12 16 20 CV B3 Ti te r (10 6 pf u/ m l) Scramble siRNA Ubiquitin siRNA * Free ubiquitin Sham Sham CVB3 CVB3 Scramble siRNA Ubiquitin siRNA (Ub)n β-actin 181.8 115.5 82.2 MW (kDa) 6 77 Figure 15. Knockdown of ubiquitin expression by siRNA reduces CVB3 replication. HeLa cells were transiently transfected with the ubiquitin siRNA or a scramble control siRNA. Twenty-four hours post-transfection, HeLa cells were infected with CVB3 or sham-infected with PBS. Cell lysates were collected at the indicated timepoints. (A). Immunoblot was performed with anti-ubiquitin and anti-β-actin (loading control) antibodies. (B). Supernatants of infected cells were collected at 7 h pi to measure CVB3 progeny virion release by plaque assay (Mean ± SE, n=4). Results represent data from three independent experiments. * p<0.05 as compared to virus titers in scramble siRNA- transfected cells. 78 CVB3 infection promotes protein ubiquitination As alluded to earlier, two successive steps are involved in protein degradation: (1) covalent attachment of ubiquitins to the target protein substrate, and (2) degradation of the polyubiquitinated protein by the 26S proteasome with the release of ubiquitin for recycling. To dissect out the role of ubiquitination and degradation in CVB3 infection, we next decided to investigate the protein ubiquitination after CVB3 infection. As shown in Figure 16A, protein ubiquitination was gradually increased along the time-course of CVB3 infection, which was accompanied by a decrease of free ubiquitin levels. Densitometric analysis further demonstrated that the increases in protein ubiquitination at 3 h, 5 h, and 7 h post-infection were statistically significant as compared to sham infection (Figure 16B). We have previously demonstrated that 26S proteasome activities were unchanged during CVB3 infection . Thus, the finding of increased accumulation of ubiquitinated proteins is likely due to enhanced protein ubiquitination as opposed to reduced proteasome activity. Decreased levels of free ubiquitin could be a direct consequence of the increased protein ubiquitination. These results suggest that enhanced ubiquitin conjugation may be a prerequisite for efficient synthesis of CVB3 viral RNA and continuation of its lifecycle. CVB3 RNA-dependent RNA polymerase 3D is ubiquitinated Interestingly, some virus RNA-dependent RNA polymerases including the sindbis virus and the turnip yellow mosaic virus RNA polymerases have been demonstrated to be phosphorylated and ubiquitinated . Even though the role of ubiquitination of these RNA polymerases in the regulation of virus replication remains to be determined, such 79 Figure 16. 10m 30m 1h 3h 5h 7h CVB3 (pi) Sh am β-actin (Ub)n 64.2 181.8 115.5 82.2 MW (kDa) VP1 Free ubiquitin A Ub iq u iti n Ex pr es si o n (N o rm al ize d to β-a ct in ) Sh am 10m 30m 1h 3h 5h 7h CVB3 (pi) Free ubiquitin Polyubiquitin 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Ex pr es si o n o f P ro te in - Ub iq ui tin Co n jug at es (N o rm al ize d to β-a ct in ) Sh am B 3h 5h 7h CVB3 (pi) 0 0.5 1.0 1.5 2.0 2.5 3.0 * & * 80 Figure 16. CVB3 infection results in increased protein polyubiquitination and decreased free ubiquitin. (A). HeLa cells were infected with CVB3 or sham-infected with PBS. At different timepoints after viral infection, cell lysates were collected and immunoblotted with anti-ubiquitin, anti-VP1 and anti-β-actin (loading control) antibodies. Protein levels of protein-ubiquitin conjugates (molecular weight starting from 82.2 kDa to approximately 230 kDa) and free ubiquitin were quantitated by densitometric analysis using NIH ImageJ program and normalized to the sham infection, which was arbitrarily set to a value of 1.0. Similar results were observed in two independent experiments. (B). Statistical analysis of protein-ubiquitin conjugates at 3 h, 5 h and 7 h after CVB3 infection. The data represent mean ± SE of five different experiments. * p<0.05; & p<0.01 as compared to protein expression in sham infection. 81 observation raises the interesting possibility that the ubiquitin/proteasome system may regulate CVB3 replication through ubiquitinating viral polymerase 3D, which is essential for initiating viral RNA replication. To examine whether coxsackieviral proteins are subjected to ubiquitination during viral infection, we performed immunoprecipitation with anti-ubiquitin antibody, followed by immunoblots using antibodies against 3Dpol and viral capsid protein VP1, respectively. As shown in Figure 17, immunoreactive bands of around 60kDa were detected in CVB3-infected cells. Non-modified 3Dpol has a molecular weight of about 53kDa, thus this observation suggests that 3Dpol likely undergoes post- translational modification by monoubiquitination. No protein ubiquitination was found for VP1 (data not shown). These results implicate that the ubiquitination process of CVB3 viral proteins might be required for successful replication of the virus. Effects of CVB3 infection on protein expression of several key enzymes involved in the process of ubiquitination and deubiquitination In trying to understand the mechanisms by which CVB3 manipulates the UPS, we examined the protein expression of several key enzymes involved in the process of protein ubiquitination and deubiquitination. We measured expression levels of ubiquitin- activating enzyme E1A/E1B, ubiquitin-conjugating enzyme Ubc H7, ubiquitin C- terminal hydrolase and two p53-related E3 ligases, human papillomavirus E6-associated protein and mouse double minute 2 homolog. However, no apparent changes were observed during the time-course of CVB3 infection (data not shown). These results indicate that the manipulation of the UPS by CVB3 is unlikely regulated by the above- 82 Figure 17. CVB3 RNA-dependent RNA polymerase 3D is ubiquitinated HeLa cells were infected with CVB3 or sham-infected with PBS for 7 h, Cell lysates were collected and immuoprecipitated (IP) with a monoclonal anti-ubiquitin antibody. Protein-ubiquitin conjugates were detected by immunoblots (IB) using a polyclonal anti- 3Dpol antibody. Immunoblot for antibody IgG was shown as loading controls. Similar results were observed in three independent experiments. IP: anti-ubiquitin IB: anti-3Dpol Figure 17. 3Dpol Sham CVB3 IgG181.8 115.5 82.2 MW (kDa) 64.2 49.2 83 examined ubiquitin-related key enzymes or molecules. Future studies will determine whether CVB3 infection targets on specific ubiquitin ligases or deubiquitinating enzymes. 4.4 Discussion In the present study, we have provided further evidence that CVB3 manipulates the UPS for its infection. CVB3 infection results in increased protein polyubiquitination and a subsequent decrease in free ubiquitin levels. Knockdown of ubiquitin and ubiquitin- mediated protein modification and/or degradation by siRNA markedly reduces CVB3 replication in HeLa cells, further supporting the essential roles of the UPS in the replication of CVB3. It is increasingly apparent that viruses can evolve various strategies to utilize the host UPS for their own benefits. The UPS has been suggested to play a critical role in the different steps of viral lifecycle, including viral entry, viral replication, maturation, viral progeny release, and latent virus reactivation [166, 212, 281]. The mechanisms that the UPS regulates viral infection involve degrading intracellular proteins or excessive viral proteins that are against efficient viral replication and modulating viral protein function through ubiquitin-mediated modification or by directly encoding ubiquitin-related enzymes . CVB3 infection stimulates protein ubiquitination without inhibition of the core 20S proteasome activity highlights the possibility that CVB3 manipulates the UPS to destabilize or modulate the host and viral proteins. Polyubiquitination and degradation of host antiviral proteins has been suggested to be a mechanism of HIV-1 replication  . We have previously identified several proteins, such as cyclin D1, p53 and β-catenin, which are downregulated through the UPS after CVB3 infection [132, 254]. 84 Destabilization of these short-lived host proteins is likely required for CVB3 viral RNA and protein synthesis in its lifecycle. Moreover, it is speculated that nonstructural viral proteins of CVB3 could also be potential targets of the UPS for degradation. Previous studies on picornavirus have shown that several viral proteins, such as encephalomyocarditis virus (EMCV) 3C protease and hepatitis A virus (HAV) 3C protease, are ubiquitinated and present in low concentrations in infected cells [284-286]. Several E3 ubiquitin ligases, such as human E3α ubiquitin ligase, have been shown to catalyze the ubiquitination of these viral proteins [285, 286]. Although the exact role of ubiquitination and subsequent degradation of nonstructural viral proteins of EMCV and HAV in infected cells remains elusive, such rapid turnover may be required for efficient viral RNA replication, viral protein synthesis and virus maturation. In addition to protein degradation, ubiquitin-modification has been suggested to be involved in the regulation of protein function. It was reported that monoubiquitination of the Gag protein of retroviruses is required for virus budding [224-226]. Depletion of free ubiquitin by proteasome inhibitors prevents Gag ubiquitination, subsequently blocks virus progeny release/budding. In addition, ubiquitination of human immunodeficiency virus type 1 Tat protein and human T-cell leukemia virus type 1 Tax protein has been shown to modulate their transactivation activities [235, 236]. We speculate that monoubiquitination is also an important machinery for post-translational modification and activation of CVB3 viral proteins. In the current study, we have shown that CVB3 RNA-dependent RNA polymerase 3D is post-translationally modified by ubiquitination, suggesting a critical role of protein ubiquitination in the regulation of viral protein functions. 85 Based on the results in this in vitro study, in combination of our previous findings that CVB3 infection promotes host protein degradation, including cyclin D1, p53 and β- catenin, a model system on the role of the UPS in CVB3 replication is proposed in Figure 18. Coxsackievirus infection facilitates host protein polyubiquitination, which subsequently increases intracellular protein degradation by the proteasome and/or viral protein modification, such as 3D, by monoubiquitination. Degradation of host antiviral proteins provides a favorable environment for virus to achieve successful replication. Knockdown of ubiquitin decreases host protein degradation and viral protein ubiquitination. Proteasome inhibition blocks host protein degradation and viral protein ubiquitination by reducing recycled ubiquitin. In conclusion, it has been demonstrated for the first time that CVB3 infection results in increased protein ubiquitination and consequent decreases in free ubiquitin levels. It was further demonstrated that protein ubiquitination is required for the completion of viral life cycle, likely through ubiquitin modification of viral polymerase. 86 Figure 18. A proposed model for UPS regulation of CVB3 replication (See text) Abbreviation: CVB3, coxsackievirus B3; Ub, ubiquitin; siRNA, small-interfering RNA; 3Dpol, coxsackievirus RNA-dependent RNA polymerase 3D. This figure is from . Proteasome Degradation Proteasome inhibitor Ubiquitination Ubiquitin siRNA Host protein Viral protein (3Dpol) Ub Ub Ub Ub Ub Ub Ub Ub Ub CVB3 Figure 18. 87 Chapter 5: Proteasome Activator REGγ Enhances Coxsackieviral Infection via Facilitating p53 Degradation In this chapter, the value of proteasome activator REGγ in CVB3 replication will be explored, meanwhile, how REGγ regulates CVB3 infection will also be investigated. 5.1 Introduction As discussed earlier, the 20S proteasome can be activated by either 19S or 11S activator, to perform ubiquitin-dependent /or -independent protein degradation, respectively. 11S proteasome REGγ is responsible for the ubiquitin-independent protein degradation of several important intracellular proteins such as cyclin-dependent kinase inhibitors p21, p16, and p19 [179, 181], and tumor suppresor p53 . Moreover, an interaction between the REGγ system and the viral proteins has recently been reported. It was shown that REGγ binds to and regulates the stability and nuclear retention of hepatitis C core protein , contributing to hepatitis C core protein-induced insulin resistance and hepatocarcinoma, and more importantly, suggesting its potential role in viral replication [211, 288]. In the previous chapter, we have found that knockdown of ubiquitin reduces viral protein synthesis and viral titres. However, such inhibition was not as potent as by proteasome inhibition, suggesting that 11S proteasome-mediated proteasomal degradation may also play a role. In this chapter, I seek to further understand the underlying mechanisms by which the UPS regulates CVB3 replication by investigating 88 the interplay between REGγ and CVB3 infection and exploring the potential mechanisms for how REGγ controls CVB3 replication. 5.2 Experimental Design In this Aim, the effect of REGγ-mediated ubiquitin-independent protein degradation on CVB3 replication was first examined. HEK293 tet-inducible REGγ stable cells were grown in the presence or absence of doxcycline (Dox) for 24 hours, followed by sham or CVB3 infection (MOI=1) for 8, 16, and 24 hours. Cell lysates were collected and expression of viral capsid protein VP1, REGγ and β-actin (loading control) was detected by Western blot. Medium of infected cells were harvested and progeny virion titers were measured by plaque assay. Next, I employed a pool of REGγ siRNA duplexes to silence the gene expression of REGγ and assessed the role of REGγ knockdown in CVB3 replication by plaque assay. HeLa and HEK293 cells were grown to 50% confluence and then transiently transfected with a pool of four REGγ siRNA duplexes (Dharmacon) at a concentration of 30nM using oligofectamine 2000 (Invitrogen). A scrambled siRNA duplex was used as a negative control. Twenty-four hours after transfection, cells were infected with CVB3 for 8, 16, and 24 hours and plaque assay was performed. The silencing efficiency was measured by Western blot analysis using anti- REGγ antibody, anti-p21 antibody, and anti-caspase-3 antibody. Caspase-3 activity was determined using a synthetic fluorogentic substrate (R&D Systems). To demonstrate whether or not the anti-viral properties of REGγ depletion is through induction of early apoptosis, the general caspase inhibitor zVAD was also applied followed for plaque assay. 89 The potential mechanism that REGγ regulates CVB3 replication was further explored in Aim 3. For doing that, I decided to focus on determining the role of the identified REGγ substrate in CVB3 replication. I speculated that REGγ-mediated proteolysis of certain host intracellular proteins may target specific aspects of the viral replication process and thus control its replication. The tumor suppressor protein p53 is a common barrier to viral replication by directly inhibiting virus transcription and through promoting premature cell death [210, 239, 289-291]. Meanwhile p53 is one of the identified REGγ substrates . To determine if p53 is involved in the regulation of REGγ on CVB3 replication, I first examined the effect of REGγ on p53 expression. HeLa and HEK293 cells at about 90% confluence were transiently transfected with a plasmid expressing Flag-tagged REGγ (pcDNA-Flag-REGγ) or control vector pcDNA for 48 hours, using lipofectamine 2000 (Invitrogen). Cell lysates were collected and expression of p53, p21, REGγ and β-actin was detected by Western blot. Effect of overexpression of p53 on CVB3 replication was next performed. HeLa cells were transiently transfected with a plasmid expression of wild type of p53 (pCMV-p53) (Clontech) or empty vector pCMV for 24 hours, followed by infection with CVB3 (MOI=1) for 16 or 24 hours, with or without the presence of zVAD (50µM). Cell lysates were analyzed by Western blot for protein expression of p53, VP1, and β-actin. Medium was collected for plaque assay. To understand the interplay between REGγ and CVB3 infection, I next examined the impact of CVB3 infection on protein expression, subcellular localization, and activation of REGγ. For protein expression, Western blot was performed for the HeLa lysates after 1, 3, 5 and 7 hours CVB3 infection. For the subcellular localization of REGγ after CVB3 infection, double-immunostaining for REGγ and VP1 was performed on 90 sham or CVB3-infected HeLa cell at 1, 3, 5, and 7 h post-infection, with or without zVAD. REGγ activity may be regulated by post-translational modifications, such as ubiquitination, phosphorylation, and sumoylation. Similar to ubiquitination, sumoylation is a process by which a small ubiquitin-like modifier (SUMO) is conjugated to the target protein, which regulates the protein’s function, especially to those nuclear proteins. To determine whether REGγ is modified by sumoylation during viral infection, Western blot was performed using anti-REGγ antibody after 1, 3, 5 and 7 hours viral infection. Then further sumoylation assay of REGγ was performed. In vitro sumoylation assay was carried out with a sumoylation assay kit according to the manufacturer’s protocol (Biomol). For in vivo sumoylation assay, sumoylated REGγ was detected by an enzyme-linked immunosorbent assay (ELISA) using the EpiQuikTM in vivo universal protein sumoylation assay kit following the manufacturer’s instruction (Epigentek). 5.3 Results Effect of REGγ knockdown or overexpression on coxsackieviral replication and virus-induced apoptosis To determine whether REGγ-mediated proteolysis plays a role in the regulation of UPS-mediated coxsackieviral replication, a pool of REGγ siRNA duplexes was employed to silence the gene expression of REGγ and assess the role of REGγ knockdown in coxsackieviral replication by plaque assay. Gene-silencing of REGγ in HeLa cells was 91 able to induce an accumulation of p21 protein, which was previously demonstrated to be a target of REGγ [179, 181]. The plaque assay results in Figure 19A demonstrated that the virus titers from REGγ-silenced cells were significantly lower than that from control siRNA-transfected cells, suggesting that knockdown of REGγ attenuates CVB3 replication. The function of REGγ in the control of cell survival/apoptosis has been increasingly recognized [179, 292]. It has been shown that REGγ deficient MEFs have markedly enhanced apoptosis as compared to the wild-type cells. To assess the effect of REGγ depletion on CVB3-induced apoptosis, experiments to measure caspase-3 cleavage and activity were performed. As shown in Figure 19B and 19C, CVB3 infection resulted in the cleavage (lane 1 in Figure 19B) and increased activity of caspase-3 (lane 3 versus lane 1, Figure 19C). Following CVB3 infection, cells with REGγ depletion had enhanced caspase-3 cleavage (lane 2 versus lane 1, Figure 19B) and increased caspase-3 activity (lane 4 versus lane 3, Figure 19C) compared with cells transfected with scramble siRNAs. It was noted that gene-silencing of REGγ did not promote increased apoptosis in mock-infected cells (lane 2 versus lane 1, Figure 19C). These results suggest that inhibition of REGγ sensitizes HeLa cells to CVB3-induced apoptosis. Similar results were observed using HEK293 cells. CVB3-induced apoptosis plays a key role in facilitating virus progeny release during late stages of viral infection. However, untimely cell death could be harmful to virus by creating an unpleasant environment for viral replication . To determine whether inhibition of CVB3 by REGγ knockdown is through induction of early apoptosis, the influence of inhibition of apoptosis on CVB3 infection was examined in both control 92 and REGγ-depleting cells. The general caspase inhibition by zVAD did not eliminate the anti-viral properties of REGγ depletion (Figure 19D). These results suggest that the effect of REGγ knockdown on CVB3 infectivity is unlikely due to enhanced apoptosis. The role of REGγ in viral replication was also assessed by REGγ overexpression. REGγ was induced in the HEK293 “tet-on” stable cell line with the addition of doxcycline. Figure 20 demonstrated that overexpression of REGγ increased viral protein synthesis (Figure 20A) and viral replication (Figure 20B) in a time-dependent manner. To exclude the possible influence of doxcycline on viral replication, an experiment to examine the effect of doxcycline incubation on virus protein expression was also performed. It was found VP1 protein expression was unchanged (data not shown), suggesting that the increased viral production in HEK293-REGγ cells is not due to the treatment of doxcycline. Collectively, these results demonstrate an important role of proteasome activator REGγ in controlling CVB3 infectivity. 93 A. B. C. CV B3 Ti te r (×1 07 pf u/ m l) REGγ Control REGγ siRNA Pro- caspase-3 β-actin CVB3 Cleaved- caspase-3 p21 Ca sp as e- 3 Ac tiv ity Figure 19. 0 1000 2000 3000 4000 5000 6000 7000 REGγ siRNA Mock CVB3 D. Control siRNA 1 2 3 4 1 2 CV B3 Ti te r (× 10 7 pf u/ m l) 0 2 4 6 8 10 12 14 16 * zVAD + CVB3 (16h) * * 0 50 100 150 200 250 300 350 * REGγ siRNA Control siRNA 8h 16h 24h CVB3) REGγ siRNA Control siRNA 94 Figure 19. Knockdown of REGγ reduces coxsackieviral progeny titers. HeLa cells were transiently transfected with the REGγ siRNA (30nM) or a scramble control siRNA for 24h, followed by mock infection or CVB3 infection (MOI=1) for various times as indicated (A) or for 16h (B, C, D). (A) Supernatants of infected cells were harvested and progeny virion titers were measured by plaque assay. Results are presented as mean ± SD (n=3). *p < 0.01 as compared to the scramble siRNA control, respectively. Similar results were observed using HEK293 cells. (B) Western blot analysis was performed to detect caspase-3, p21, REGγ and β-actin (loading control). (C) Caspase-3 activities were measured using a synthetic fluorogenic substrate and results are expressed as mean ± SD (n=3). *p < 0.01 compared to siRNA control. (D) HeLa cells were transfected with REGγ or control siRNAs for 24h, and then infected with CVB3 for 16h in the presence of zVAD (50µM). Plaque assay results are shown as mean ± SD (n=3). *p < 0.01 compared to scramble siRNA control. 95 - + - + - + DOX CV B3 Ti te r (× 10 7 pf u/ m l) # A. B. Figure 20. VP1 REGγ β-actin Mock CVB3 16h CVB3 24h +Dox - Dox 0 50 100 150 200 250 # CVB3 16h CVB3 24h 96 Figure 20. Overexpression of REGγ promotes CVB3 replication. HEK293 tet-inducible REGγ cells were grown in the presence or absence of doxcycline (Dox) for 24h, followed by mock or CVB3 infection (MOI=1) for various times as indicated. (A) Cell lysates were collected and expression of viral capsid protein VP1, REGγ and β-actin (loading control) was detected by Western blot. (B) Supernatants of infected cells were harvested and progeny virion titers were measured by plaque assay. Results are presented as mean ± SD (n=3). #p < 0.001 as compared to the control cells without Dox induction. 97 Overexpression of REGγ decreases p53 levels To further explore the molecular mechanisms by which REGγ regulates CVB3 infection, we decided to focus on determining the role of the identified REGγ substrate in CVB3 growth. We speculate that REGγ-mediated proteolysis of certain host intracellular proteins may target specific aspects of the viral replication process and thus control its replication. The tumor suppressor protein p53 has been suggested to be a common barrier to viral replication by directly inhibiting virus transcription and through promoting premature apoptosis [210, 239, 240, 289-291]. Recent study demonstrates a role of the REGγ-proteasome pathway in regulating the stability of p53 . It was reported that REGγ facilitates p53 degradation by promoting MDM2-mediated p53 ubiquitination . Consistent with this report, Figure 21 showed that transient transfection of HeLa cells (Figure 21A) or HEK293 cells (Figure 21B) with Flag-tagged REGγ expression vector reduced p53 and p21 levels. 98 Figure 21. - REGγ p21 β-actin p53 Endogenous REGγ Exogenous (Flag-REGγ) 1.00 0.47 1.00 0.65 1.00 1.15 1.00 2.18 A. B. p21 β-actin p53 Endogenous REGγ Exogenous (Flag-REGγ) - REGγ 1.00 0.24 1.00 8.25 1.00 1.16 1.00 0.38 99 Figure 21. Overexpression of REGγ decreases p21 and p53 levels. (A) HeLa cells or (B) HEK293 cells were transiently transfected with Flag-tagged REGγ expression vector or control plasmid pcDNA for 48h. Cell lysates were collected and expression of p53, p21, REGγ and β-actin (loading control) was detected by Western blot. Protein expression was quantitated by densitometric analysis using National Institutes of Health ImageJ 1.41o, and normalized to the control vector-transfected cells, which was arbitrarily set to a value of 1.0. Relative protein levels were listed below each panel. “-“, empty vector control. 100 Overexpression of p53 reduces CVB3 replication and attenuates the pro-viral function of REGγ Several viruses have been shown to inactivate p53 during early viral infection for their own benefits via different mechanisms [210, 289, 291]. The results in Figure 22A showed that CVB3 infection resulted in increased viral protein synthesis, accompanied by decreased levels of p53 and p21. We have previously reported that proteasome inhibition reduces CVB3 replication [252, 253]. Figure 22B demonstrated that inhibition of p53 and p21 degradation during CVB3 infection by proteasome inhibitor lactacystin was associated with decreased viral infectivity, suggesting a potential link between the p53 pathway and viral replication. To delineate the potential functions of p53 degradation on CVB3 replication, p53 was overexpressed in HeLa cells, which were then exposed to CVB3 for various times. As shown in Figure 23, CVB3 infection led to a reduction of p53 protein expression in a time-dependent manner (lanes 3 and 5 versus lane 1 in Figure 23A left panel, and lane 2 versus lane 1 in Figure 23B left panel) which is consistent with the observation in Figure 22. Overexpression of p53 overcame the suppression induced by CVB3 infection (Figure 23A left panel, lanes 4 and 6 versus lanes 3 and 5, respectively), resulting in significant reduction of viral protein expression (Figure 23A left panel, lanes 4 and 6 versus lanes 3 and 5, respectively) and virus titers (Figure 23A right panel). These results indicate a mechanism by which REGγ regulates viral replication via regulating p53 levels. To further determine whether p53 inhibits viral replication by promoting early apoptosis, we treated the cells with zVAD and examined the effect of apoptosis inhibition on viral 101 Figure 22. p21 0 0 1 5 10 20 lactacystin (µM) CVB3 VP1 p53 Mock β-actin 5 h 7 h1 h 3 h Mock CVB3 Mock CVB3 Mock CVB3 Mock CVB3 β-actin VP1 p21 p53 A. B. 102 Figure 22. Blockage of p21 and p53 degradation by proteasome inhibitor lactacystin is associated with decreased virus protein expression. (A) HeLa cells were mock-infected or infected with CVB3 (MOI=10) for different time courses as indicated. Western blot analysis was carried out for detection of p53, p21, VP1, and β-actin. (B) HeLa cells were infected with CVB3 (MOI=10) in the presence of increasing concentrations of lactacystin as indicated. Cell lysates were collected at 7h post-infection for Western blot analysis of p21, p53, VP1, and β-actin. 103 β-actin VP1 - p53 - p53 - p53 A. B. CV B3 Ti te r (´1 07 pf u /m l) # p53 Figure 23. 1 2 3 4 5 6 Pro- caspase-3 Cleaved- caspase-3 - p53 - CVB3Mock 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Ab so rb an ce - p53 C. CVB3 16h CVB3 24h p53 - - p53 β-actin VP1 CVB3Mock - zVAD zVAD 1 2 3 Mock CV B3 Ti te r (´1 07 pf u /m l) # CVB316h CVB3 24h 0 2 4 6 8 10 12 14 16 18 0 50 100 150 200 250 300 350 400 p53 - p53 - zVAD + CVB3 (16h) # 104 Figure 23. Overexpression of p53 inhibits CVB3 infection. (A) HeLa cells were transiently transfected with a plasmid expressing p53 or empty vector pCMV for 24h, followed by infection with CVB3 (MOI=1) for 16 or 24h. Cell lysates were analyzed by Western blot for protein expression of p53, VP1, and protein loading control β-actin (left panel). Supernatants of infected cells were harvested and progeny virion titers were measured by plaque assay (right panel). Results are presented as Mean ± SD (n=3). #p < 0.001 compared to vector control cells. (B) HeLa cells were transfected with p53 construct or empty vector for 24h, and then infected with CVB3 for 16h in the presence of zVAD (50µM). Western blot (left panel) and plaque assay (right panel) were performed to examine VP1 expression and virus titers, respectively. Results are shown as Mean ± SD (n=3). #p < 0.001 compared to vector control. (C) HeLa cells were transiently transfected with p53 or empty vector for 48h. Left panel: cell viability was determined by the MTS assay (Mean ± SD, n=3). Right panel: Western blot analysis for the cleavage of caspase-3. HeLa cells infected with CVB3 were used as positive control. “-“, empty vector control. 105 infection. As shown in Figure 23B, inhibition of apoptosis did not prevent the inhibitory effects of p53 on viral protein expression (left panel) and virus titers (right panel). Cell viability assay and Western blot analysis were also performed to examine cell death and apoptosis following overexpression of p53. Figure 23C showed that overexpression of p53 for 48h did not induce increased cell death (left panel) and apoptosis (right panel) as compared to vector control. Collectively, our results suggest that p53 appears to inhibit CVB3 replication by a direct mechanism independent of its regulatory role of initiating apoptosis. Finally, we examined whether overexpression of p53 can attenuate the effect of REGγ on viral replication. HEK293-REGγ inducible cells were transiently transfected with p53 or control vector for 24h in the presence or absence of doxcycline, and then infected with CVB3 for 16h. Consistent with the results shown in Figure 20, REGγ overexpression markedly enhanced CVB3 VP1 expression and virus titer (Figure 24, lane 2 versus lane 1). However, the positive effect of REGγ on viral replication was significantly attenuated by p53 overexpression (lane 4 versus lane 2), supporting a role of REGγ in regulating CVB3 infection via destabilizing p53. 106 Figure 24 - + - + DOX REGγ - - + + p53 VP1 β-actin p53 1 2 3 4 CV B3 Ti te r (×1 07 pf u/ m l) 0 50 100 150 200 250 # 107 Figure 24. Overexpression of p53 attenuates the effect of REGγ on promoting CVB3 replication. HEK293 tet-inducible REGγ cells were grown in the presence or absence of doxcycline (Dox) as indicated for 24h, followed by transient transfection with either p53 (“+”) or empty vector (“-”). Twenty-four hours later, cells were infected with CVB3 (MOI=1) for 16h. Upper panel: supernatants of infected cells were harvested and progeny virion titers were measured by plaque assay. Results are presented as mean ± SD (n=3). #p < 0.001 as compared to the vector control with induction of Dox (lane 2). Lower panel: cell lysates were collected for detection of viral capsid protein VP1, REGγ, p53, and β- actin (loading control) by Western blot. 108 CVB3 infection leads to cytoplasmic relocalization of REGγ To understand the interaction between REGγ and CVB3 infection, the impact of CVB3 infection on protein expression, subcellular localization, and activation of REGγ were examined next. Figure 25A showed that the protein expression levels of REGγ appeared to be unchanged throughout the course of virus infection. The cellular localization of REGγ after CVB3 infection was further examined. Double- immunostaining for REGγ and viral protein VP1 was performed on mock or CVB3- infected HeLa cells at 1h, 3h, 5h, and 7h post-infection. As shown in Figure 25B and 25C, REGγ was exclusively localized in the nucleus in mock-infected cells (top panel). However, following 5h (Figure 25B) or 7h (Figure 25C) viral infection, REGγ was largely redistributed to the cytoplasm in CVB3-infected cells (green-stained cells), whereas in non-infected cells (green-negative cells), REGγ remained in the nucleus (middle panel). The bottom panels in Figure 25B and 25C showed that inhibition of apoptosis by zVAD did not prevent REGγ redistribution, suggesting that cytoplasmic translocation of REGγ is unlikely a consequence of CVB3-induced apoptosis. It should be noted that detection of VP1 protein was not very sensitive when it was at a relatively low level. The expression of VP1 was undetectable by immunostaining until 5h post- infecetion, consistent with the Western blot results shown in Figure 22A. Thus, cytoplasmic relocalization of REGγ at 5h and 7h post-infection in virus-infected cells were only shown (Figure 25B and 25C). However, REGγ redistribution likely occurred earlier, similar to what was observed by Western blot in Figure 22A that expression of p53 and p21 was downregulated at 3h post-infection, prior to the detection of VP1 protein expression (i.e. 5h post-infection). 109 A. REGγ β-actin Mock 1h 3h 5h 7h CVB3 B. Figure 25. DAPIREGγ OverlayCVB3 VP1 CVB3 5h Mock CVB3 5h + zVAD 110 C. DAPIREGγ OverlayCVB3 VP1 CVB3 7h Mock CVB3 7h + zVAD 111 Figure 25. CVB3 infection leads to redistribution of REGγ. (A) HeLa cells were either mock infected or infected with CVB3 (MOI=10) for different times. Western blot was performed for detecting REGγ and β-actin. (B, C) HeLa cells were infected with CVB3 (MOI=10) for 5h (B) or 7h (C) in the presence or absence of zVAD (50µM). Double-immunocytochemical staining was carried out for examining the expression and localization of REGγ (red) and viral protein VP1 (green). The nucleus was stained with DAPI (blue). Arrows denote cells without or with low levels of viral protein expression. The yellow staining in the merged image indicates colocalization of these two proteins. It is noteworthy that REGγ is redistributed to the cytoplasm in CVB3- infected cells (green-positive cells), whereas it remains in the nucleus of non-infected cells (green-negative cells). 112 CVB3 infection promotes REGγ sumoylation REGγ activity may also be regulated by post-translational modifications, such as ubiquitination, phosphorylation, and sumoylation. By Western blot analysis, a higher molecular weight protein band above the regular REGγ band was detected starting at 3h post-infection (Figure 26A). This result suggests a potential post-translational modification of REGγ after CVB3 infection. The molecular weight of the higher band (~45kDa) seemed to correspond in size to the sumoylated REGγ. It was further shown that general caspase inhibition did not block the appearance of this band, suggesting that this modification of REGγ is not due to virus-induced apoptosis. Small ubiquitin-related modifier proteins (SUMO) are a family of small proteins that are structurally similar to ubiquitin . Protein modification by sumoylation is directed by an enzymatic cascade catalyzed by three enzymes . SUMO E1- activating enzyme is a heterodimeric complex consisting of Aos1 and Uba2. Ubc9 is the only identified SUMO E2-conjugating enzyme. At least three classes of SUMO E3- ligases have been reported, RanBP2, PIAS and the Polycomb protein Pc2. A recent report shows that CVB5 infection results in cytoplasmic redistribution of SUMO-1 and UBC9 . To verify the potential of REGγ sumoylation, the in vitro and in vivo sumoylation assays were performed. As shown in Figure 26B, in the presence of SUMO E1 (Aos/UBa2), SUMO E2 (UBC9), and ATP, REGγ can be sumoylated to form multiple sumoylated REGγ. REGγ sumoylation was further confirmed by an in vivo sumoylation ELISA assay by overexpressing SUMO-1 in HEK293-REGγ stable cells either in the absence or presence of doxcycline induction (Figure 26 C, lane 3 versus lane 1(-Dox), 113 A. Sumoylated REGγ 40kDa 30kDa Figure 26 Mock 1h 3h 5h 7h 7h REGγ - - - - zVAD CVB3 B. - + ATP 40 50 60 10 30 80 Di-SUMO SUMO 20 15 100 160 260 (SUMO)1-REGγ (SUMO)2-REGγ (SUMO)3-REGγ (SUMO)4-REGγ MW (kDa) C. IgG REGγ Ab so rb an ce & 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 * 1 2 3 4 5 6 7 8 CVB3 Mock IgG REGγ -Dox +Dox * * 114 Figure 26. CVB3 infection promotes REGγ sumoylation. (A) HeLa cells were infected with CVB3 (MOI=10) for different times in the presence or absence of zVAD (50µM). Western blot analysis was performed to detect the expression of REGγ and β-actin. (B) In vitro sumoylation assay was performed on purified REGγ protein according to the manufacturer’s instruction (Biomol). Following in vitro reaction, sumoylated proteins were detected by Western blot using anti-SUMO antibody. (C) HEK293-REGγ stable cells were treated with Dox or without Dox as indicated for 48h, followed by transient transfection with SUMO-1 for additional 48h. After 20h of mock or CVB3 infection (MOI=1), cell extracts were harvested for an in vivo ELISA sumoylation assay. The data displayed are mean ± SD (n=3). *p < 0.01 compared to mock or IgG controls (lane 3 versus lane 1, lane 4 versus lane 2, and lane 8 versus lane 6). &p < 0.05 compared to mock IgG control (lane 7 versus lane 5). 115 lane 7 versus lane 5 (+Dox)). Importantly, it was shown that CVB3 infection significantly enhanced REGγ sumoylation (Figure 26C, lane 4 versus lane 2 (-Dox), lane 8 versus lane 6 (+Dox)). 5.4 Discussion The major findings of this study are as follows: 1) p53 negatively regulates CVB3 replication; 2) REGγ promotes CVB3 replication, at least in part, through facilitating p53 degradation; 3) CVB3 infection enhances REGγ sumoylation which may result in its nuclear export and subsequent p53 degradation in the cytoplasm. The tumor suppressor protein p53 is a transcription factor which plays a central role in regulating cell growth arrest and apoptosis in response to DNA damage . Many viruses have evolved different strategies to inactivate p53 to prevent early apoptosis, allowing for effective viral replication [210, 289, 291]. In addition, it was reported that p53 can directly interfere with the propagation of several viruses, such as human immunodeficiency virus type-1 , simian virus , and encephalomyocarditis virus . CVB3 infection leads to a dramatic downregulation of p53 protein, suggesting that CVB3 may have evolved strategies to regulate p53 function and stability to permit efficient viral replication. We have previously found that cardiomyocytes and HEK293 cells which express higher levels of p53 produce much lower yields of virus as compared to HeLa cells in which the p53 expression is relatively low. This observation implicates a potential inhibitory function of p53 in the control of coxsackievirus replication. Furthermore, in the study of poliovirus, a close family member of coxsackievirus, 116 knockdown of endogenous p53 by siRNA in U2OS cells has been shown to result in higher viral replication . In the current study, the role of p53 in coxsackievirus infection was investigated by overexpressing p53 in HeLa cells. The results provide direct evidence that p53 functions as an anti-viral protein against CVB3 replication. Although the mechanism of the anti-viral action of p53 has not yet been fully defined, results in the present study suggest that p53 appears to inhibit virus replication by a direct mechanism, independent of its regulatory role of initiating apoptosis. HeLa cells are human cervical cancerous cells with relative lower level of p53 expression, probably due to the existence of specific p53 ubiquitin E3 ligase, such as E6/E6-AP. To exclude the possible cell type-specific effect of REGγ on p53 regulation and viral infection, we have repeated the experiment using HEK293 cells which have normal level of p53 and similar results was obtained in this study. The current study provides evidence showing that REGγ-mediated p53 proteolysis may contribute, as least in part, to its pro-viral function. However, the potential roles of other host protein targets of REGγ in regulating coxsackieviral infectivity can not be excluded. In addition, future investigation will explore the possibility that REGγ may directly regulate CVB3 protein processing. Coxsackievirus or poliovirus infection has been shown to induce cytosolic translocation of several host nuclear proteins, such as nuclear protein La , Sam68 , and polypyrimidine tract binding protein . In the current study, REGγ has been added to this list. Regarding the mechanism by which CVB3 infection triggers the subcellular redistribution of REGγ, one possibility may be related to the disruption of the nucleo-cytoplasmic trafficking pathways. It has been reported that poliovirus infection 117 leads to the degradation of the nuclear pore complex protein Nup153 and p62, resulting in the blockage of nuclear import of nucleo-cytoplasmic shuttle proteins . The other possible explanation may be due to increased sumoylation modification of REGγ during CVB3 infection which leads to its nuclear export. Although sumoylation has been most often implicated in promoting nuclear import , there is evidence that sumoylation can also serve as a signal for cytoplasmic translocation of some protein substrates, such as TEL , Smad3 , Med , MEK1 , and heterogeneous nuclear ribonucleoproteins M and C . REGγ has been suggested to be a potential nucleo- cytoplasmic shuttling protein . However, the mechanisms regulating REGγ nuclear import/export have not yet been fully characterized. The role of sumoylation modification in such regulation warrants further investigation. REGγ is not a previously recognized substrate for SUMO modification. Here we provide both in vitro and in vivo evidence that REGγ can be sumoylated and that such post-translational modification is enhanced during CVB3 infection. However the exact sumoylation site(s) and the SUMO E3 responsible for REGγ sumoylation have not been identified in the current study. Interestingly, protein RanBPM and PIAS, two known SUMO E3s, have been reported to physically associates with REGγ , suggesting a possible function for them as SUMO E3 ligases for REGγ sumoylation. For the in vivo sumoylation assay of REGγ, the universal protein sumoylation assay ELISA kit provided by Epigentek was used, instead of immunoprecipitation/Western blot, which is the traditional approach for measuring protein sumoylation. Sumoylation is a post-translational modification on protein. Typically, only a small fraction of protein is SUMOylated at a given time and this modification is rapidly reversed by the action of 118 deSUMOylating enzymes. The ELISA sumoylation kit provides more sensitive method than traditional immunoprecipitation/Western blot assay. Figure 27 shows how this ELISA kit works. The significance of cytosolic REGγ in relation to coxsackievirus replication is still unclear. Given that coxsackievirus replication takes place exclusively in the cytoplasm, cytoplasmic localization of REGγ may allow for easier access to virus proteins and closer interaction with host proteins which control viral replication. For example, it has been shown that efficient degradation of p53 by proteasome occurs in the cytoplasm [308, 309]. Thus nuclear export of REGγ may provide a more effective way to interact with and promote p53 degradation in the cytoplasm. REGγ has been reported to be required for nuclear retention and degradation of hepatitis C virus core protein . We expect that cytosolic translocation of REGγ may also render an opportunity for its direct interaction with viral proteins and thus enhance viral protein processing. In summary, the results in this study demonstrate that proteasome activator REGγ plays an important role in facilitating coxsackieviral replication. As illustrated in Figure 28, CVB3 infection results in increased sumoylation and cytoplasmic translocation of REGγ which is associated with augmented p53 degradation. An anti-viral activity of p53 against CVB3 replication was also be reported here. The current study demonstrates that CVB3 infection promotes REGγ-mediated proteolysis of p53 which may enhance its own replication by reducing the inhibitory influence of p53 on viral replication. 119 Figure 27. In vivo sumoylation assay using an ELISA kit. The ELISA plate was first coated with anti- REGγ antibody or IgG (as control), then nuclear extract from cells overexpressing REGγ and SUMO1 was applied on the plate. Sumoylated REGγ will be detected by anti-SUMO antibody and shows the yellow color. This figure is modified from Epigentek website. Anti- REGγ (REGγ) Anti- SUMO Yellow color Figure 27. 120 Figure 28. A proposed mechanism by which REGγ enhances CVB3 infectivity. Following CVB3 infection, REGγ is sumoylated and exported from the nucleus. Cytoplasmic translocation of REGγ facilitates proteasomal degradation of tumor suppressor protein p53, which subsequently enhances CVB3 infection by suppressing the inhibitory effect of p53 on viral replication. Figure 28. CVB3 REGγ CVB3 replication REGγ SUMO REGγ SUMO p53 Nucleus Cytoplasm p53 degradation via proteasome Nuclear export 121 Chapter 6: Conclusion and Future Directions 6.1 Conclusion CVB3 is the most prevalent pathogen for viral myocarditis and its sequelae DCM, which can cause sudden death and heart failure in children. Currently, there is no vaccine available for viral myocarditis; the only treatment available for DCM is heart transplantation [38-41]. The UPS is the major intracellular protein degradation system that catalyses the rapid degradation of abnormal proteins and short-lived regulatory proteins, and involves in a variety of fundamental cellular processes [160, 161]. Previously, it has been demonstrated that the UPS is associated with CVB3 replication [132, 253, 254]. Inhibition of the UPS by proteasome inhibitor has been applied for the treatment of several diseases . Of the most promise is the application of proteasome inhibitors in cancer therapy . In the current study, the ability of proteasome inhibitor in treating diseases was extended to viral myocarditis. Proteasome inhibitor MLN353 was shown, for the first time, to decrease CVB3 replication and protect CVB3-induced myocarditis in mice. This finding reveals a novel mechanism of coxsackievirus pathogenesis and suggests that manipulation of the UPS may provide a therapeutic option against viral myocarditis. During this in vivo study, on the other hand, the potential toxicity of proteasome inhibitor was recognized, probably because the general inhibition of proteasome activity may affect those cellular functions the UPS involved. Thus, another inhibitory method of the UPS, ubiquitin siRNA, was introduced. Similar to proteasome inhibitors, application 122 of ubiquitin siRNA decreased CVB3 replication in cells, suggesting that ubiquitin siRNA may also be a therapeutic means for CVB3 induced myocarditis. Comparing to proteasome inhibition, however, the inhibitory effect of ubiquitin siRNA was not as potent as by proteasome inhibitors, indicating that the ubiquitin-independent proteasomal degradation also plays a role in CVB3 infection. REGγ is a proteasome activator to regulate proteasomal activity for the ubiquitin- independent protein degradation, which has been implicated and associated with viral replication . In the current study, the importance of REGγ in CVB3 replication was demonstrated for the first time. Furthermore, how REGγ regulates CVB3 replication was investigated and successfully shown. These findings promote the value of REGγ as a potential therapeutic target within the UPS for CVB3-induced myocarditis. After CVB3 infection, virus interacts with the host cell. A better understanding of the interaction between virus infection and the host cell may provide new insights into viral replication, viral pathogenicity, and may lead to a new avenue for therapeutic intervention for virus-induced disease. In the light of the UPS, the interaction between CVB3 and the host cells was investigated in the current study. Indeed, ubiquitin modification or protein degradation by the UPS is a mechanism for controlling the function and availability of regulatory protein in the cells; it also provides a platform for many different viruses to achieve successful viral infection . Virus can utilize this system for degrading those intracellular proteins that are against viral infection and replication or modifying their own function either directly, by encoding their own ubiquitinating/deubiquitinating enzymes, or indirectly, by use of endogenous molecules of the ubiquitin system. Meanwhile, people may speculate 123 that the host can also use the UPS to affect viral replication by inducing direct viral protein degradation or by modifying viral protein. The RNA-dependent RNA polymerase of some viruses has been reported to be ubiquitinated . However, ubiquitination of CVB3 viral protein has not been clarified so far. In this dissertation, for the first time, 3D, the RNA-dependent RNA polymerase of CVB3, was demonstrated to be ubiquitinated during CVB3 infection. Although function of this monoubiquitination of 3D is not clear, this finding confirms the speculation and further illustrates the importance of the UPS in CVB3 replication. In this dissertation, I present my work on the details of the interaction between CVB3 infection and the host UPS and the potential mechanisms involved, using both in vitro and in vivo models. A summary of the major findings from this thesis project is listed below: 1) Proteasome inhibitor protects CVB3-induced myocardial damage in mice - MLN353 treatment attenuates CVB3-induced myocardial injury in mice. 2) Inhibition of UPS decreases CVB3 replication – Application of proteasome inhibitor or siRNA of ubiquitin dramatically decreases CVB3 replication in cells. 3) CVB3 infection stimulates accumulation of protein-ubiquitination without inhibition of the core 20S proteasome activity - CVB3 infection promotes protein-ubiquitination and results in increased accumulation of protein-ubiquitin conjugates in both cells and mouse heart. 4) During CVB3 replication process, viral RNA-dependent RNA polymerase 3D is mono-ubiquitinated. 124 5) Like the ubiquitin-dependent protein degradation, REGγ-mediated ubiquitin- independent protein degradation is important for CVB3 replication. CVB3 infection induces the sumoylation and translocation of REGγ that are required for CVB3 replication. Based on the results and findings in current dissertation, I conclude that the ubiquitin/proteasome system plays a critical role in the pathogenesis of CVB3- induced myocarditis. 6.2 Future directions In the current study, proteasome inhibition has been demonstrated to attenuate myocardial injury at 9 days post-infection in mice. This study indicates that temporal blockage of the UPS may be beneficial during the acute viral infection and inflammatory stages of myocarditis, when the UPS is utilized to promote viral replication and induce immune-meidated pathogenesis. However, prolonged inhibition of protein degradation in the cardiac remodeling stage with impairment of proteasome function may cause further myocardial damage resulting in heart failure . This is consistent with recent clinical reports that long-term treatment with proteasome inhibitior, Velcade, in cancer patients increases the incidence of cardiac failure [310, 311]. . Meanwhile, my results confirmed the importance of the ubiquitin-dependent proteasome degradation in the control of CVB3 replication. However, further investigation to identify more specific targets of the UPS during CVB3 infection, for example, the ubiquitin ligase(s) involved in the process of specific protein ubiquitination, will allow for more precise targeting of drug therapy and development for viral myocarditis. 125 The field of siRNA has rapidly developed into a highly promising approach to alleviate disease pathology. This technique is well-studied to treat viral infections, and several examples of virus infection can be inhibited by specific siRNA both in vitro and in vivo . In the current study, I showed the inhibitory effect by siRNA of ubiquitin on CVB3 replication. Further studies will determine whether inhibition of the UPS by siRNA targetings on the specific molecules involved in the process of ubiquitination, for example, siRNA of ubiquitinating enzyme E1, E2, or E3, will provide a more specific treatment option for CVB3 myocarditis. CVB3 viral protein 3D was demonstrated to be ubiquitinated during CVB3 infection in this thesis study. This ubiquitination of 3D may modulate viral protein function for efficient viral replication in cells. In the same study, viral capsid protein VP1 showed non-ubiquitination. Like 3D, CVB3 viral protein 2A and 3C are also important for viral replication. They are responsible for the cleavage of host proteins, such as eIF4G, and for starting the viral replication by cleaving the viral polyprotein in cells [90, 101- 104]. Thus these viral proteins are also potential targets for inhibiting viral replication. Further investigation on the ubiquitination of other CVB3 viral protein(s) needs to be clarified and is important for understanding the UPS function in CVB3 replication. Proteasome activator REGγ is an important mediator for CVB3 replication through the ubiquitin-independent proteolysis. During viral infection, REGγ was found to be sumoylated, and this sumoylation is seemly required for CVB3 replication. However, the exact sumoylation site(s) of REGγ have not been identified. Using SUMOplotTM analysis program (Abgent), six putative sumoylation sites are predicted. Further studies will be necessary to verify these predictions, which are important to understand the exact 126 functions of REGγ sumoylation on CVB3 replication. In addition, REGγ mediates degradation of some important host proteins, such as cyclin-dependent kinase inhibitors p21, p16, and p19 [179, 181]. Here, in this dissertation, I have demonstrated that REGγ regulates CVB3 replication through facilitating p53 degradation. However, I cannot rule out the possibility that other proteins may also be involved in this process. Identifying more substrate proteins of REGγ will further illustrate the function of this proteasome activator in viral diseases. The biological significance of the UPS in the pathogenesis of viral myocarditis and its progression to DCM has been uncovered gradually . To date, therapeutic manipulation of host protein degradation systems in viral myocarditis becomes attractive, however, the complex interaction between the host and the virus makes it difficult for drug development. Understanding the precise functional and regulatory mechanisms of the UPS at each disease stage of viral myocarditis may guide the future therapeutic strategies. System-like approaches, such as ubiquitomics, degradomics, and RNAi screens, will be necessary to identify more specific modulators and targets of the UPS in viral myocarditis and DCM pathogenesis. 127 Chapter 7: Materials and Methods Cell cultures HeLa cells - HeLa cells (American Type Culture Collection) were grown and maintained in complete medium [Dulbecco's modified Eagle's media (DMEM) supplemented with 10% heat-inactivated newborn calf serum (NCS) (Invitrogen)]. HL-1 cell line - HL-1 cell line is transformed murine cardiac muscle cells established from an AT-1 mouse atrial cardiomyocyte tumor lineage (a generous gift from Dr. William C Claycomb at the Louisiana State University Medical Center, New Orleans), LA). Cells were plated onto flasks as described previously  and maintained in Claycomb media from JRH Biosciences (Lenexa, KS) supplemented with 10% fetal bovine serum, 100 µg/ml penicillin/streptomycin, 0.1 mM norepinephrine (Sigma) in ascorbic acid and 2mM L-glutamine (Life Technologies). HEK293 tet-inducible REGγ stable cells – HEK293 cells with stably overexpressing REGγ under the control of a tet-on promoter was previously established  and overexpression of REGγ was induced by addition of doxcycline (1µg/ml) HEK293 tet-inducible REGγ cells. Virus and in vitro viral infection CVB3 was propagated in HeLa cells and stored at -80°C. Virus titer was routinely determined by a plaque assay prior to infection. For Aim 1, HL-1 cells were preincubated with different concentrations of proteasome inhibitor MLN353 (molecular weight: 382.26; from Millennium 128 Pharmaceuticals, Inc.) for 30 minutes. Cells were then infected at a multiplicity of infection (MOI) of 100 with CVB3 or sham infected with phosphate-buffered saline (PBS) for 1 hour, washed with PBS, and placed in Claycomb media containing fresh inhibitor. For Aim 2, HeLa cells were grown in complete medium to 70-80% confluence, and then infected at an MOI of 10 with CVB3 or sham-infected with PBS for 1 h in serum- free DMEM. Cells were then washed with PBS and cultured in serum-free medium. For inhibition experiments, HeLa cells were infected with CVB3 for 1 h, washed with PBS, and then incubated with DMEM containing various concentrations of inhibitors MG132 or lactacystin (Calbiochem). For Aim 3, HeLa cells and REGγ stable cells were mock-treated with PBS or infected with CVB3 for 1h in serum-free DMEM at different MOI as specified in the Figure legends. The cells were then washed with PBS and cultured in complete DMEM for various times. For proteasome or apoptosis inhibition, the cells were infected with CVB3 in the presence of absence of different concentrations of proteasome inhibitor lactacystin (Calbiochem) or the general caspase inhibitor benzyloxycarbonyl-Val-Ala- Asp-fluoromethylketone (zVAD.fmk) (BD Biosciences). Antibodies The monoclonal anti-β-actin, anti-ubiquitin antibodies and anti-GAPDH antibodies were purchased from Sigma-Aldrich. The monoclonal anti-VP1 antibody was obtained from DakoCytomation. The ubiquitin siRNA, scramble control siRNA, horseradish peroxidase-conjugated secondary antibodies, the polyclonal anti-E6-AP, the monoclonal mouse anti-p53 (DO-1), the anti-caspase-3, and the anti-p21 (F-5) antibodies were 129 obtained from Santa Cruz Biotechnology. The proteasome inhibitors, MG132 and lactacystin, the UCH L1 inhibitor (LDN-57444) and the UCH L3 inhibitor (4,5,6,7- Tetrachloroindan-1,3-dione), the polyclonal anti-ubiquitin and anti-E1A/E1B antibody were obtained from Calbiochem. The polyclonal anti-3Dpol antibody was a generous gift from Dr. Karin Klingel (University Hospital Tuebingen, Germany). The polyclonal anti- UbcH7 was obtained from Chemicon. The polyclonal anti-UCHL1 was purchased from Abgent. The rabbit polyclonal anti-REGγ antibody was purchased from the Zymed Laboratories. Animals and in vivo viral infection For Aim 1, myocarditis-susceptible A/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). A total of 60 male A/J mice at age 4-5 weeks were randomized to four groups: sham+vehicle (n=10), sham+MLN353 (n=10), virus+vehicle (n=20) and virus+MLN353 (n=20). Mice were either infected intraperitoneally with 105 plaque forming units (PFU) of CVB3 or sham infected with PBS. Virus- or sham-infected mice were administered the proteasome inhibitor MLN353 subcutaneously (0.02 mg/kg, once a day for 3 days, i.e. one day prior to virus infection, 3 and 6 days post-infection) or vehicle (PBS). Mice were sacrificed on day 9 post-infection and infected heart was harvested for further analysis. All procedures were approved by the Animal Care Committee at the University of British Columbia (Vancouver, Canada). 130 Plasmid DNA and small-interfering RNA (siRNA) transfection For plasmid transfection in Aim 3, HeLa cells at ~90% confluence were transiently transfected with a plasmid expressing REGγ (pcDNA-Flag-REGγ)  or a construct encoding wildtype p53 (pCMV-p53) (Clontech) using lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. The empty vectors were transfected as a control. For siRNA transfection in Aim 2 and 3, HeLa cells were grown to 50% confluency and then transiently transfected with ubiquitin-specific siRNA (200nM, Santa Cruz) or a pool of four REGγ siRNA duplexes (Dharmacon) at a concentration of 30nM, using oligofectamine according to the manufacturer’s suggestion (Invitrogen). A scramble siRNA at the corresponding concentration was used as a control. The silencing efficiency was detected by immunoblot analyses using the anti-ubiquitin antibody or anti- REGγ antibody. After 24 h of transfection, cells were infected with CVB3 as indicated. Western blot analysis Cell or tissue lysates were harvested with lysis buffer (20mM Tris-HCl [pH 8.0], 100mM NaCl, 1mM EDTA, and 0.5% NP-40) containing proteasome inhibitor cocktail (Roche) as previously described . Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% of nonfat dry milk solution containing 0.1% Tween 20 for 1h. The membrane was then incubated for 1h with the primary antibody, followed by incubation with a horseradish peroxidase- conjugated secondary antibody for another 1h. Immunoreactive bands were visualized with an enhanced chemiluminescence detection system (GE Healthcare). 131 Plaque assay CVB3 titer in mouse heart homogenates (Aim 1) or cell supernatant (Aim 2 and 3) was determined on monolayers of HeLa cells by an agar overlay plaque assay in triplicate as described previously . Briefly, samples were serially diluted and overlaid on monolayer of HeLa cells. After 1 h incubation, medium was replaced with complete medium containing 0.75% agar. Cells were incubated for 72 h, then fixed with Carnoy's fixative (75% ethanol-25% acetic acid), and stained with 1% crystal violet. Plaques were counted and viral titer was calculated as plaque forming unit (PFU) per milliliter. Viral RNA in situ hybridization For Aim 2, HeLa cells were grown and maintained on two-chamber culture slides (Becton Dickinson Labware). Subconfluent cells were infected with either PBS or CVB3 (MOI=10). Following 1 h of incubation at 37°C, cells were washed with PBS and replenished with complete medium in the absence and presence of MG132. HeLa cells were incubated for an additional 6 h. The culture slides were then washed gently with PBS, fixed with formalin buffer for 15 min, and then air-dried at room temperature. Culture slides were then subjected to in situ hybridization assays to detect the sense- strand of CVB3 genomic RNA as previously described . Immunoprecipitation For Aim 2, cells were lysed using the above-described lysis buffer with freshly added 20 mM iodoacetamide. A total of 500µg of cell lysates were incubated with a monoclonal anti-ubiquitin antibody (1:100) at 4°C overnight, followed by 2 h incubation 132 with protein G-agorose beads (Amersham). Immunocomplexes were washed five times with the lysis buffer containing 20mM iodoacetamide, and then boiled for 5 min in the 2 × non-reducing sample buffer which lacks both β-mercaptoethanol and DTT, but with addition of 20mM iodoacetamide. After centrifugation, the precipitated proteins were separated by SDS-PAGE. Ubiquitin conjugates were analyzed by immunoblot using polyclonal anti-3Dpol antibody. Caspase-3 activity assay For Aim 3, HeLa cells transfected with REGγ or control siRNAs were infected with CVB3 for 18h, cell lysates were collected and caspase-3 activities were measured using a synthetic fluorogenic substrate (R & D Systems) following the manufacturer’s instruction. Cell viability assay For Aim 2 and 3 in vitro studies, cell viability assay was performed using the 3,4- (5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS, Promega) as previously described . Following treatment, cells were incubated with MTS solution for 2h and absorbance was measured at a wave length of 490 nm using an enzyme-linked immunosorbent assay plate reader. Morphological changes of cells were visualized by phase contrast microscopy. Histological grading and immunohistochemistry For Aim 1, mid ventricular portions of heart specimens were formalin-fixed, paraffin-embedded, and 4µm sections were cut and stained with hematoxylin and eosin 133 (H&E). Sections were graded blindly by an experienced pathologist for the severity of myocarditis based on the myocardial lesion area, cellular vacuolization, calcification, necrosis, and inflammatory infiltration as previously described [258, 259], with the following scales: 0, no or questionable presence; 1, limited focal distribution; 2-3, intermediate severity; and 4-5, coalescent and extensive foci over the entirety of the transversely sectioned ventricular tissue. Sections were also submitted for immunohistochemical staining. Briefly, sections were dewaxed and rehydrated, followed by antigen unmasking by heating. After blocking, sections were incubated with primary antibody (anti-E1A/E1B) overnight at 4°C, and then secondary antibody for 30 minutes at room temperature, followed by incubation with ABC and DAB reagents (Cell Signaling), and hematotoxylin staining. Sections were then dehydrated and mounted with coverslips. Proteasome activity assay For Aim 1 animal study, fresh heart homogenates, prepared as described above but in the absence of protease inhibitors, were used to measure proteasome activity as previously described . Briefly, ten micrograms of heart homogenates were added to an assay buffer (20 mM Tris-HCl [pH8.0], 1 mM ATP, and 2 mM MgCl2). The mixture was placed at room temperature for 10 minutes, and then incubated with 75 µM of synthetic fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, Calbiochem) at 30 °C for 1 hour. The fluorescence product AMC in the supernatant was measured at a 465 nm emission wavelength using a fluorometer. 134 Immunocytochemical staining For Aim 3, cells grown on glass slides were fixed with 4% paraformaldehe, permeabilized with 0.2% Triton-X100, and double labeled by indirect immunofluorescence with anti-REGγ and anti-VP1, followed by incubation with AlexaFluor 488 conjugated anti-rabbit or AlexaFluor 594 anti-mouse IgG (Molecular Probes), respectively. Nuclei were stained with 4’, 6 diamidino-2-phenylindole (DAPI) and cells were imaged and analyzed with a Leica SP2 AOBSTM confocal fluorescence microscope. In vitro and in vivo sumoylation assay For Aim 3, In vitro sumoylation assay was carried out with a sumoylation assay kit according to the manufacturer’s protocol (Biomol). In brief, 200nM purified recombinant REGγ (#BML-PW9875-0100, Biomol) was mixed with SUMO E1 (Aos1/Uba2), SUMO E2 (Ubc9), and SUMO in a reaction buffer in the presence or absence of Mg2+-ATP. The reaction mixture was incubated at 30°C for 1h and then quenched with SDS–PAGE loading buffer. The samples were separated by SDS–PAGE and Western blot analysis was performed with anti-SUMO antibody. For in vivo sumoylation assay, HEK293-REGγ inducible cells were treated with doxcycline for 48h, followed by transient transfection with a construct expressing SUMO-1 (pCMV3T-HA-SUMO-1), a generous gift from Dr. Louis Flamand at the Laval University, for another 48h. After 20h of mock or CVB3 infection (MOI=1), cell extracts were collected in NETN buffer and sumoylated REGγ was detected by an enzyme-linked immunosorbent assay (ELISA) using the EpiQuikTM in vivo universal protein 135 sumoylation assay kit following the manufacturer’s instruction (Epigentek). Briefly, equal amounts of proteins from the cell extracts were added to the strip wells which are pre-coated with either anti-REGγ antibody or IgG, and incubated in SUMO assay buffer for 1h at room temperature. 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Proteasome Activator REGγ enhances Coxsackieviral Infection via Facilitating p53 Degradation. J Virol. 2010; 84(21): 11056-66 2. Liu J, Yu G, Zhao Y, Zhao D, Wang Y, Wang L, Liu J, Li L, Dang Y, Wang C, Gao G, Long W, Lonard DM, Tsai M, Luo H, Li X. Regγ Promotes Cancer Development by Inactivating the Tumor Suppressor p53. J Cell Sci (in press). 3. Gao G, Zhang J, Si X, Wong J, Cheung C, Luo H. Proteasome Inhibition Attenuates Coxsackievirus-Induced Myocardial Damage in Mice. Am J Physiol Heart Circ Physiol. 2008; 295(1): H401-8 4. Si X, Gao G, Wong J, Wang Y, Zhang J, Luo H. Ubiquitination Is Required for Effective Replication of Coxsackievirus B3. PLoS One. 2008; 3(7) e2585 5. Gao G and Luo H. The Ubiquitin/Proteasome Pathway in Viral Infection. Can J Physiol Pharmacol. 2006; 84(1):5-14 6. Wong J, Zhang J, Si X, Gao G, Mao I, McManus B, Luo H. Autophagosome Supports Coxsackievirus B3 Replication in Host Cells. J Virol. 2008; 82(18): 9143- 53 7. Wong J, Zhang J, Si X, Gao G, Luo H. Inhibition of the Extracellular Signal- Regulated Kinase Signaling Pathway Is Correlated with Proteasome Inhibitor Suppression of Coxsackievirus Replication. Biochem Biophys Res Commun. 2007; 358(3): 903-907 8. Wong J, Zhang J, Gao G, Esfandiarei M, Si X, Wang Y, Yanagawa B, Suarez A, McManus BM, Luo H. Liposome-Mediated Transient Transfection Reduces Cholesterol-Dependent Coxsackievirus Infectivity. J Virol Methods. 2006; 133(2): 211-8 9. Si X. Luo H, Morgan A, Zhang J, Wong J, Yuan J, Esfandiarei M, Gao G, Cheung C, McManus BM. Stress-Activated Protein Kinases Are Involved in Coxsackievirus B3 Viral Progeny Release. J Virol. 2005; 79(22): 13875-81 156 B) Book Chapters Gao G, Yang D, McManus BM, Luo H. Chapter "Host Signaling Responses to Coxsackievirus Infection” in book “RNA Viruses: Host Gene Response to Infection”. World Scientific Publishing. New Jersey, London and Singapore, page 525-546, 2008 C) Abstracts Gao G, Zhang J, Wong J, Mao I, Luo H. Proteasome Activator REGγ Regulates Coxsackievirus Replication. 1st Conference: Proteomics of Protein Degradation and Ubiquitin Pathways. June, 2010, Vancouver, Canada. Gao G, Wong J, Zhang J, Mao I, Shravah J, Luo H. Role of Proteasome Activator REGγ in Coxsackievirus Replication. YI Forum Canada, May 2010. Gao G, Zhang J, Si X, Wong J, Luo H. Protective Effect of Proteasome Inhibitor on Coxsackievirus-Induced Myocarditis in Mice. 5th International Congress of Pathophysiology. June 2006, Beijing, China. Gao G, Zhang J, Si X, Wong J, Cheung C, Luo H. Proteasome Inhibition Attenuates Coxsackievirus-Induced Myocardial Damage in Mice. YI Forum Canada, April 2006.
UBC Theses and Dissertations
Role of the ubiquitin/proteasome system in coxsackievirus induced-myocarditis Gao, Guang 2010
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