@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Pathology and Laboratory Medicine, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Chau, David Hau Wing"@en ; dcterms:issued "2009-11-23T21:48:00Z"@en, "2004"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "Coxsackievirus B3 (CVB3)-induced myocarditis is a common heart disease in children and young adults. This viral infection can cause severe cardiac injury and patients may experience progressive heart failure, which may ultimately lead to dilated cardiomyopathy (DCM). Since the development of viral myocarditis involves complex interactions between host cells and the virus, the mechanisms by which CVB3 causes myocarditis and progression to DCM are still not well understood. The small CVB3 genome contains 11 genes, of which the proteases 2A (2A[sup pro]) and 3C (3C[sup pro]) are known to play critical roles in the virus life cycle by processing the viral polyprotein and in viral pathogenesis by cleaving the host proteins. Recent studies have demonstrated that overexpression of picornaviral protease 2A[sup pro] or 3C[sup pro] leads to caspase-3 activation and apoptosis. However, the molecular mechanisms that link these viral proteases to the induction of host cell death remain unclear. My dissertation addresses this issue by identifying cellular proteins that are cleaved and/or activated by these CVB3 proteases and the subsequent apoptotic pathways induced by these cleavages. In order to achieve this goal, full length CVB3 protease genes 2A and 3C were cloned into a eukaryotic expression vector using a PCR-based strategy. The resulting plasmids were then transiently transfected into HeLa cells. The transfected samples were subjected to Western blot analyses to detect cleavage (activation) of various cellular proteins. Upon protease expression, cell morphological alterations and reduction in cell viability are observed in both 2A[sup pro]- and 3C[sup pro]-transfected cells. Functional evaluation of 2A[sup pro] or 3C[sup pro] expression has also shown that caspase-3 and -8 are activated and their substrates, poly(ADP-ribose) polymerase (PARP) and Bid, are cleaved in these transfected cells. These results indicate that the expression of CVB3 2A[sup pro] or 3C[sup pro] in mammalian cells is sufficient to induce cell apoptosis through a caspase-dependent pathway. When other apoptotic pathways were explored, Western blot analyses of Bcl-2 family members demonstrated that only 3C[sup pro] but not 2A[sup pro] can up-regulate expression of Bax, a pro-apoptotic protein. However, expression of Bcl-2 protein, an anti-apoptotic member of Bcl-2 family protein, remains unchanged in both 2A[sup pro]- and 3C[sup pro]-transfected cells. Up-regulation of Bax and the presence of truncated Bid (tBid) further contribute to the release of cytochrome c from the mitochondria and activation of caspase-9. Finally, cleavage of host transcription factor, cyclic AMP responsive element binding protein (CREB) and translation initiation factors, eukaryotic translation initiation factor 4GI (elF4GI) and NAT1, by 2A[sup pro] or 3C[sup pro] are also observed. These proteolytic activities are accompanied by a severe inhibition of host mRNA and protein synthesis, which also contributes to the viral protease-induced cell death. In conclusion, the data suggest that the mechanism of apoptosis induced in CVB3 2A[sup pro]- or 3C[sup pro]-transfected HeLa cells is likely through multiple converging pathways. Firstly, both 2A[sup pro] and 3C[sup pro] can induce HeLa cell apoptosis through a caspase-dependent pathway. Secondly, 2A[sup pro] and 3C[sup pro] can also induce intrinsic mitochondria-mediated pathway through up-regulation of Bax and release of cytochrome c from mitochondria. Finally, cleavage of host transcription and translation initiation factors by 2A[sup pro] or 3C[sup pro] results in the inhibition of host protein expression, which further enhances apoptotic cell death."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/15553?expand=metadata"@en ; dcterms:extent "10556867 bytes"@en ; dc:format "application/pdf"@en ; skos:note "COXSACKIEVIRUS B3 PROTEASES 2A AND 3C INDUCE APOPTOTIC CELL DEATH THROUGH A MITOCHONDRIA-MEDIATED PATHWAY AND CLEAVAGE OF HOST FACTORS FOR TRANSCRIPTION AND TRANSLATION INITIATION by D A V I D H A U W I N G C H A U B . S c , S imon Fraser Universi ty, 2000 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department of Patho logy and Laboratory Med ic ine ; Facu l ty of Med ic ine W e accept this thesis as conforming to the required s tandard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A © David Hau Wing C h a u , 2004 FACULTY OF GRADUATE STUDIES ' THE UNIVERSITY OF BRITISH COLUMBIA Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Name of Author (please print) Date (dd/mm/yyyy) ntie of Thesis^ Cot^tbtfilj)rus> &3 firtitates 2 A Twi^us /•wfr Asb^ryj Degree: /W,Jc- Year: if Department of PATIIX I o & y i A ^ i L^o^^hr^) /U^>(?cfL^__ The University of British Columbia U Vancouver, BC Canada grad.ubc.ca/forms/?formlD=THS page 1 of 1 last updated: 2Q-M-04 ABSTRACT Coxsackievi rus B 3 (CVB3)- induced myocarditis is a common heart d isease in children and young adults. This viral infection can cause severe cardiac injury and patients may experience progressive heart failure, which may ultimately lead to dilated cardiomyopathy ( D C M ) . S ince the development of viral myocarditis involves complex interactions between host cells and the virus, the mechanisms by which C V B 3 causes myocarditis and progression to D C M are still not well understood. The small C V B 3 genome contains 11 genes, of which the proteases 2 A ( 2 A p r o ) and 3 C ( 3 C p r o ) are known to play critical roles in the virus life cycle by processing the viral polyprotein and in viral pathogenesis by cleaving the host proteins. Recent studies have demonstrated that overexpress ion of picornaviral protease 2 A p r o or 3 C p r 0 leads to caspase-3 activation and apoptosis. However , the molecular mechanisms that link these viral proteases to the induction of host cell death remain unclear. My dissertation addresses this issue by identifying cellular proteins that are c leaved and/or activated by these C V B 3 proteases and the subsequent apoptotic pathways induced by these c leavages . In order to achieve this goal , full length C V B 3 protease genes 2 A and 3 C were cloned into a eukaryotic expression vector using a P C R - b a s e d strategy. The resulting plasmids were then transiently transfected into H e L a cells . The transfected samples were subjected to Western blot analyses to detect c leavage (activation) of various cellular proteins. Upon protease expression, cell morphological alterations and reduction in cell viability are observed in both 2 A p r 0 - and 3C p r o - t ransfec ted cells . Functional evaluation of 2 A p r o or 3 C p r o expression has also shown that caspase-3 and -8 are activated and their substrates, poly(ADP-ribose) polymerase ( P A R P ) and Bid , are c leaved in these transfected cells. These results indicate that the expression of C V B 3 2 A p r o or 3 C p r o in mammalian cells is sufficient to induce cell apoptosis through a caspase-dependent pathway. W h e n other apoptotic pathways were explored, Western blot analyses of Bcl-2 family members demonstrated that only 3 C p r o but not 2 A p r o can up-regulate expression of Bax, a pro-apoptotic protein. However, expression of Bcl-2 protein, an anti-apoptotic member of Bcl-2 family protein, remains unchanged in both 2 A p r o - and 3C p r o - t ransfec ted cells. U p -regulation of Bax and the presence of truncated Bid (tBid) further contribute to the release of cytochrome c from the mitochondria and activation of caspase-9 . Finally, c leavage of host transcription factor, cyclic A M P responsive element binding protein ( C R E B ) and translation initiation factors, eukaryotic translation initiation factor 4GI (elF4GI) and N A T 1 , by 2 A p r o or 3 C p r o are also observed. These proteolytic activities are accompanied by a severe inhibition of host m R N A and protein synthesis, which also contributes to the viral protease-induced cell death. In conclusion, the data suggest that the mechanism of apoptosis induced in C V B 3 2 A p r o - or 3C p r o - t ransfec ted H e L a cells is likely through multiple converging pathways. Firstly, both 2 A p r 0 and 3 C p r o can induce H e L a cell apoptosis through a caspase-dependent pathway. Secondly, 2 A p r o and 3 C p r o can also induce intrinsic mitochondria-mediated pathway through up-regulation of Bax and release of cytochrome c from mitochondria. Finally, c leavage of host transcription and translation initiation factors by 2 A p r o or 3 C p r o results in the inhibition of host protein express ion, which further enhances apoptotic cell death. - iv -TABLE OF CONTENTS Abstract ii Table of Contents v List of Tables and Figures ix List of Abbreviations xi Acknowledgements xiii C h a p t e r O n e : In t roduc t ion 1.1 General information 1 1.1.1 Family of Picornaviridae and genus Enterovirus 1 1.1.2 Coxsackievirus: Role of Coxsackievirus B3 in myocarditis 5 1.2 Molecular genetics of CVB3 7 1.2.1 Genome sequence information and organization 7 1.2.2 Virus replication cycle 8 1.2.2.1 Viral receptors 8 1.2.2.2 Viral transcription and translation 8 1.2.2.3 Functional roles of proteases 2A and 3C in viral post-translational processes 10 1.3 Overview of cell death 14 1.3.1 Apoptosis: Cellular and molecular characteristics 14 1.3.2 Biochemical aspects of cell death 15 1.3.2.1 Caspases and caspase activation 15 - V -1.3.2.2 Effector caspases, cell surface receptor activation and mitochondrial signaling in regulation of cell death 15 1.4 Pathogenesis of CVB3 19 1.4.1 Cytopathic effects and apoptotic cell death induced by CVB3 19 1.4.2 Viral proteases in inducing cell injury 20 1.4.2.1 Cleavages of host proteins by viral proteases 20 i) . Viral 2A p r o 20 ii) . Viral 3C p r o 24 1.4.2.2 Functional significance of viral 2A p r o and 3C p r o in inducing apoptosis 32 1.5 Research focus and project rationale 34 Chapter Two: Hypothesis and Specific Aims 2.1 Hypothesis and specific aims 41 Chapter Three: Experimental Design, Materials and Methods 3.1 Cloning of CVB3 protease genes 2A and 3C into an eukaryotic expression vector 42 3.1.1 Cloning of protease genes 2A and 3C 42 3.1.2 Transformation of E. coli (DH5oc) and sequence analysis ....42 3.2 Transient transfection and cell culture conditions 43 3.3 Analysis of proteins by polyacrylamide gel electrophoresis 43 3.4 Immunoblot analysis 44 - vi -3.5 Cel l viability a s say 45 3.6 Ce l l culture and Wes te rn blot detection of cytochrome c release 45 3.7 Caspase -9 activity a s say 46 3.8 Statistical analysis of M T S assay 47 3.9 Limitations 47 C h a p t e r Fou r : R e s u l t s a n d D i s c u s s i o n 4.1 Ce l l death induced by C V B 3 2 A p r o and 3 C p r o 50 4.1.1 Overexpress ion of 2 A p r o or 3 C p r o induces morphological alterations...50 4.1.2 Overexpress ion of 2 A p r o or 3 C p r a reduces cell viability 50 4.1.3 C leavage of translation initiation factors induced by C V B 3 2 A p r a and 3 C p r o 53 4.1.3.1 Overexpress ion of 2 A p r o or 3 C p r o induces c leavage of e lF4GI . . . . 53 4.1.3.2 Overexpress ion of 3 C p r o induces c leavage of NAT1 53 4.1.4 C leavage of transcription factor induced by C V B 3 2 A p r o and 3 C p r o 57 4.1.4.1 Overexpress ion of 2 A p r o or 3 C p r o induces down-regulation of C R E B 57 4.1.5 C a s p a s e activation and c leavage of their respective substrates 59 4.1.5.1 Overexpress ion of 2 A p r o or 3 C p r o induces activation of caspase-3 and c leavage of P A R P 59 4.1.5.2 Overexpress ion of C V B 3 2 A p r o or 3 C p r o induces activation of caspase-8 and c leavage of Bid 62 - vii -4 . 1 . 6 A l t e ra t i on of e x p r e s s i o n of B c l - 2 f a m i l y m e m b e r 6 5 4 .1 .6 .1 O v e r e x p r e s s i o n of 3 C p r 0 u p - r e g u l a t e s e x p r e s s i o n of B a x , but not B c l - 2 6 5 4 . 1 . 7 O v e r e x p r e s s i o n of 2 A p r o o r 3 C p r o l e a d s to c y t o c h r o m e c r e l e a s e f r o m m i t o c h o n d r i a 6 8 4 . 1 . 8 O v e r e x p r e s s i o n of 2 A p r o o r 3 C p r o i n d u c e s c a s p a s e - 9 ac t i va t i on 7 0 Chapter Five: Discussion, Conclusions, and Future Directions 5.1 D i s c u s s i o n 7 2 5.2 S u m m a r y of r esu l t s 7 9 5 .3 C o n c l u s i o n s 81 5.4 Fu tu re d i r e c t i o n s 8 2 5.4.1 C l o n i n g of C V B 3 p r o t e a s e g e n e s 2 A a n d 3 C into a p r o k a r y o t i c e x p r e s s i o n v e c t o r 8 2 5 .4 .2 In vitro d i rec t c l e a v a g e a s s a y 8 3 5 .4 .3 W o r k C o m p l e t e d 8 3 5.4.3.1 C o n s t r u c t i o n of p l a s m i d s e x p r e s s i n g C V B 3 2 A p r o o r 3 C p r o 8 3 5 .4 .3 .2 O v e r e x p r e s s i o n a n d pur i f i ca t ion of p ro te i ns 8 3 References 8 6 - viii -LIST OF TABLES AND FIGURES T a b l e s : T a b l e l . Amino acid sequence homology among the proteins of coxsackie B viruses ( C V B ) and two other picornaviruses, poliovirus (PV) and human rhinovirus (HRV) 31 Table 2. List of primary antibodies used for Western blot analyses 49 F i g u r e s : Figure 1. Schemat ic diagram of the picornavirus caps id 4 Figure 2. The rate of cardiovascular diagnoses per 1,000 viral infections 6 Figure 3. Overview of the coxsackievirus replication cycle 12 Figure 4. G e n e organization and post-translational c leavage of C V B 3 polyproteins by 2 A p r o and 3 C p r o 13 Figure 5. Simplified schematic diagram summarizing cell surface signaling and mitochondrial signaling pathways leading to apoptosis 18 Figure 6. Summary of major host protein c leavages and downstream effects induced by viral 2 A p r o and 3 C p r 0 30 Figure 7. Structural homology between e lF4GI and N A T 1 , and function of the truncated NAT1 when cells undergo apoptosis 37 Figure 8. A model scheme illustrating the contribution of NAT1 in the presence of an apoptotic stimulus 38 Figure 9. Morphological alterations of H e L a cells express ing C V B 3 2 A p r o or 3 C p r o 51 Figure 10. M T S cell viability a s say 52 F igu re l 1. Analys i s of e lF4GI in H e L a cells transfected with C V B 3 2 A p r o or 3 C p r o gene 55 Figure 12. Immunoblot analysis of NAT1 c leavage 56 Figure 13. Down-regulation of C R E B in both C V B 3 2 A p r o - and 3C p r o - t ransfected H e L a cells 58 Figure 14. C leavage of procaspase-3 in gene-transfected H e L a cells 60 Figure 15. C leavage of P A R P in gene-transfected H e L a cells ......61 Figure 16. C V B 3 3 C p r o express ion induces activation of caspase-8 63 Figure 17. C V B 3 2 A p r o and 3 C p r o induce c leavage of Bid 64 Figure 18. Levels of Bax expression in C V B 3 2 A p r o - or 3C p r 0 - t ransfec ted H e L a cells 66 Figure 19. C V B 3 2 A p r o and 3 C p r o do not alter Bcl-2 expression in 2 A p r o - and 3C p r o - t ransfec ted H e L a cells 67 Figure 20. C V B 3 2 A p r o or 3 C p r o induces cytochrome c release from mitochondria 69 Figure 21 . Caspase -9 activity a s say in C V B 3 2 A p r o - or 3 C p r o - e x p r e s s i n g cells 71 Figure 22. Prediction of c leavage sites of 2 A p r o and 3 C p r o on e lF4GI and NAT1 77 Figure 23. Overexpress ion and purification of the recombinant C V B 3 3 C p r o using a prokaryotic express ion system 84 LIST OF ABBREVIATIONS 2 A p r o Protease 2 A 3 C p r o Protease 3 C 3 C D p r o Protease 3 C D 3 D p o l Polymerase 3D Apaf-1 Apoptotic protease activating factor-1 C 9 i Caspase -9 inhibitor, L E D H - C H O C A R Coxsackievi rus and adenovirus receptor C R E B cyclic A M P - r e s p o n s i v e element-binding protein C V B 3 Coxsackievi rus B3 D A F Decay-accelerat ing factor D A P 5 Death-associated protein 5 D C M Dilated cardiomyopathy D F F D N A fragmentation factor D T T Dithiothreitol E C L Enhanced chemiluminescence e l F 4 A Eukaryotic translation initiation factor 4 A e l F 4 E Eukaryotic translation initiation factor 4 E e l F 4 F Eukaryotic translation initiation factor 4 F e lF4GI Eukaryotic translation initiation factor 4GI elF4GII Eukaryotic translation initiation factor 4GII F A K Foca l adhesion kinase G S T Glutathione S-transferase - xi -I P T G Isopropyl - p-D-thiogalactopyranoside I R E S Internal r ibosome entry site moi Multiplicity of infection m R N A Messenge r R N A M T S 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium Oct-1 Octamer binding transcription factor-1 P A B P Poly(A) binding protein P A G E Polyacrylamide gel electrophoresis P A R P Poly(ADP-r ibose) polymerase P C R Polymerase chain reaction pi Post-infection pt Post-transfection P T B Poly-pyrimidine tract binding protein S D Standard deviation. S D S Sodium dodecyl sulphate tBid Truncated Bid T B P TATA-b ind ing protein T N F - a Tumor necrosis factor-a T R A I L Tumor necrosis factor-related apoptosis-inducing ligand t R N A Transfer R N A U T R Untranslated region X I A P X-l inked inhibitor of apoptosis protein - xii -ACKNOWLEDGEMENTS I would like to thank Dr. Decheng Yang, my thesis supervisor, for giving me the opportunity to work in his laboratory and providing me with hours of supervision and advices. He always motivates and encourages me throughout my research project. He has been a role model for me, and his dedication and commitment to science will always inspire me throughout my life. I wish to thank the members of my advisory committee, Dr. Wan Lam, Dr. Bruce McManus and Dr. Delbert Dorscheid, for their time, insights and contributions towards my research project. I would also like to thank the members in the laboratory for making the last few years so enjoyable. I would like to especially thank Huifang Zhang, Dr. Ji Yuan, and Dr. Paul Cheung for their contributions and comments that have helped me throughout my research project from the beginning to the end. I am forever indebted to my parents and sister for teaching me the importance of patience, hard working and motivation. Their continuous love and support gave me strength to overcome all my difficulties. Lastly, I would like to thank Lally Chan for all her kindly support and comfort throughout my graduate study. Without her love, support, and faith during the good times and difficult times this thesis would have never been materialized. Her strength and confidence in my success gave me encouragement I needed to follow my dreams to the end. - xiii -Chapter One: Introduction 1.1 General information 1.1.1 Family of Picornaviridae and genus Enterovirus The word Picornaviridae describes the small s ize (pico) of this group of virus and the type of nucleic acid (RNA) that constitutes the viral genome. The picornaviruses are non-enveloped viruses with a single-stranded R N A genome of positive polarity (from which R N A acts directly as the template for translation of viral proteins). The R N A genomes vary in length from 7209 to 8450 nucleotides and contain both 5' and 3' untranslated regions (UTRs) that are involved in regulation and initiation of viral transcription and translation. The 5' U T R s of picornaviruses are long (vary from 624 to 1199 nucleotides) and highly structured, whereas the 3' U T R s are short (vary from 47 to 125 nucleotides) and contain secondary structure responsible for viral R N A synthesis. The genome of picornaviruses is infectious because it can be translated upon entry into the host cell in order to produce all the viral proteins required for viral replication. Picornavirus virions are spherical in shape with a diameter of about 30 nm. The virus particles lack a lipid envelope but contain a protein shell, called caps id , which surrounds the naked R N A genome and renders the viral particle insensitivity to organic solvents. The caps ids of picornaviruses are composed of four structural proteins: V P 1 , V P 2 , V P 3 , and V P 4 , and a combination of 60 of these structural proteins are arranged in an icosahedral lattice (Rueckert et al., 1969) (Figure 1). The genome is covalently linked at the 5'-uridylylate moiety to a protein called V P g (virion protein, genome linked) through an 04-(5'-uridylyl)-tyrosine linkage (Flanegan et al., 1977; Lee et al., 1977). Al l picornavirus genomes are linked to V P g , which varies in s ize from 22 to 24 amino acid residues. V P g is encoded by a single viral gene in all picornaviruses except the genome of foot-and-mouth disease virus, which encodes three V P g genes (Forss et al., 1982). E v e n though V P g is expressed in every picornavirus, it is not required for infectivity of the viral R N A . In the case of poliovirus, the removal of V P g from the viral R N A by proteinase treatment does not lead to a reduction in specific infectivity of the viral R N A (Racaniel lo, 2001). V P g is removed from virion R N A by a host protein cal led unlinking enzyme (Ambros et al., 1978) and is present on nascent R N A chains of the replicative intermediate R N A and on the negative-stranded R N A s (Pettersson et al., 1978), which has led to the suggestion that viral R N A synthesis uses V P g as a \"primer\" (Nomoto etal., 1977; Pettersson et al., 1978). However , the role that V P g plays in viral R N A synthesis during picornaviral infection is still unclear. S ince 1898 hundreds of picornavirus serotypes have been discovered. The family Picornaviridae is comprised of six genera that are classified based on acid stability and buoyant density (Stanway, 1990). These include the aphthovirus, cardiovirus, enterovirus, hepatovirus, parechovirus, and rhinovirus, which all contain viruses that infect vertebrates. The genus enterovirus was initially named for their resistance to low pH and their replication in the alimentary tract. Enteroviruses are very small , approximately 20-30 nm in size, capable of withstanding low gastric pH, and are transmitted by the fecal-oral route. Many structures are conserved within this genus, namely the existence of the p -s t rand anti-parallel (3-sandwich of V P 1 - V P 4 ; however, the viral caps id proteins and loops connecting the (3-strands are different (Muckelbauer et al., 1997). This genus includes poliovirus (3 serotypes), coxsackievirus groups A and B (23 serotypes), echovirus (28 serotypes), human enterovirus (4 serotypes), and many non-human enteric viruses. Many similarities exist within the enteroviruses and classification of this group of viruses is based on their ability to cause similar d isease in murine models and their similar growth in cell cultures. Most of the history of enteroviruses is on poliovirus as this virus was the first enterovirus to be discovered (Landsteiner et al., 1909). In 1948, coxsackievirus group A wa s first isolated from the feces of paralytic children in Coxsack ie , N e w York, during a poliomyelitis outbreak (Dalldorf et al., 1948). In the following year, coxsackievirus group B wa s isolated from ca se s of aseptic meningitis (Melnick et al., 1949). This group of coxsakieviruses produced a general ized infection in newborn mice, presenting with myositis as well as involvement of the brain, pancreas, heart, and brown fat. Figure 1. S chemat i c d iagram of the p icornav i rus c a p s i d . Diagrammatic representation of the organization showing the pseudoequivalent packing arrangement of the caps id proteins, V P 1 , V P 2 , and V P 3 domains in a 60-subunit shell, of picornaviruses in an icosahedron. V P 4 is on the interior of the caps id and is not visible from the external face. (Adapted from Racanie l lo , 2001; Arnold etal., 1987). - 4 -1.1.2 Coxsackievirus: Role of Coxsackievirus B3 in myocarditis Enterovirus has been considered as an etiologic agent of viral myocarditis in humans. Coxsackievirus B3 (CVB3) as well as other coxsackieviruses of group B and group A, has been implicated in acute cardiac disease. Myocarditis is a very frequent autopsy finding in children who die of overwhelming coxsackievirus infection. In the World Health Organization global surveillance of viral diseases from 1975 to 1985, the coxsackievirus B group was ranked the number one causal virus group in the category of cardiovascular diseases, with approximately 35 cardiovascular diagnoses per 1,000 documented virus infections (Figure 2). This is followed by influenza viruses and many other enteroviruses, such as coxsackie A virus and poliovirus. The most frequent incidents of CVB-induced myocarditis occur in young adults, primarily between 20 and 39 years of age, with a higher prevalence among men (Woodruff et al., 1980). As much as 30% of human acquired dilated cardiomyopathy (DCM) is associated with an enteroviral infection of the heart, especially with coxsackievirus B infection (Baboonian et al., 1997). Recent studies have shown that dystrophin, a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary DCM, is proteolytically cleaved by CVB3 protease 2A (2Apro), which leads to functional impairment and morphological disruption in both cultured myocytes as well as intact mouse hearts infected with CVB3 (Badorff et al., 1999; Badorff et al., 2000). Such findings allow us to propose that the cleavage of dystrophin initiates a cascade of events during CVB3 infection that contributes to the pathogenesis of DCM. F i g u r e 2 . The rate of cardiovascular diagnoses per 1,000 viral infections. T h e resu l t of the g l o b a l s u r v e i l l a n c e o n v i ra l i n fec t i ons re l a ted to c a r d i o v a s c u l a r d i s e a s e s f r o m 1 9 7 5 to 1 9 8 5 c o n d u c e d by the W o r l d H e a l t h O r g a n i z a t i o n . T h e c o x s a c k i e v i r u s B g r o u p is the n u m b e r o n e c a u s a t i v e a g e n t in m y o c a r d i t i s . (G r i s t e r a / . , 1993 ) . 1.2 Molecular genetics of CVB3 1.2.1 Genome sequence information and gene organization CVB3 is a non-enveloped, single-stranded, positive polarity RNA virus. Its genome is approximately 7.4 kb long, which contains a single open reading frame flanked by 5' and 3' UTRs. The 5' UTR is unusually long (741 nucleotides) and contains an internal ribosome entry site (IRES) where the translation of viral RNA is initiated via a cap-independent IRES-mediated mechanism. The 3' UTR, on the other hand, is 99 nucleotides long, followed by a poly(A) segment. Both 5' and 3' UTRs can form highly ordered structures thereby regulating viral translation and transcription (Melchers et al., 1997; Yang etal., 1997). During infection, the coxsackieviral genome serves as a template to synthesize a single long polyprotein using the host cell translational machinery. This polyprotein is subsequently cleaved during translation, so that the full-length product is not observed. Cleavage is carried out by virus-encoded proteases to yield 11 to 12 final mature structural and non-structural proteins that are essential for viral replication, assembly, release, and re-infection. The detailed steps of post-translational process of viral polyprotein will be discussed later in Section 1.2.2.3. 1.2.2 Virus replication cycle 1.2.2.1 Viral receptors C V B 3 initiates infection of cells by first attaching to the host cell membrane through a host cell receptor, cal led coxsackievirus and adenovirus receptor ( C A R ) . It is a 46 k D a protein which not only binds to C V B 3 , but also mediates viral R N A entry into cells for viral replication. Other than C A R , C V B 3 also binds to another cell surface protein cal led decay-accelerat ing factor (DAF) or C D 5 5 , which is a member of the complement cascade . However, the interaction between C V B 3 and D A F is not sufficient for infection, it may require cxvp6-integrin as a coreceptor (Shafren et al., 1995). O n c e C V B 3 has attached to the cellular receptor, the viral R N A enters into the host cells and serves as a template for viral translation using the host translational machinery. 1.2.2.2 Viral transcription and translation Viral translation takes place entirely in the cytoplasm. Following attachment of the virus to the cell receptor, the R N A genome is uncoated, a process that involves structural changes in the caps id . Once the positive-stranded viral R N A enters the cytoplasm, it is immediately translated to produce viral proteins that are essential for viral R N A replication, assembly and release (Figure 3). Efficient translation initiation of most eukaryotic messenger R N A s ( m R N A s ) involves RNA-prote in and protein-protein interactions at both the 5' and 3' ends of the transcript through a cap-dependent mechanism. The recruitment of a 4 0 S ribosomal subunit and the recognition of the 5' 7-methyl guanosine ( m 7 G p p p N ) capped end of the m R N A require the recruitment of the eukaryotic initiation factor 4 F (e lF4F) complex, comprised of e l F 4 E , e l F 4 A and e l F 4 G , which associates with the multi-subunit e lF3 complex. The poly(A) tail serves as a translational enhancer and interacts with the 5' cap to initiate translation. This associat ion between the cap and poly(A) tail requires the poly(A) binding protein ( P A B P ) that binds to the N-terminus of e l F 4 G (Le et al., 1997; W a k i y a m a et al., 2001). However, the coxsackieviral R N A is not capped with a 7-methyl guanosine component as found in all eukaryotic transcripts. Instead, it is covalently linked to the virus-encoded protein V P g , as previously mentioned. Due to its uncapped but polyadenylated genome, the viral R N A employs an alternative mode of translation initiation through a novel mechanism. This cap-independent translation mechanism requires interaction between the r ibosome and the specific sequence element I R E S within the 5' U T R . The I R E S directs the landing of the small r ibosomal subunit within the 5' U T R of the viral R N A to initiate translation. Viral R N A replication begins by synthesis of a negative-stranded intermediate using the input positive strand R N A as a template. One of the viral polyprotein products, polymerase 3 D (3D p o 1 ) , is an RNA-dependen t R N A polymerase which is essential for this viral transcription process. The negative strand intermediate then serves as a template for multiple rounds of transcription to produce numerous progeny genomes . Then the progeny R N A is packaged into a mature caps id and is released from the cell (Figure 3). However , the exact mechanisms of viral release are unknown. Many picornaviruses are released as the host cell loses its integrity and lyses. Other picornaviruses, such as hepatitis A virus, are released from cells in the absence of cytopathic effects. Usually, a single replication cycle lasts five to ten hours, depending on the particular virus, temperature, pH level, type of host cell, and multiplicity of infection (moi). 1.2.2.3 Functional roles of proteases 2A and 3C in viral post-translational processes Like other enteroviruses, the entire coding region of CVB3 is a single open reading frame encoding a single, long, and continuous polypeptide (approximately 200 kDa). The coding region of CVB3 starts from nucleotide 741 and ends at 7299. This single polyprotein is translated using host cell machinery and then processed by virally-encoded proteases to generate 3 precursors P1, P2, and P3 (Figure 4). Structural proteins (VP1-VP4) are encoded in P1, while non-structural proteins (2A-2C and 3A-3D) are encoded in domains P2 and P3, respectively. The precursor P1 is encoded at the N-terminal end of the polyprotein and 2A p r o is encoded immediately downstream of the last capsid protein VP1. This protease cleaves the polyprotein at the P1-P2 junction in cis or as a co-translational event, and cleaves the cellular proteins in trans (an intermolecular mechanism). Viral 2A p r o autolyses between the C-terminus of the capsid protein VP1 and the N-terminus of 2A itself, which releases the P1 precursor (capsid protein) from the P2-P3 precursors (replicative domains). This \"primary\" cleavage occurs very rapidly while the polyprotein chain is still nascent -10-and is the most significant event in the separation of the structural from the non-structural polypeptide domains . In addition, 2 A p r o a lso recognizes a second c leavage site on the 3 C D precursor protein that gives rise to the products 3 C and 3D ' instead of the viral protease 3 C ( 3 C p r o ) and polymerase 3 D p o 1 ; however, this c leavage of protease 3 C D ( 3 C D p r o ) appears to be strain specific. Only some polioviruses utilize this pathway but not all, and the c leavage products are not essential for viral replication (Ryan et al., 1997; Krauss l ich et al., 1988). This \"secondary\" c leavage in the formation of 3 C and 3D' may permit the virus to modulate express ion of its caps id proteins and replication enzymes (Kraussl ich et al., 1988). Most of the remaining c leavages of the picornaviral polyprotein are mediated by the viral enzyme 3 C p r 0 . This protease catalyzes eight or nine c leavages within the polyprotein in order to release other viral proteins (Figure 4). Figure 3. Overview of the coxsackievirus replication cycle. Virus binds to the C A R and D A F receptors (1) and the genome is uncoated (2). V P g is removed from the viral R N A and the resulting R N A is then translated (3). The polyprotein is c leaved nascently to produce individual viral proteins (4). R N A synthesis occurs in ves ic les . Viral (+) strand R N A is transcribed by the viral R N A polymerase to form full-length (-) strand R N A (5), which is then copied to produce additional (+) strand R N A (6). During infection, newly synthesized (+) strand R N A is translated to produce additional viral proteins (7). Later in infection, the (+) strands enter the morphogenetic pathway (8). Newly synthesized virus particles are released from the cell by lysis (9). (Adapted from Racaniel lo , 2001). - 1 2 -< CO o Q . Q CO O CO DO CO < CO O CM DO CM 151 | C R Cu > CO CL > CM > CL > H r (0 > (0 u o i_ a o co a> \"(A o k CM a. > c \"55 o a. > o IDL Q CO O CO CO CO < CO a CM CQ CM < CM ^_ a. > CO CL > CM CL > CL > 0 CO CL Q CO (A a> * J 'co o ro > ro o o o k Q. o co CM CL I) O (A i_ 3 O C '53 *-> o i_ a >. o Q . o co OJ CL > < CO 0 O CM CA C \"53 o a ro J _ O 3 i_ <-» (A • C o CQ CM < CM CL > 4 CO CL > CM CL > (A C \"3 2 a. ro 3 •4-1 o 3 i_ *-> (0 T t CL > CL ^ 0) CD £ ~ S* CD O co CO > _ CO ^ 0 o g ^ CO CM -~ _ CO > o n o CO 3 c ca \"5 CD 2 r -Q. . > . CO II CD CO 0 CQ O c 2 o CO 0 D5 CO > CO _0 o -4—. c 0 C5 CT 0 CO 3 CO ** o 0 CM O __r Q.\"c5 — . CO 0 -*—' r-§1 CO c= CO O C c 0 E \"5 c CO CO c 0 .E 1 P Q- 0 2 0 CJ x: +-* -*—> g 0 c co to CO 0 co o 0 0 C £ 0 rn co vJ 0 T3 O O . C T j - 0 0 c 3 O CD O) E 2 0 CD ro o CO o 0 o g 0 I' C 0 2 to o =5 Q. ® >< E §.8> M _ CO o w < = CN 0 o 2 CO P; C O E 0 0 sz CO 1.3 Overview of apoptotic cell death Apoptosis , also referred to programmed cell death, is a genetically controlled process which is required to maintain the integrity and homeostasis of a multicellular organism (Jacobson et al., 1997; Nagata , 1997). It is an intrinsic self-eliminating mechanism which eliminates cells during development or during the continuous generation of the immune repertoire. It is also required in balancing cell division, maintaining the constancy of tissue mass , removing cells injured by genetic defects, aging, disease, or exposure to noxious agents (Golstein et al., 1991; Schwartz , 1998; Sa ikumar et al., 1999). 1.3.1 Apoptosis: Cellular and molecular characteristics Morphologically, cells undergoing apoptosis demonstrate cell shrinkage, nuclear/cytoplasmic condensation, membrane blebbing, fragmentation into membrane bound apoptotic bodies, and membrane alterations that eventually lead to phagocytosis of the affected cells. Biochemical ly, apoptotic cells are characterized by a reduction in the mitochondrial t ransmembrane potential, production of reactive oxygen species , externalization of phosphatidylserine residues in membrane bilayers, selective proteolysis of a subset of cellular proteins, and the degradation of D N A into internucleosomal fragments (Wyllie et al., 1984; Hockenbery et al., 1993; Lazebnik et al., 1994; Martin et al., 1995; Gottlieb et al., 1996; Z a m z a m i et al., 1996). - 14-1.3.2 Biochemical aspects of cell death 1.3.2.1 Caspases and caspase activation Apoptosis is regulated by a series of biochemical events (Saikumar et al., 1999). It is now clear that caspases , a family of aspartic acid-directed proteases, are key effector molecules in apoptotic cell death. C a s p a s e s are synthesized in the cytosol of mammal ian cells as inactive zymogens , which become active through intracellular ca spase cascades (Cohen, 1997). Once activated, ca spases cleave a large subset of specific downstream substrates, including poly(ADP-ribose) polymerase ( P A R P ) (Lazebnik et al., 1994), inhibitor of caspase-act ivated D N a s e (ICAD) (Liu et al., 1997) , focal adhes ion kinase (FAK) (Wen et al., 1997), gelsolin (Kothakota et al., 1997), lamin A (Takahashi et al., 1996a), and many others. S u c h c leavages result in disruptions of normal cellular processes and cause cellular morphological and structural changes . More importantly, caspase-dependent c leavage of specific proteins induces the abatement of survival pathways, such as the extracellular signal regulated kinase, which can interfere with the apoptotic response (Widmann et al., 1998). 1.3.2.2 Effector caspases, cell surface receptor activation and mitochondrial signaling in regulation of cell death Apoptosis can be triggered by two distinct pathways, one initiated by extracellular signals and the other by intracellular signals. These two independent pathways converge at the activation of downstream effector caspases , such as caspase-3 , -6, and -7. The first major pathway involves -15-ligation of the death receptors by their ligands, such as ligation of F a s L and tumor necrosis factor-a (TNF-a) with their respective receptors, resulting in the recruitment of adaptor proteins and procaspase-8 molecules to the receptors to transactivate caspase-8 . The activated caspase-8 will either activate the downstream caspases directly or induce direct c leavage of Bid , a member of the Bcl-2 family, to yield a truncated Bid (tBid). Upon c leavage, tBid can then translocate from the cytosol to the mitochondrial membrane, resulting in the release of cytochrome c, which amplifies the death signals. Mitochondrial release of cytochrome c can also be triggered by various cellular stresses, such as D N A damage, toxins, and A T P depletion. The released cytochrome c, which binds to apoptotic protease activating factor-1 (Apaf-1), activates procaspase-9 to active caspase-9 . This initiator ca spase will c leave and activate the downstream effector caspases , such as caspase-3 , inducing cell apoptosis (Figure 5). The second major pathway, the intercellular/intrinsic pathway, could be directly stimulated when cells expose to various apoptotic stimuli or stress as mentioned above. This pathway is regulated by the Bcl-2 family proteins. The Bcl-2 family members consist of both anti-apoptotic proteins (such as Bcl-2, B c l -xL, and Bcl-w) which inhibit ca spase activation, as well as pro-apoptotic proteins (such as Bax, Bak, and Bid) which promote caspase activation. Anti-apoptotic members act not only by direct binding to Apaf-1 to inactivate it, but also by stabilizing mitochondrial membranes to inhibit the release of cytochrome c. O n the other hand, a pro-apoptotic member like Bax translocates to mitochondria - 16-and releases cytochrome c by selectively permeabilizing the outer mitochondrial membrane (Eskes era/ . , 1998). Unlike Bax, Bid requires proteolysis by caspase -8 for its pro-apoptotic function (mechanism as descr ibed above). S u c h caspase -8-mediated c leavage of Bid may be a mechanism des igned to amplify downstream effector events during death-receptor-mediated apoptosis (Saikumar era/ . , 1999) (Figure 5). - 17-S? o O ro = \"co < 3 CJ .SP «- \"O CO (A b C Ja CO co c re te CL i_ \" Vi Q . £ 1 3 CL JD CO •D ^ TJ C o CO o £ sz CO > o CD CO ~c c <0 CD o co co CL o CD W S O) • I CO = ro ® i - aS tf) CO CO o CL 0 CO t tf) O CB o O CD *- 0 5 .h= o 0 5 JC > T ~ o CO c -= ® .2 75 CD m CO CD 2 CO C tf) o CD a> co >,.> O 0 JD \" K ; CO CJ CO o> 2 £2 E c N 0 m i_ u u E 2 1 o o 3 St tf) 0 o r ^ *-> ± 3 Q. CO O O CO CL 1 co - Q r— C5 T _ O 75 CJ C *1 o .2 Q CO CO tH . i i 0 CO CO 1 o \"D J= c 8 CO CD CO S -J2 ro € id 0 g co E co S.8 E . 2 > O CO O CO o to o a 0.4= (0 co co o CJ CO -g — CO CD. 2- SP ro ^ §. E .E 0 ro § o — CO 0 LO tf) >» ro SI EP to co c o co E o w L. / \\ to SZ ~ CO o CL o Q . CO - t i c ro >• E 00 L L a CJ O CO 1.4 Pathogenesis of CVB3 1.4.1 Cytopathic effects and apoptotic cell death induced by CVB3 Apoptosis can also be considered as a host defence mechanism against virus infections to limit viral replication and to prevent the virus from spreading to surrounding uninfected cells. A n increasing number of viruses is known to induce apoptosis at the late stages of infection (Cuff et al., 1996; Tsunoda et al., 1997a&b; Carthy et al., 1998; Girard ef al., 1998). Severa l picornaviruses, including coxsackievirus and poliovirus, have been demonstrated to induce apoptotic cell death (Tolskaya et al., 1995; Carthy et al., 1998; Ghadge et al., 1998). Previous studies from our laboratory observed C V B 3 - i n d u c e d cytopathic effects in an in vitro H e L a cell model (Carthy et al., 1998) and virus-induced direct injuries in multiple susceptible organs, including the myocardium, in infected mice (Carthy, 2002). At day three post-infection (pi) of mouse hearts, coagulation necrosis and cytopathic effects were observed, followed by immune cell infiltration and cardiac remodeling at day nine and day 30 pi, respectively (Carthy, 2002). Biochemical ly, the 32 kDa procaspase-3 is found to be c leaved and activated following degenerative morphological changes observed in infected H e L a cells. This c leaved caspase-3 is proteolytically active and further c leaves a number of substrates, including P A R P and I C A D (Carthy ef al., 1998). T h e s e proteolytic activities result in alterations of normal cellular homeostasis and cellular morphological changes . Upon virus infection, apoptosis of infected cells can also be induced by cytotoxic T cells through the F a s / F a s L pathway and by granzyme B through - 1 9 -caspase activation (Saikumar et al., 1999). In previous studies, we have demonstrated that caspase-3 is activated in CVB3- infec ted H e L a cells eight h pi (Carthy et al., 1998). This result suggests that ca spase activation occurs after infection. Furthermore, recent studies have shown that overexpression of the polioviral 2 A p r o or 3 C p r 0 alone leads to caspase-3 activation and apoptosis (Barco et al., 2000; Goldstaub et al., 2000). However, further studies are required since it is still unknown whether caspase-3 is directly activated by these viral proteases or indirectly activated through the different death cascades , such as the death receptor-mediated and/or intrinsic mitochondria-mediated pathway. 1.4.2 Viral proteases in inducing cell injury 1.4.2.1 Cleavages of host proteins by viral proteases i). Viral 2 A p r o 2 A p r o is a small cysteine protease, approximately 17 k D a , which shares homology with the bacterial trypsin-like serine protease (Ryan et al., 1997; Y u et al., 1991). The frans-cleavage activity of 2 A p r 0 involves processing an increasing list of host cellular proteins, including e l F 4 G (Bovee et al., 1998a&b; Novoa et al., 1999) and other translation initiation factors (Figure 6). Over the past decade, extensive studies have been performed on the 2 A p r o frans-cleavage of e l F 4 G . Investigations have demonstrated that purified recombinant 2 A p r 0 of coxsackievirus B4 and human rhinovirus type 2, as well as poliovirus, can c leave e l F 4 G at Arg485-Gly486 directly in vitro (Lamphear et al., 1993; Bovee et al., 1998a&b). C leavage of e l F 4 G dysregulates host cell metabolism by abruptly - 2 0 -halting host protein synthesis. During viral infection, complete c leavage of e l F 4 G occurs very rapidly, approximately two h pi. This c leavage event and subsequent abatement of host protein synthesis lead to the abolishment of cap-dependent cellular m R N A translation and the initiation of cap-independent viral m R N A translation. Interestingly, earlier studies have shown that host protein synthesis is suppressed by 50% or less after poliovirus infection, despite complete e l F 4 G cleavage (Lloyd et al., 1987; Pe rez et al., 1992). These findings suggested that complete inhibition of host protein synthesis may require proteolysis of other factors for translation initiation. Recently, a novel human homologue of e l F 4 G , called elF4GII (the original e l F 4 G was renamed e lF4GI) , has been reported as the second c leavage target required for complete inhibition of host protein synthesis (Gradi et al., 1998a&b). elF4GII appears to be functionally equivalent to e l F 4 G I , but is only 4 6 % identical at the amino acid level and has a 56% overall similarity to e l F 4 G I (Gradi et al., 1998a). Both poliovirus and human rhinovirus 2 A p r 0 were found to cleave elF4GII, with s lower kinetics as compared to c leavage of e lF4GI , and the c leavages result in the complete shutoff of host protein synthesis (Svitkin et al., 1999; Goldstaub et al., 2000). Thus , c leavage of both e lF4GI and elF4GII appears to be required for the abolishment of host protein synthesis after virus infection. These requirements may explain several earlier reports documenting the lack of correlation between e lF4GI c leavage and the inhibition of cellular m R N A translation after enterovirus infection. Most significantly, e lF4GI and elF4GII c leavages may trigger host cell apoptosis either by inhibiting the cap--21 -dependent translation of cellular m R N A s that encode proteins required for maintaining cell viability or by enhancing cap-independent translation of cellular m R N A s , such as c-myc and Apaf-1 (Johannes et al., 1998), which contain I R E S and encode a pro-apoptotic (death-associated) protein. Another host protein target of 2 A p r o is the P A B P (Joachims et al., 1999; Kerekatte et al., 1999) (Figure 6). The c leavage of P A B P occurs at the C -terminal domain (Met490-Gly491) of human, separating the N - (recognition motifs) and C-terminus (homodimerization domain). This c leavage can be mediated by both poliovirus 2 A p r o and coxsackievirus (B3 and B4) 2 A p r o . The resulting N-terminal c leavage product of P A B P was found to be less efficient in promoting translation than the full-length P A B P . Furthermore, P A B P c leavage does not occur during infection in the presence of guanidine-HCI, an inhibitor which prevents host translation obstruction. This data suggests that c leavage of P A B P may play a role in altering host protein synthesis upon virus infection. It is also important to note here that P A B P is involved in translation initiation in lower eukaryotes via its interaction with the poly(A) tail of m R N A s . Poly(A) tail serves as a translational enhancer and interacts with the 5' cap to initiate translation in yeast (Preiss et al., 1998). This associat ion between the cap and poly(A) tail requires the binding of P A B P to the N-terminus of e lF4GI (Imataka et al., 1998). Bes ide translation initiation factors, host proteins involved in transcription are also proteolytic targets of 2 A p r o , such as TATA-b ind ing protein (TBP) (Das et al., 1993; Yalamanchi l i et al., 1997a) (Figure 6). T B P plays an essential role in promoting R N A polymerase II- and Ill-mediated transcriptions. T B P has been -22-demonstrated to be c leaved directly at the N-terminal domain (Tyr35-Gly36) by poliovirus 2 A p r o . Surprisingly, this c leavage of T B P does not alter its transcriptional activity and the R N A polymerase ll-mediated transcription is not inhibited. This suggests that T B P c leavage by 2 A p r 0 may not be important in inhibiting host transcription. Recently, the functional roles of 2 A p r 0 in viral pathogenesis through c leavage of cytoskeletal proteins have been documented (Figure 6). Dystrophin is a cytoskeletal protein which connects the internal F actin-based cytoskeleton to the p lasma membrane where it binds to the p-dystroglycan component of the dystrophin-glycoprotein complex (Badorff et al., 2000). Mutations in dystrophin that result from premature termination of translation can cause human X-l inked D C M . C V B 3 has been postulated to cause D C M ; however, the pathological mechanism remains unknown. Dystrophin has been reported to be c leaved by C V B 3 2 A p r o directly in vitro and in vivo (Badorff et al., 1999; Badorff er al., 2000). Dystrophin contains four hinge segments that are access ib le for proteolytic c leavage and C V B 3 2 A p r o - m e d i a t e d cleavage was found to be at the site (human: amino acid 2434; mouse: amino acid 2427) in the hinge 3 region. C l e a v e d dystrophin can lead to functional impairment and morphological disruption of heart muscle cells. However, the detailed pathogenic mechanism by which enterovirus infection begets D C M needs to be studied further. Cytokeratin 8 is another structural protein c leaved by 2 A p r o (Seipelt et al., 2000) (Figure 6). Cytokeratin 8 is a member of the intermediate filament family that forms the cytoskeleton together with actin filaments and microtubules. -23-Col lapse of the intermediate filament network leads to the deterioration of mechanical support and the stability of cells. C leavage of cytokeratin 8 by 2 A p r o of human rhinovirus type 2 was found to be at Se r14-Gly15 and results in the removal of 14 amino acids from the N-terminal domain. This c leavage is highly specific s ince other intermediate filament proteins, such as cytokeratin 18 and vimentin, are not c leaved by 2 A p r o . C leavage of cytokeratin 8 is speculated to destabilize the host cell and thus promote the spread of the virus. However, the molecular basis of cytokeratin 8 c leavage in contributing to the virus pathogenesis has not been elucidated. ii). Viral 3CPRO Another cysteine protease found in coxsackievirus is the 3 C p r o . In addition to processing the viral polyprotein, 3 C p r o (20 kDa) also specifically c leaves a number of host proteins, including factors in both translation and transcription and proteins that regulate host cell survival and death. A major host protein c leaved by viral 3 C p r o is the L a autoantigen (Figure 6). L a is a cellular protein that has been demonstrated to stimulate internal r ibosomal entry, a mechanism by which picornaviruses initiate translation of its genomic R N A . Other physiological functions of L a protein include transfer R N A (tRNA) processing, synthesis of polymerase II transcripts, and formation of s n R N P complexes as well as ribosomal binding (Gottlieb et al., 1989a&b; Peek et al., 1996; Pel l izzoni er al., 1996). C leavage of L a by the poliovirus 3 C p r o occurs in the C-terminal region (between Gln358 and Gly359) (Shiroki et al., - 2 4 -1999), and such modification causes L a to relocate into the cytoplasm from the nucleus. This c leavage is believed to help improve the translation efficiency of the virus. Recently, L a autoantigen has been demonstrated to bind to the 5' U T R of C V B 3 R N A genome (Cheung et al., 2002; R a y et al., 2002). S u c h a specific interaction between the L a autoantigen and the C V B 3 R N A is believed to promote viral translation. Another major host protein c leaved by viral 3 C p r o is the poly-pyrimidine tract binding protein (PTB) (Figure 6). P T B is found to be pertinent to picornaviral translation initiation (Kaminski et al., 1995; Hunt et al., 1999). It has been shown to bind viral 5' U T R and cooperate with the L a autoantigen in optimal and accurate translation initiation of the viral genome (Toyoda et al., 1994). C leavage of the P T B by poliovirus 3 C p r o occurs in three different locations which generate multiple peptides that appear to inhibit IRES-dependent translation (Back et al., 2002). It has been suggested that the c leavage event promotes a molecular switch from viral protein express ion to viral genome replication (Back et al., 2002). However, the molecular mechanisms that signal the appropriate time of transition v ia the 3 C p r o remains unknown. C leavage of e lF4GI and P A B P by viral 2 A p r o has been propose d to cause severe translation inhibition in virus-infected cel ls . However , these c leavages have been shown to be insufficient for complete translation inhibition, and only lead to a partial translation shutoff (Bonneau et al., 1987; Pe rez et al., 1992). A recent study has demonstrated that polioviral 3 C p r 0 cleaves P A B P and removes the C-terminal domain (Kuyumcu-Mart inez et al., -25-2004), which interacts with several translation initiation factors, such as the translation initiation factor e l F 4 B . C leavage of P A B P by 3 C p r o inhibits translation of endogenous m R N A and reporter R N A . This 3 C p r o - m e d i a t e d translation inhibition is poly(A)-dependent and is as effective as complete c leavage of e lF4GI and elF4GII by 2 A p r 0 . This c leavage event has raised the functional significance of P A B P in the regulation of translation and has suggested that enteroviruses use a dual strategy for host translation shutoff, requiring both c leavage of P A B P by 3 C p r o and c leavage of e lF4GI and elF4GII by 2 A p r 0 3 C p r o inhibits host protein express ion not only at the translational level, but also at the transcriptional level. Many early studies have been conducted to elucidate the mechanism of transcriptional inhibition and a number of transcription factors have been demonstrated to be c leaved directly by viral 3 C p r o . One of these key proteins is T B P (Berk, 1999; Pugh , 2000; Ge iduschek et al., 2001) (Figure 6). T B P has been shown to be directly c leaved by 3 C p r o during poliovirus infection, resulting in an inhibition of R N A polymerase ll-mediated transcription (Yalamanchil i et al., 1996). Unlike 2 A p r o , this inhibition indicates that T B P c leavage by 3 C p r o is more important in the regulation of host transcription than proteolysis by 2 A p r o . Another pivotal player in 3 C p r o - m e d i a t e d transcription inhibition is the cyclic A M P (cAMP)-respons ive element (CRE)-binding protein, C R E B , (Figure 6). It is c leaved by 3 C p r o between amino acid residues 172 and 173 (Yalamanchil i et al., 1997b), resulting in the separation of its D N A binding domain from the transcription activation domain. Recently, C R E B has also been demonstrated to - 2 6 -have an anti-apoptotic effect upon induction of phosphorylation at serine residue 133 by insulin-like growth factor-l in a phosphatidylinositol-3-OH-kinase-dependent and mitogen-activated protein kinase-dependent manner (Mehrhof et al., 2001). A variety of stimuli, including those regulating the intracellular levels of c A M P and C a 2 + , growth factors, and cellular stress, induce C R E B activation leading to the up-regulation of Bcl -2 (Riccio et al., 1999; Mehrhof et al., 2001). Al so , there have been reports of caspase-mediated degradation of C R E B during neural cell apoptosis (Francois et al., 2000). Thus , the c leavage of C R E B by 3 C p r 0 may disrupt not only the R N A polymerase ll-mediated transcription but also a variety of cellular processes that are involved in cell survival and death. As ide from the transcription factor C R E B , 3 C p r 0 a lso c leaves Octamer binding transcription factor-1 (Oct-1) (Figure 6), a host protein involved in both R N A polymerase II- and Ill-mediated transcription (Yalamanchil i et al., 1997c; Ge iduschek et al., 2001). Oct-1 has been implicated in the up-regulation of inducible nitric oxide synthase, an enzyme that catalyzes the synthesis of nitric oxide (Sawada et al., 1997; Lee er al., 2001). Thus , c leavage of Oct-1 by 3 C p r o may alter the production of N O and hence protect the virus against early immune responses . However , further investigation is needed to verify the link and the functional significance of the 3 C p r 0 c leavage of Oct-1 in the overall pathogenesis of the virus. Other than the R N A polymerase ll-mediated transcription, 3 C p r o a lso inhibits R N A polymerase Ill-mediated transcription through c leavage of the transcription factor IMC (TFIIIC) (Figure 6). TFIIIC is a large complex, containing -27-five subunits of 240, 110, 100, 80, and 60 kDa , and binds the B-box internal promoter element of t R N A genes . TFIIIC is c leaved and inactivated by 3 C p r o during poliovirus infection (Shen et al., 1996). Shen et al. demonstrated in vitro that an N-terminal 83 k D a domain of the largest subunit (240 kDa) associates with the 110 k D a subunit to generate the TFIIIC D N A binding domain. Although the D N A binding activities of the c leavage products are unchanged, 3 C p r o cleavage of TFIIIC inactivates its transcriptional activity. S ince the c leaved forms of TFIIIC that bind to the B-box promoter element appear to lack the 100, 80, and 60 k D a subunits and are defective for transcription, it suggests that at least one of these subunits is required for TFIIIC's function in transcription. Al l of these c leavages by both picornaviral 2 A p r o and 3 C p r o lead to a decrease in cellular transcription and translation, resulting in the down-regulation of the express ion of many host genes and proteins. S u c h c leavages also lead to an imbalance in cellular homeostasis , which ultimately may lead to host cell death and myocardial remodeling. However, many of the c leavages descr ibed above were studied in other picornaviral models, such as poliovirus and human rhinovirus, and there is currently no documented evidence showing that C V B 3 will perform the same c leavages or process the same set of translation and transcription factors. More importantly, the amino acid sequence homologies of the coxsackieviral proteins are highly conserved among coxsackie B viruses but not as highly conserved to the respective proteins of other picornaviruses (Table 1). Table 1 shows that both C V B 3 2 A p r o and 3 C p r o share over 90% homologies with their counterparts of other coxsackie B viruses; however, they share only -28-60% and 50% homologies with poliovirus (Enterovirus) and human rhinovirus (Rhinovirus), respectively. Therefore, it remains to be elucidated whether C V B 3 2 A p r o and 3 C p r o will a lso affect cellular homeostasis by processing similar target proteins that are involved in viral pathogenesis. -29-CD jy j _ _ W _ LL O i C_ I s ' 03 D) £ a _ ro O ^ > c w O CO CD +—< _2 Q-< O < CD CM CO Table 1 . Amino acid sequence homology among the proteins of coxsackie B viruses (CVB) and two other picornaviruses, poliovirus (PV) and human rhinovirus (HRV). (Taken from Jenkins et al., 1 9 8 7 ; Klump et al., 1 9 9 0 ) . Homology between* Protein CVB3:CVB1 CVB3:CVB4 CVB3:PV1 CVB4:PV3 CVB4:HRV14 VP4 9 7 9 4 6 5 7 1 6 1 VP2 8 3 8 0 5 1 5 6 5 7 VP3 8 1 7 9 5 6 5 6 4 7 VP1 7 7 6 9 3 9 4 5 3 6 2 A p r o 94 92 58 59 44 2B 9 8 9 9 5 0 5 1 5 6 2C 9 8 9 8 6 2 6 3 5 9 3A 9 8 9 3 5 2 5 1 4 8 3B/VPg 9 1 9 1 7 7 7 3 4 1 3 C p r o 99 98 55 61 53 3 D p o l 9 6 9 7 8 0 7 4 6 6 Sequence identities are expressed as percentages. -31 -1.4.2.2 Functional significance of viral 2 A p r o and 3 C p r o in inducing apoptosis Death and lysis of cells during virus infection facilitates the release of the virus progeny (Carrasco, 1995). However, premature induction of cell death upon virus infection will severely limit virus production, reducing the spread of the progeny in the host. Therefore, the degree of host cell lysis and death should be controlled in a timely manner. A n increasing number of viruses is known to induce apoptosis in an active fashion at late stages of infection. This process may provide an efficient way to help the virus spread to neighboring cells, to protect progeny viruses from host immune defenses, and to avoid an inflammatory response (Teodoro et al., 1997; O 'Br ien , 1998). Apoptos is induced by the picornaviruses remains an interesting and important observation because the persistence of picornaviral infection may be linked to the ability of the virus to propagate in cells with minimal elicitation of immune responses . Further investigations into the molecular mechanisms by which 2 A p r o and 3 C p r o lead to cellular apoptosis in a time-specific manner can yield insights potentially relevant to the treatment and understanding of persistent picornaviral infections. At the present time, very little is known concerning specific coxsackieviral protease(s) that is/are directly responsible for inducing apoptosis, but the involvement of both viral 2 A p r o and 3 C p r o has been strongly implicated. Within the Picornaviridae family, recent studies have demonstrated that express ion of the polioviral 2 A p r o or 3 C p r o alone leads to caspase-3 activation and apoptosis (Barco et al., 2000; Goldstaub et al., 2000). Moreover , recent studies -32-have shown that transient expression of 2 A p r a or 3 C p r o of enterovirus 71 results in the c leavage of the D N A repair enzyme P A R P (Li et al., 2002), indicating that viral proteases may directly or indirectly activate caspases , which in turn leads to apoptotic responses . A s previously mentioned, viral 2 A p r 0 and 3 C p r o may induce apoptotic cel l death through c leavages of a number of host cellular proteins involved in transcription, translation initiation, and cytoskeletal organization. There is also a suggestion that these viral proteases may mediate delayed apoptotic responses through a number of unknown host protein c leavages , such as the degradation of p53 (Weidman et al., 2001). A recent report demonstrated that p53 can also induce apoptosis by perturbations of the mitochondria, resulting in changes in the trans-membrane potential, cytochrome c release, and caspase activation (Regula et al., 2001). Thus , the c leavage of p53 by the viral protease(s) may be yet another strategy that viruses use to modulate host responses and, thereby, enhance viral propagation (Weidman et al., 2001). In conclusion, investigation of the mechanism by which C V B 3 induces apoptotic cell death by viral protease express ion will provide a better understanding of the pathogenesis in the induction and progression of C V B 3 -induced myocarditis. -33-1.5 Research focus and project rationale C V B 3 - i n d u c e d myocarditis and its sequela , D C M , are major heart d iseases in children and young adults (Woodruff, 1980; Grist et al., 1993; Hosenpud et al., 1994). Molecular techniques from our laboratory and many others have identified and localized viral R N A in the infected mouse heart and other organs using in situ hybridization, all of which indicates that C V B 3 plays an important role in these d iseases . However, the molecular mechanisms, by which C V B 3 causes initiation of myocarditis and progression to D C M , are not well understood. A s a result, there are still no virus-specific preventive and therapeutic procedures available at the present time for protecting humans against such virus-induced heart muscle d iseases . The development of viral myocarditis is a complicated interaction between host and virus. Earlier studies have suggested that the mechanisms of viral myocarditis include direct myocyte injury by C V B 3 (Lodge et al., 1987; C h o w et al., 1992; M c M a n u s et al., 1993) and immune- or autoimmune-mediated damage of the heart induced by viral infection, typically with inflammation. Previous studies carried out on mice with severe combined immunodeficiency have shown that C V B 3 is capable of lytic damage to myocytes (Chow et al., 1992; M c M a n u s et al., 1993). A l so , our previous data on the activation of various caspases by CVB3-infect ion and subsequent induction of cytopathic effect at nine h pi on H e L a cells suggest that C V B 3 induces cell death directly (Carthy et al., 1998). In previous work using a differential m R N A display technique, our laboratory had identified 28 genes that were either up- or down-regulated in the -34-CVB3-infec ted mouse heart (Yang et al., 1999). The gene express ion patterns of 15 of the 28 genes were further confirmed by either Northern hybridization or R T -P C R . Thus, these genes may have potential functions in contributing to the development of viral myocarditis. A m o n g these genes, N A T 1 , also called death-associa ted protein 5 (DAP5) , is one of the most interesting candidates involved in d isease induction. NAT1 is a 97 k D a protein which is found to be down-regulated in the CVB3- infec ted mouse heart and possesses unique structural and functional characteristics. It is predominantly expressed in atrial and ventricular myocytes and plays important roles in the postnatal development of the heart (Pak et al., 1999). More importantly, it is a structural homologue of e l F 4 G I (Figure 7). The N-terminal part of NAT1 has 39% identity and 6 3 % similarity to the central region of e lF4GI , while its C-terminal part is less homologous to the corresponding region of e lF4GI , suggesting that it may possess unique functional properties (Imataka et al., 1997; Levy-Strumpf et al., 1997; Kimchi , 1998). NAT1 m R N A has no A U G initiation codon and translation starts at G U G codon to produce a 97 k D a protein. This protein lacks the N-terminal region of e lF4GI , which is responsible for associat ion with cap-binding protein e l F 4 E . A s a result, NAT1 performs as a translation repressor in a normal cell environment. In a recent study, ca spase was found to cleave NAT1 at a conserved site to yield a novel C -terminal truncated 86 k D a protein that promotes cap-independent translation. Another recent study has shown that translation rate of NAT1 in apoptotic cells w as selectively maintained, while the translation rate of other cellular proteins in -35-apoptotic cells was reduced by 60 to 70% with the degradation of the cap-dependent translation mediators, e lF4GI and elF4GII. These results suggested that NAT1 is a caspase-act ivated translation factor, which mediates cap-independent translation, at least for its own R N A , in cells undergoing apoptosis (Figure 7). There is an increasing number of reports indicating that many eukaryotic m R N A s can also initiate their translation v ia a cap-independent mechanism. Recen t transfection-based experiments in cell cultures indicated that expression of the truncated 86 kDa protein in cells stimulated protein translation from the I R E S s of death-associated proteins, such as c - M Y C and A P A F - 1 , and anti-apoptotic proteins, such as X-l inked inhibitor of apoptosis protein (XIAP) which is an effective mammalian inhibitor-of-apoptosis protein whose mechanism of action involves direct inhibition of caspae-3 and -7. (Holick era / . , 1999; Henis-Korenbli t et al., 2000; Henis-Korenbli t et al., 2002) (Figure 8). A s previously mentioned, e lF4GI can be c leaved by C V B 3 2 A p r 0 and e lF4GI shares structural homology with N A T L This raises the possibility that NAT1 may be c leaved by C V B 3 proteases, and the c leavage products may promote the translation of a specific unidentified subset of m R N A s in a cap-independent manner and thus contribute to the apoptotic process (Figure 8). -36-2 A p r o PABP elF4E elF3/4A elF4A Tt 9Wffl elF4GI 63% T NAT1/DAP5/p97 Caspase Cells undergo apoptosis J W i i i i f i Caspase-cleaved NAT1 (86 kDa) Cap-independent translation via IRES Figure 7. Structural homology between elF4GI and NAT1 and function of the truncated NAT1 when cells undergo apoptosis. The binding domains of various proteins are shown and the same domain found on both e lF4GI and NAT1 is indicated by the same pattern. The similarity of the central region of e lF4GI to those of NAT1 is indicated. The 2 A p r o and ca spase c leavage sites are indicated by arrows. W h e n cells undergo apoptosis, the truncated NAT1 generated through caspase c leavage is found to promote cap-independent translation via the I R E S . (Holcik era/ . , 2000; Henis-Korenbli t etal., 2002). -37-Apoptotic Stimulus * \\ -(^) | Caspase activation NAT1/p97 IRES AH. { MYC < \" APAF-1 <-XIAP <-NAT1/p86 A U G Stimulation of IRES-mediated translation DAP5 mRNA c-myc mRNA Apaf-1 mRNA Pro-apoptotic IRESs XIAP mRNA I Anti-apoptotic IRESs Figure 8. A model scheme illustrating the contribution of NAT1 in the presence of an apoptotic stimulus. This diagram shows the contribution of NAT1 specifically to the balance between cell death and survival in the presence of an apoptotic trigger through IRES-mediated translation. Positive and negative feedback are marked by plus and minus signs, respectively. (Henis-Korenblit et al., 2002). -38-Another interesting gene that was found to be down-regulated in the CVB3-infec ted mouse heart is C R E B (Yang et al., 1999). This gene encodes a 43 k D a basic leucine zipper transcription factor which plays a critical role in regulating gene expression in response to a variety of extracellular signals, including nerve growth factor in neurons and antigen receptor cross-linking in T-lymphocytes (Arias et al., 1994; Kwok era/ . , 1994). The transcriptional activity of C R E B is dependent upon phosphorylation of a critical serine133 residue by protein kinase A . The transcriptionally active C R E B is present in cardiac myocytes. Experiments using rat hearts have shown that chronic stimulation with the p-adrenergic agonist isoproterenol, a model that mimics the hyperadrenergic state, appears to play a role in the development of human heart failure as a result of a reduction of the level of C R E B m R N A (Muller et al., 1995a&b). Thus, it is suggested that C R E B may be an important regulator of cardiac myocyte gene expression and plays a role in regulating cardiac myocyte function or survival . Furthermore, transgenic mice with heart-specific overexpression of a non-phosphorylatable dominant-negative C R E B - m u t a n t (serine133 replaced by alanine) developed four-chamber dilation and severe heart failure within a few weeks after birth (Fentzke et al., 1998). These results suggest that an inhibition of the C R E B - m e d i a t e d gene transcription is associa ted with the phenotype of the final s tages of various cardiac diseases , such as ischemic or idiopathic D C M and eventually leads to altered gene regulation in human heart failure. However, little is known about the transcriptional activation mediated by C R E B and the c A M P -dependent signaling pathway in cardiomyocytes and about the mechanisms - 3 9 -leading to alterations of these regulations. Interestingly, proteolytically c leaved dystrophin by C V B 3 2 A p r o can lead to functional impairment and morphological disruption of heart muscle cells, which can lead to the development of D C M . This raises the possibility that viral proteases may also process C R E B during infection. S u c h c leavage couples with C R E B m R N A down-regulation in the CVB3- infec ted mouse heart may contribute to or further enhance the initiation and progression of viral myocarditis into D C M . A m o n g the members of the Picornaviridae family, recent studies have demonstrated that express ion of the viral proteases 2 A p r o or 3 C p r o alone leads to caspase-3 activation and apoptosis (Barco et al., 2000; Goldstaub et al., 2000). A s mentioned in chapter one, a wide subset of factors involved in transcription, translation, and cytoskeleton organization are also processed by these two proteases, which may further promote apoptosis. Overexpress ion of viral 2 A p r o or 3 C p r o in H e L a cells may induce cell death by i) c leavage of translation and transcription initiation factors, ii) activation of caspases , and/or iii) altered express ion of anti- or pro-apoptotic molecules. Clearly, elucidating the relationship between the viral proteases and apoptosis will highlight novel mechanisms linking translational control to cell survival/death pathways in C V B 3 -infected heart d iseases . -40-Chapter Two: Hypothesis and Specific Aims 2.1 Hypothesis and specific aims In this project, I chose to study the role of C V B 3 protease genes 2 A and 3 C and their potential substrates in promoting cell death. The central hypothesis of my thesis work is that coxsackieviral proteases 2A and 3C directly and/or indirectly induces apoptotic cell death via activation of caspases and proteolysis of host transcription and translation factors. This hypothesis was tested in H e L a cell cultures ( A T C C , Rockvil le , M D , U S A ) . The specific aims are as follows: 1. To clone C V B 3 2 A p r o or 3 C p r 0 gene into pCI-neo vector and then transiently transfect H e L a cells . 2. To measure H e L a cell viability by M T S assay . 3. To detect c leavage of endogenous transcription factor, C R E B , and translation initiation factors, e lF4GI and N A T L 4. To detect c leavage of procaspases-3 and -8 and their respective substrates, P A R P and Bid . 5. To detect altered expression of the Bcl -2 family members , such as Bcl-2 and Bax. 6. To detect the release of cytochrome c from mitochondria into the cytosol and the activity of activated caspase-9 . -41 -Chapter Three: Experimental Design, Material and Methods 3.1 Cloning of CVB3 protease genes 2A and 3C into an eukaryotic expression vector 3.1.1 Cloning of protease genes 2A and 3C G e n e s encoding C V B 3 2 A p r o and 3 C p r o were first prepared by polymerase chain reaction ( P C R ) using C V B 3 full-length c D N A ( p S T 1 8 - C V B 3 plasmid) as template. The primer sequences used for amplifying 2 A gene were forward 5'-C A A T G G G A C A A C A A T C A G G G - 3 ' and reverse 5'-A G T C C T G C A G T C A C T G T T C C A T T - 3 ' , while the primer sequences used for amplifying 3 C gene were forward 5' - A G G C A A G C T T A A A T G C A A G G C C C T G -3' and reverse 5 ' - T A A A G T C G A C T T A A C C T T T C T C - 3 ' . The amplified P C R fragments were ligated into the T A vector (Invitrogen). The resulting T A plasmids containing the protease genes were then digested with E c o R I and the released fragments were ligated into a pCI-neo vector (Promega) that had been treated with E c o R I and dephosphorylated with 2 units of calf intestinal phosphatase (Promega). The new constructs were named as pCI-neo(2A) and pCI-neo(3C), respectively. 3.1.2 Transformation of E. coli (DH5a) and sequence analysis E a c h of the ligation products of the two constructs, pCI-neo(2A) and pCI-neo(3C), were added to 50 uL of competent cells (DH5oc) and iced for 15 min. Then the samples were heat-shocked at 4 2 ° C for 2 min and immediately iced for another 5 min. After incubation on ice, 1 mL of L B medium was added to each of - 42 -the samples and then incubated at 3 7 ° C for 1 h with shaking. Var ious volumes of samples were then plated on agar plates containing ampicillin and incubated overnight at 3 7 ° C . Individual colonies were selected and grown in L B medium with ampicillin. D N A was extracted and the sequences of the inserts were determined by D N A sequencing performed at the U B C Biotechnology Laboratory ( N A P S units). 3.2 Transient transfection and cell culture conditions H e L a cells (Rockville, M D , U S A ) were grown in 35-mm plates to 60% confluence and then transfected using Lipofec tamine™ transfection reagent (Invitrogen), with 1.5 pg of plasmid D N A [pCI-neo(2A) or pCI-neo(3C)] according to the manufacturer's protocol. Vector (pCI-neo)-transfected H e L a cells were used as a parallel control. Al l the samples were incubated at 3 7 ° C in a humidified 5% C O 2 atmosphere and harvested at different timepoints post-transfection (pt) to obtain homogenized cell lysates, which were a s se s sed by Wes te rn blot analyses . 3.3 Analysis of proteins by polyacrylamide gel electrophoresis Cel l cultures were washed twice with 1x P B S and harvested at various timepoints pt. Who le cel l lysates were prepared in lysis buffer [20 m M T r i s - H C L (pH8.0); 150 m M N a C l ; 1% Naniodet-P40; 10% glycerol] at 4 ° C for 20 min and centrifuged at 14,000 rpm at 4 ° C for 20 min. The supernatants were heated at 9 0 ° C for 10 min in 6x Laemmli sample buffer [50 m M Tris-HCI (pH 6.8), 100 m M dithiothreitol, 2% sodium dodecyl sulphate (SDS) , 0 .1% bromophenol blue, 10% -43-glycerol] to obtain reduced conditions. Al l samples were then applied to and separated by 9-15% SDS-polyacry lamide gel electrophoresis ( P A G E ) . G e l s were run at 80 V for 20 min and then at 100 V for 90 min, and the proteins were transferred onto nitrocellulose membranes ( H y b o n d ™ E C L ™ ; A m e r s h a m Pharmac ia Biotech) at 30 V overnight or at 100 V for 2 h. Then membranes were blocked in blocking buffer (1x P B S containing 0.1% Tween 20 and 5% skim milk powder) at 4°C overnight. 3.4 I m m u n o b l o t a n a l y s i s After blocking overnight, the membranes were incubated with the respective primary antibody (Table 2) for 2 h at room temperature. The membranes were washed 6 times with washing buffer (1x P B S containing 0.1% Tween 20) and then incubated with horseradish peroxidase conjugated goat secondary antibody to mouse/rabbit immunoglobulin G (BD Biosciences) for 1 h at room temperature. The membranes were again washed 6 times with washing buffer. Then blots were visual ized by enhanced chemiluminescence ( E C L ; A m e r s h a m Pharmacia Biotech). The luminescence reaction was performed using equal volumes of solution A (100 m M Tris-HCI [pH 8], 5 m M H 2 0 2 ) and solution B (2.5 m M luminal, 78 m M luceferin). The membranes were incubated in the above mixed solution for 2 min at room temperature and were then exposed to X-ray film (Amersham). Al l Western blot analysis results were obtained from triplicate experiments. - 4 4 -3.5 Cell viability assay To determine the effects of C V B 3 2 A p r o and 3 C p r o express ions on H e L a cell viability, M T S cell viability a s say was performed using an assay kit from Promega following the manufacturer's instructions. Ce l l lines and the parallel control culture (vector-alone-transfected cells) were treated with M T S [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and phenazine methosulfate at different timepoints, and submitted for absorbancy measurement at 490 nm. The number of live cells of the transfected cells and the control cells were determined according to the absorbancies corrected with the background reading. The corrected absorbance was expressed relative to that of the vector-alone-transfected cells at each respective timepoint pt (to reflect the percent viable cells of each of the transfected samples relative to the number of viable cells of the control at each of the timepoint pt). 3.6 Cell culture and Western blot detection of cytochrome c release For total cell lysates, cell were w a s h e d twice in cold 1x P B S and resuspended in 1 mL of cold lysis buffer [20 m M Tris (pH 8), 137 m M N a C l , 10% glycerol, 1% Nonidet P-40, 1 m M phenylmethylsulfonyl fluoride, 10 ug/ml aprotinin] per 5 6 - c m 2 culture area. After 20 min on ice, the supernatant was collected following centrifugation at 10,000 x g. For cytosolic extracts, cells were washed twice in cold 1x P B S and resuspended in 1 mL of ice cold buffer [250 m M sucrose, 20 m M H E P E S (pH 7.4), -45-10 m M KCI , 1.5 m M M g C I 2 , 1 m M E G T A , 1mM E D T A , 1 m M Dithiothreitol (DTT), supplemented with 1 m M phenylmethylsulfonyl fluoride and 10 ug/ml aprotinin] per 5 6 - c m 2 culture area. Ce l l s were gently disrupted by 20 strokes with the B pestle of a Kontes dounce homogenizer. The supernatant was collected following centrifugation at 10,000 x g and further centrifuged at 100,000 x g for 1 h at 4 ° C in a B e c k m a n Optima ultracentrifuge using a TI-100 rotor. Ce l l lysate protein concentration was* determined by the B C A method (Bio-Rad) . Samples were separated by S D S - P A G E and proteins were transferred onto a nitrocellulose membrane (Hybond E C L , A m e r s h a m Biosc iences) . Following blocking and incubation with primary and secondary antibodies (Table 2), horseradish peroxidase conjugated secondary immunoglobulins were detected using the enhanced chemiluminescence ( E C L ) method (Amersham Biosc iences) and exposed to Hyperfilm (Amersham Biosc iences) . 3.7 Caspase-9 activity assay The activity of caspase-9 was measured using the B D ApoAler t C a s p a s e -9/6 Fluorescent Kit (Clontech) following the manufacturer's instructions. Equa l number of cells (1x10 6 cells) were lysed with lysis buffer provided from kit and centrifuged at 14,000 x g for 10 min. The cellular extracts were collected and mixed with reaction buffer containing D T T (10 mM) and then incubated with specific fluorescent substrates L E H D - A M C of caspase-9 in the presence and absence of its specific inhibitor, L E D H - C H O , for 1 h at 3 7 ° C . H e L a cells treated - 4 6 -with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for 3 h at a final concentration of 150 ng/mL were used as a positive control for the caspase-9 activity assay kit, while vector-transfected H e L a cells and samples treated with specific caspase-9 inhibitor were used as negative controls. Absorbance of samples was measured on a fluoromicroplate reader with a 360-nm excitation filter and 465-nm emiss ion filter using cell lysis buffer for background calibration. Quantification and normalization of caspase-9 activities were calculated following the manufacturer's instructions and various publications, and fold increase of caspase-9 activity for each of the samples was presented (Balachandran et al., 2000; Zhang et al., 2002; Fukazawa et al., 2003; K a n d a s a m y et al., 2003). 3.8 Statistical analysis of MTS assay All values are presented as mean ± standard deviation (SD). Statistical significance was evaluated using the Student's t test for paired comparisons , with p<0.05 considered statistically significant. 3.9 Limitations In our previous studies, infection of cell cultures with the entire virus would likely complicate the interpretation regarding the contribution of the individual viral protease to cellular effects. Therefore, in an attempt to understanding the roles of these viral proteases in inducing cell death, transient express ion of the individual viral protease in H e L a cells will be used to investigate and identify the cellular - 4 7 -effects of these proteases on the host. However, as expression of 2 A p r o and 3 C p r o are likely to be toxic to cells, all attempts to express these two proteases by means of transient transfection are subjected to variations in the proportion of successfully transfected cells and may result in a mixed cell population with different levels of transfection efficiency. - 4 8 -Table 2 . List of primary antibodies used for Western blot analyses. (Information taken from product catalogues of Santa C r u z Biotechnology, Inc, B D Biosc iences , and I M G E N E X ) . Primary antibodies Species Molecular Protein cross-reactivity* weight (kDa) detected Source* Company Bax H , M , R 2 2 M, monoc lona l Santa Cruz Biotechnology Bcl-2 H , M , R 2 8 M, monoc lona l Santa Cruz Biotechnology Bid H 15 , 2 2 Rt, polyclonal Santa Cruz Biotechnology Caspase -3 H , M 17 , 32 M, monoc lona l Santa Cruz Biotechnology Caspase -8 H , M , R 2 0 M, monoc lona l Santa Cruz Biotechnology C R E B H 4 3 M, monoc lona l Santa Cruz Biotechnology Cytochrome c H , M , R 15 M, monoc lona l B D Biosciences e lF4GI H 2 2 0 M, monoc lona l B D Biosciences N A T 1 H , M 9 7 M, polyclonal IMGENEX PARP H 8 9 , 113 M, monoc lona l Santa Cruz Biotechnology * H = Human M = Mouse R = Rat Rt = Rabbit -49-Chapter Four: Results 4.1 Cell death induced by CVB3 2 A p r o and 3 C p r o 4.1.1 Overexpression of 2 A p r o or 3 C p r o induces morphological alterations Cel l morphological changes were observed at different timepoints pt with overexpress ion of C V B 3 2 A p r o or 3 C p r o in H e L a cells as observed under a phase-contrast microscope. A s shown in Figure 9, the appearance of cell shrinkage and loss of cell adherance began at 24 h pt and 48 h pt in 2 A p r 0 - and 3 C p r o -transfected cells, respectively. B y 72 h pt, most cells have died as evidenced by unadhered, floating particles in the cell culture media, particularly in the case of 2 A p r o 4.1.2 Overexpression of 2 A p r o or 3 C p r o reduces cell viability To determine the percentage of cell death induced by C V B 3 protease express ion, cell viability was measured using the M T S assay kit (Promega). A s shown in Figure 10, the percent viable cells rapidly decreased upon transient express ion of 2 A p r o or 3 C p r 0 . At 72 h pt, the percent cell viability of 2 A p r o - or 3 C p r o - e x p r e s s i n g cells was approximately 4 4 % and 68%, respectively, as compared to control cells . - 5 0 -CD C L LO D CO L L CO Figure 10. M T S c e l l v i ab i l i t y a s s a y . Percent of viable cells of 2 A p r o - or 3 C p r o -transfected cells was plotted against indicated timepoints pt. H e L a cells were transfected and cultured in a 6-well plate. At 72 h pt, approximately 4 4 % and 6 8 % cells appear viable in the 2A p r o - exp re s s ing and 3 C p r 0 - e x p r e s s i n g cells, respectively, relative to the vector-transfected control cells. Bars shown are ± S D of the mean from triplicate experiments; * indicates P<0.05. - 5 2 -4.1.3 Cleavage of translation factors induced by CVB3 2 A p r o and 3 C p r o 4.1.3.1 Overexpression of 2 A p r o or 3 C p r o induces cleavage of elF4GI e lF4GI is a key factor in cap-dependent translation initiation. T o determine whether C V B 3 2 A p r o and 3 C p r o can affect e lF4GI integrity and thus influence translation initiation of the host cells, Western blot was performed using homogenized cell lysates of H e L a cells transfected with either 2 A p r 0 - or 3 C p r o -expressing plasmids and the signals were analyzed with mouse ant i-elF4GI antibody. Figure 11 demonstrates that 220 k D a e lF4GI was c leaved by 2 A p r o and 3 C p r o at 72 h pt. However, different c leavage products of e lF4GI appeared in 2 A p r o - and 3C p r o - t ransfec ted cells . 2A p r o - t ranfected cells produced a 100 k D a product which is analogous to the c leavage product found in the CVB3- infec ted cells, while 3C p r o - t ransfected cells appeared to produce more than one proteolytic product of various molecular weights, ranging from 100 k D a to 120 kDa . 4.1.3.2 Overexpression of 3 C p r o induces cleavage of NAT1 N A T 1 , also cal led p97 or death-associated protein 5, is a newly identified protein that shares structural homology with e lF4GI (Levy-Strumpf et al., 1997). The 86 k D a c leavage product of NAT1 by ca spase was found to promote cap-independent translation of many pro-apoptotic proteins, such as c -Myc and Apf-1 , and anti-apoptotic proteins, such as X I A P (Henis-Korenblit et al., 2002). To determine whether C V B 3 infection can induce c leavage of N A T 1 , H e L a cells were infected with C V B 3 at a moi of 10 and harvested at different timepoints pi. The CVB3- infec ted H e L a cell lysates were a s s e s s e d by Wes te rn blot analyses -53-using anti-NAT1 antibody. Figure 12A demonstrates that the c leavage of full-length NAT1 (97 kDa) was observed as early as 5 h pi to give a - 5 5 k D a product. Then we further investigated whether the c leavage of NAT1 could be attributed to the activity of C V B 3 3 C p r o . To this end, H e L a cells were transiently transfected with 3 C p r o - e x p r e s s i n g plasmid, pCI-neo(3C). Immunoblot analysis revealed that NAT1 was c leaved and yielded two products ranging from 50 k D a to 55 kDa in the transfected cells (Figure 12B). The presence of an 86 k D a protein was also observed in Figure 12B, which indicated that caspase(s) was/were activated in the transfected cells. This observation strongly suggests that C V B 3 3 C p r o plays an important role in apoptotic cell death. -54-C V B 3 -in fec ted V e c t o r 2 A p r o 3 C p r o k D a C l e a v e d •^ggMMwp ' ^~~*^ ^0 e lF4GI 72 72 72 H o u r s pt Figure 11. A n a l y s i s of e lF4GI in H e L a ce l l s t r a n s f e c t e d w i t h C V B 3 2 A p r o o r 3 C p r o gene . Cel ls harvested at different timepoints pt were ana lyzed by Western blotting using a mouse monoclonal antibody against human e l F 4 G I . C V B 3 -infected H e L a cells (8 h pi) were used as positive control and vector-transfected cells were used as negative control. -55-^ C V B 3 Jurkat Sham 30 ' 1 3 5 7 9 h p i k D a B C V B 3 -J u r k a t in fec ted V e c t o r 3 C p r o NAT1-* k D a H o u r s pt Figure 12. I m m u n o b l o t a n a l y s i s of NAT1 c l e a v a g e . H e L a cells were infected with C V B 3 (A) or transfected with pCI-neo(3C) (B). Total proteins were prepared from the infected or transfected cells harvested at the indicated timepoints pi/pt and subjected to Western blot analysis of NAT1. CVB3- infected H e L a cells (9 h pi) were used as positive control, while Jurkat cell lysate, s h a m -infected H e L a cells, and empty vector-transfected cells were used as negative controls. - 5 6 -4.1.4 Cleavage of transcription factor induced by CVB3 2 A p r o and 3 C p r o 4.1.4.1 Overexpression of 2 A p r o or 3 C p r o induces down-regulation of C R E B C R E B is a 43 k D a transcription factor which is considered to be an important regulator of cardiac myocyte gene expression and plays a role in regulating cardiac myocyte function and survival . To determine whether C V B 3 2 A p r o and 3 C p r 0 can influence host gene express ion by affecting C R E B protein regulation, Western blot was performed using H e L a cells transfected with either 2 A p r o - or 3 C p r o - e x p r e s s i n g plasmids and the signals were analyzed with mouse a n t i - C R E B antibody. Figure 13 demonstrates that express ion of 2 A p r 0 or 3 C p r o induced down-regulation of C R E B as early as 24 h pt, as compared to the non-transfected and vector-transfected H e L a cell lysates. These results indicate that both C V B 3 2 A p r o and 3 C p r o can influence cellular m R N A transcription by down-regulation of the transcription factor C R E B via proteolytic c leavage, which in turn can affect the translation of host cellular proteins, leading to the enhancement of apoptotic responses . -57-2 A p r o H o u r s pt H e L a V e c t o r 24 48 72 k D a G A P D H 3 C p r o H o u r s pt H e L a V e c t o r Figure 13. D o w n - r e g u l a t i o n of C R E B in b o t h C V B 3 2 A p r o - a n d 3 C p r o -t r ans fec t ed H e L a c e l l s . Cel l extracts were prepared and detected for C R E B express ion levels at the indicated timepoints pt. Leve l s of C R E B protein were found to be down-regulated in both transfected cells beginning at 24 h pt. However, C R E B express ion returned to basal level at 72 h pt in 3C p r o - t rnasfec ted cells. G A P D H was used as an equal loading control. Non-transfected H e L a cells and empty vector-transfected H e L a cells (72 h pt) were used as negative controls. - 5 8 -4.1.5 Caspase activation and cleavage of their respective substrates 4.1.5.1 Overexpression of 2 A p r o or 3 C p r o induces activation of caspase-3 and cleavage of PARP Caspases , a family of cysteine proteases, play an essential role in the process of apoptosis (Salvesen et al., 1997; Hengartner, 2000). It has been shown that these caspases process several key structural proteins which result in the systematic and orderly disassembly of the cells . A s shown in Figure 14, the transient express ion of C V B 3 2 A p r o or 3 C p r 0 could induce procaspase-3 (32 kDa) c leavage as evidenced by an increase of the c leavage product (17 kDa). Activation of caspase-3 was further verified by the c leavage of its substrate, P A R P , a nuclear protein involved in D N A repair. The 113 k D a P A R P was c leaved 14 h pi with C V B 3 into 24 k D a (not shown) and 89 k D a fragments (Figure 15), a process which represents a specific marker for ca spase activation. P A R P c leavage was detected in both 2 A p r o - and 3 C p r 0 - e x p r e s s i n g cells, suggesting that caspase-3 was activated by both 2 A p r o and 3 C p r o expression (Figure 15). -59-C V B 3 -infected V e c t o r 2 A p r o 2 A p r o V e c t o r 3 C p r o P r o c a s p a s e - 3 C a s p a s e - 3 «- 17 72 24 48 72 72 H o u r s pt Figure 14. C l e a v a g e of p r o c a s p a s e - 3 in gene - t r ans fec t ed H e L a c e l l s . Ce l l s were transfected with C V B 3 2 A p r o or 3 C p r 0 gene and harvested at the indicated timepoints pt. C leavage of procaspase-3 w as determined by Wes te rn blot analysis using mouse monoclonal caspase-3 antibody. CVB3- in fec ted H e L a cells (8 h pi) and vector-transfected cells were used as positive and negative controls, respectively. - 6 0 -CVB3-infected 2 A p r o Hours pt Vector 24 48 72 P A R P - * Cleaved —• PARP kDa 113 89 CVB3-infected 3 C p r o Hours pt Vector 24 48 P A R P - * Cleaved —• PARP 72 kDa 113 89 Figure 15. Cleavage of PARP in gene-transfected HeLa cells. Cel l s were transfected with C V B 3 2 A p r o or 3 C p r o gene and harvested at the indicated timepoints pt. C l e a v a g e of P A R P was determined by Wes te rn blot analysis using mouse monoclonal P A R P antibody. CVB3-infec ted H e L a cel ls (14 h pi) and vector-transfected cells (72 h pt) were used as positive and negative controls, respectively. -61 -4.1.5.2 Overexpression of 2 A p r o or 3 C p r o induces activation of caspase-8 and cleavage of Bid To determine the activity of other caspases that could activate caspase-3 , c leavage of procaspase-8 was detected using the C V B 3 2 A p r o - or 3 C p r 0 -expressing cells . Resul ts obtained from immunoblot analysis showed a significant prolonged increase in the 20 k D a procaspase-8 c leavage product in 3C p r o - t ransfected cells . In 2A p r o - t ransfec ted cells, the procaspase-8 c leavage product peaked at 24 h pt and returned to basal level thereafter (Figure 16). Activation of caspase-8 was then a s se s sed in 2 A p r o - or 3 C p r o - e x p r e s s i n g cells by the c leavage of its substrate B i d . Figure 17 shows that Bid (22 kDa) was c leaved and yielded the tBid (15 kDa) in both 2 A p r o - and 3 C p o r - e x p r s s i n g cells. The presence of tBid indicated that caspase-8 was activated upon both 2 A p r o and 3 C p r o expression in H e L a cells. - 6 2 -2 A P r o Hours pt CVB3-3 C p r o Hours pt CVB3-F i g u r e 16. CVB3 3C P R O expression induces activation of caspase-8. H e L a c e l l s w e r e t r a n s f e c t e d w i th p C I - n e o ( 2 A ) or p C I - n e o ( 3 C ) a n d h a r v e s t e d a t the i n d i c a t e d t i m e p o i n t s pt. C l e a v e d p r o - c a s p a s e - 8 p ro te in l e v e l s w e r e d e t e r m i n e d by W e s t e r n blot a n a l y s i s u s i n g a n t i b o d y a g a i n s t t he 2 0 k D a c l e a v a g e p roduc t . N o t e that c l e a v a g e i n d u c e d by 2 A p r o e x p r e s s i o n r e a c h e d h i g h e s t l e ve l 2 4 h pt a n d re tu rned to b a s a l l e ve l therea f te r . C V B 3 - i n f e c t e d ce l l e x t r a c t (14 h pi) a n d e m p t y v e c t o r - t r a n s f e c t e d ce l l ex t rac t (72 h pt) w e r e u s e d a s p o s i t i v e a n d n e g a t i v e c o n t r o l s , r e s p e c t i v e l y . - 6 3 -2 A P r o Hours pt CVB3-infected Vector 24 48 72 k D Bid -* i . — — — -^ 22 Cleaved —• 15 Bid CVB3-3 C p r o Hours pt infected Vector 24 48 72 kD Bid-* 1 «• 22 Cleaved-*— • 15 Bid F i g u r e 17 . CVB3 2Apro and 3Cpro induce cleavage of Bid. H e L a c e l l s w e r e h a r v e s t e d at the i n d i c a t e d t i m e p o i n t s pt. C e l l e x t r a c t s w e r e p r e p a r e d a n d d e t e c t i o n of B i d c l e a v a g e in 2 A p r a - o r 3 C p r o - e x p r e s s i n g c e l l s w a s p e r f o r m e d by W e s t e r n blot. C V B 3 - i n f e c t e d (12 h pi) H e L a ce l l ex t r ac t a n d e m p t y v e c t o r -t r a n s f e c t e d ce l l ex t r ac t (72 h pt) w e r e u s e d a s p o s i t i v e a n d n e g a t i v e c o n t r o l s , r e s p e c t i v e l y . -64-4.1.6 Alteration of expression of Bcl-2 family member 4.1.6.1 Overexpression of 3 C p r o up-regulates expression of Bax, but not Bcl-2 The Bcl-2 family proteins include both anti-apoptotic, such as Bcl-2 and Bcl -xL, and pro-apoptotic, such as Bax and Bid , members . To determine whether C V B 3 protease expression can induce altered express ion of Bcl-2 and Bax, Western blot analyses were conducted using 2 A p r o - or 3C p r o - t ransfected H e L a cell lysates. Figure 18 shows that expression of Bax in 3 C p r o - e x p r e s s i n g cells was significantly increased but not in 2 A p r o - e x p r e s s i n g cel ls . However , Figure 19 demonstrates that both C V B 3 2 A p r o and 3 C p r o did not induce changes in the expression of Bcl-2 , indicating that apoptotic cell death was triggered by C V B 3 3 C p r 0 through activation of the pro-apoptotic Bcl-2 family protein Bax rather than suppression of the anti-apoptotic protein Bcl -2 . -65-Figure 18. Levels of Bax expression in CVB3 2AP R O- or 3Cpro-transfected HeLa cells. Ce l l extracts were prepared and detected for B a x protein levels by Western blot at the indicated timepoints pt. Bax up-regulation started 24 h pt and significantly increased by 72 h pt in the transfected cells . CVB3- in fec ted H e L a cells (14 h pi) and vector-transfected cells (72 h pt) were used as positive and negative controls, respectively. - 6 6 -CVB3-infected Vector 2 A p r o Hours pt Bcl-2 GAPDH—• Figure 19 CVB3 2 A p r o and 3 C p r o do not alter Bcl-2 expression in 2 A p r o -and 3Cpro-transfectd HeLa cells. Total protein extracts were prepared at indicated timepoints pt and the Bcl-2 expression w a s ana lyzed by Western blotting. CVB3- in fec ted (14 h pi) and empty vector-transfected H e L a cells (72 h pt) were used as controls. - 6 7 -4.1.7 Overexpression of 2 A p r o or 3 C p r o leads to cytochrome c release from mitochondria It has been reported that the presence of tBid and up-regulation of Bax can promote cytochrome c release from the mitochondria during apoptosis in many cellular systems. Thus, my next experiment was to test whether the overexpression of 2A p r 0 or 3 C p r 0 can induce cytochrome c release from mitochondria. Western blot to detect cytochrome c was performed using cellular extracts prepared from cytosolic fractions and whole cell lysates. Figure 20 demonstrates cytochrome c release from mitochondria in both 2A p r 0 - and 3C p r o -transfected HeLa cells. This indicates that CVB3 2A p r o and 3 C p r o expression can lead to cytochrome c redistribution in the transfected cells. -68-C V B 3 -infec ted V e c t o r 2 A p r o 3 C p r o H e L a k D a 72 72 72 H o u r s pt Figure 20. C V B 3 2 A p r o o r 3 C p r o i n d u c e s c y t o c h r o m e c re lease f r o m m i t o c h o n d r i a . H e L a cells were transfected with 2 A p r o or 3 C p r 0 gene. Ce l l extracts were prepared from cytosolic and whole cell lyses at 72 h pt. Cytochrome c was found in the cytosolic fraction of 2 A p r o - or 3 C p r o - e x p r e s s i n g cells . CVB3- infec ted (14 h pi) H e L a cells were used as a positive control, while vector-transfected cells as well as non-transfected cells were used as negative controls. - 69 -4.1.8 Overexpression of 2 A p r o or 3 C p r o induces caspase-9 activation As previously mentioned, caspase-9, which serves as an upstream caspase to activate the downstream caspase-3, is activated following the release of cytochrome c from mitochondria. To confirm whether the release of cytochrome c detected in both 2A p r o- and 3C p r o-transfected cells activates procaspase-9, caspase-9 activity assay were performed. Figure 21 shows that caspase-9 activities were increased upon 2A p r 0 or 3C p r o expression in HeLa cells as compared to the controls. Also, results obtained from the samples after application of caspase-9 inhibitor (C9i) showed that the specificity of this assay. This activation of caspase-9 indicates that an intrinsic mitochondria-mediated pathway was also activated upon 2A p r o or 3C p r 0 expression, in addition to a caspase-dependent pathway through activation of caspase-8 and -3. -70-Figure 21 . Caspase-9 activity assay in CVB3 2Apro- or 3C p r o -express ing cel ls. Activity of caspase-9 was measured using the ApoAler t Kit following the manufacture's instructions. Ce l l extracts (72 h pt) were prepared and incubated with specific caspase-9 substrate ( L E H D - A M C ) in the presence and absence of specific inhibitor of caspase-9 ( L E H D - C H O , C9i) . TRAIL-treated H e L a cells (3 h post induction) were used as positive control, while empty vector-transfected H e L a cells (72 h pt) and protease-transfected samples treated with C 9 i were used as negative controls. Caspase -9 activities were measured and calculated as per the manufacturer's instructions (Clontech); bars represents ± S D of the mean from triplicate experiments; * indicates P<0.05. -71 -Chapter Five: Discussion, Conclusions, and Future Directions 5.1 Discussion • It has been known that many viral infections can induce host cell apoptosis. This process is considered as a host defence mechanism to limit viral replication and prevent virus spread to re-infect surrounding cells (Zhang et al., 2002; Zhang et al., 2003). Thus , this response is obviously beneficial to the host against viral infection. However , the overall cost will be huge if the dying cel ls cannot be regenerated or regenerated at a very low rate, such as the cardiomyocytes (Anversa et al., 2002). Thus, study of the molecular mechanism of CVF33-induced cardiomyocyte apoptosis and development of strategies to prevent cardiomyocyte damage are important in cardiovascular research. Picornaviral proteases, such as those found in polioviruses and enteroviruses, have been reported to play an important role in inducing host cell apoptosis (Barco et al., 2000; Golds taub et al., 2000; Kuo et al., 2002; Li et al., 2002). However, the molecular mechanisms of how these proteases induce apoptosis are still unclear. More importantly, the 2 A p r o and 3 C p r o of the cardiovirulant virus, C V B 3 , has not been studied in the context of apoptosis. Here, experiments were conducted to elucidate the death signal transduction cascade induced by the C V B 3 2 A p r o and 3 C p r o express ion. In C V B 3 2 A p r o - or 3C p r o - t ransfected cells, protease expression was found to induce reduction of cell viability and induction of apoptotic cell death. I a lso demonstrated that C V B 3 2 A p r o and 3 C p r 0 can promote apoptosis by activation of caspase-8 and up-regulation of pro-apoptotic members of Bcl-2 family, Bax . A l so , down-regulation -72 -of protein expression of host transcription factor, C R E B , and cleavage of translation initiation factors, elF4GI and NAT1, would further enhance the apoptotic cell death. A s mentioned in chapter one, the induction/initiation of apoptotic pathways can be either caspase-dependent or caspase-independent . In the caspase -dependent pathway, upstream caspases , such as caspase-8, are activated v ia ligation of the cell membrane death receptors by their ligands. Once this initiator caspase is activated, it will either activate the downstream effector caspase -3 or induce cleavage of its substrate Bid . In the present study, I have shown that caspase -3 was activated and further c leaved its substrate P A R P in both 2 A p r o -and 3C p r o - t ransfected cells through activation of caspase -8. Moreover, I also demonstrated that the activation of caspase -8 was associa ted with c leavage of Bid in both 2 A p r 0 - and 3C p r o - expres s ing cells. Al l these data suggest that 2 A p r o and 3 C p r o can induce apoptosis through a caspase-8-dependent pathway, which can subsequently activate effector caspase -3 most likely via direct c leavage by caspase -8. Apoptosis can also be triggered through an intrinsic mitochondria-mediated pathway. The key regulatory components in this mitochondria-mediated apoptotic event are the Bcl-2 family proteins. In the functional evaluation of the consequences of 2 A p r 0 and 3 C p r o transfection, up-regulation of Bax expression wa s found only in the 3C p r o - expres s ing cells, but both C V B 3 2 A p r 0 and 3 C p r o did not induce changes of Bcl-2 express ion. S u c h up-regulation of Bax and 3 C p r o - induced c leavage of Bid could further contribute to the release -73-of cytochrome c from mitochondria. In this study, I also showed the release of cytochrome c from mitochondria and this release could further activate the caspase-9 , which in turn could activate downstream caspase-3 . These results suggest that apoptotic cell death was triggered by C V B 3 3 C p r o through activation of pro-apoptotic members of Bcl-2 family rather than suppress ing expression of anti-apoptotic members . In the investigation of the detailed molecular mechanism of C V B 3 2 A p r o and 3 C p r o - i n d u c e d apoptosis, we must take into account that these proteases also c leave a variety of host translation factors. e lF4GI is a key player in initiating eukaryotic cellular protein synthesis. It is a component of the cap-binding complex (elF4F), which leads to the initiation of cap-dependent translation. C leavage of e lF4GI results in the disruption of the e l F 4 F complex and inhibition of cap-dependent translation. It was demonstrated that the recombinant 2 A p r 0 of poliovirus and human rhinovirus can cleave e lF4GI directly (Sommergruber era/ . , 1994; Bovee era/ . , 1998a&b; Gradi et al., 1998; N o v o a et al., 1999). It has been proposed that the inhibition of cap-dependent translation is a major mechanism of the execution phase of apoptosis, which leads to rapid cell death (Clemens era/ . , 1998; Mar i ssen era/ . , 1998). In this report, c leavage of e lF4GI was found in both C V B 3 2 A p r 0 - and 3C p r 0 - t ransfected cells . However , 2 A p r o and 3 C p r o appeared to have different c leavage sites on e l F 4 G I , which was evidenced by the different c leavage products obtained in 3 C p r o - e x p r e s s i n g cells. There are a few potential reasons for this divergent phenomenon. One of the possibilities is that e lF4GI can serve - 7 4 -as a substrate for caspase-3 as caspase-3 is activated upon 3 C p r o expression. However, I did not observe similar c leavage products in 2A p r o - t ransfec ted cells, although caspase-3 was also activated by 2 A p r 0 express ion. Therefore, this possibility is unlikely. The other possibility is that e l F 4 G I is directly processed by 3 C p r o to produce the c leavage product of approximately 120 k D a . From in silico prediction ( N e t P i c o R N A V 1 . 0 , Prediction of Picornavirus Protease Sites, ht tp: / /www.cbs.dtu.dk/services/NetPicoRNA), it has been shown that e l F 4 G I contains only one potential c leavage site for 2 A p r 0 but eight for 3 C p r 0 (Figure 22A) . The molecular weight of the predicted c leavage product by 2 A p r 0 is approximately 100 kDa , while two of the potential c leavage sites, Gln424 and Gln465, by 3 C p r 0 yield two c leavage products of approximately 120 and 125 k D a , respectively (Figure 22A). Therefore, the second possibility is a more sound hypothesis because the predicted c leavage products are similar in size to the ones observed in the Western blot analysis . Although both proteases were found to cleave e lF4GI , only the 100 k D a c leavage product was found in CVB3- infec ted H e L a cells. This may be because the 120 k D a cleavage product generated by 3 C p r 0 was further processed by 2 A p r o to yield the 100 k D a product which was then detected by the antibody against e lF4GI at the C-terminus, or the activity of 3 C p r 0 towards e lF4GI is much lower than that of 2 A p r o in the viral infected cells. A l so , previous studies have demonstrated that complete c leavage of e lF4GI by viral 2 A p r 0 takes an average of two h after infection (Bovee et al., 1998a&b). Therefore, the 100 k D a would be expected to be the only c leavage product found in CVB3- infec ted H e L a cells . -75-Another factor involved in regulating host translation is N A T L In a recent study, NAT1 was reported to be c leaved by caspase(s) at a conserved site to yield a novel C-terminal truncated 86 k D a protein that promoted cap-independent translation of death associa ted proteins including itself, suggesting that NAT1 may play a role in apoptosis modulation (Henis-Korenblit et al., 2000; Henis -Korenblit et al., 2002). Here, I demonstrated that the c leavage of NAT1 occurred in both CVB3- infec ted and C V B 3 3C p r 0 - t ransfected cells. However, the two c leavage products (ranging from 50 to 55 kDa) of NAT1 were found to be smaller than the c leavage product (86 kDa) by caspase(s) (Figure 22B) . This may be because NAT1 can be c leaved directly by 3 C p r o in the transfected cells, or the activity of 3 C p r o in CVB3- infec ted cells is higher than that in 3C p r o - t ransfected cells . From in silico prediction, it has been shown that NAT1 contains three potential c leavage sites for 3 C p r 0 but none for 2 A p r o , and the molecular weight of the predicted c leavage products by 3 C p r o are approximately 55 kDa . Therefore, it suggests that NAT1 may be a target protein directly c leaved by 3 C p r 0 in 3 C p r o -express ing cells to give the 50 to 55 k D a products and the larger 86 k D a c leavage product obtained by Western blot analysis in Figure 12 may be residual products from caspase activation as a result of 3 C p r o transfection. This truncated 86 k D a protein may be further processed by 3 C p r 0 to yield the small c leavage product, as shown in the Western blot. These observations strongly suggest that C V B 3 3 C p r 0 plays an important role in promoting cell death. Whether these c leavage products perform the same function as the truncated 86 k D a protein in promoting the cap-independent translation remains to be elucidated. - 7 6 -^ Potential cleavage T sites for 2 A p r o Potential cleavage sites for 3 C p r o elF4GI (1404a.a -60 -95 w \\ * * * * \\ kDa •• • • • + • • • 220 GInlOO Gln108 Gln218 Gln424 Glu465 Arg490 Gln547 Glu631 Gln1318 -54 -100 kDa -50 -120 kDa -45 -125 kDa kDa caspase B NAT1 1 * * * cleavage site kDa (907a.a.) • • •• # • 97 Glu152 Gln419 Gln433 Gln444 D E T D 7 9 0 Gln889 -50 -55 -45 kDa kDa 86 kDa Figure 22. Prediction of cleavage sites of 2A P R O and 3C P R O on e lF4GI and NATL Full length of protein sequence of e lF4GI (A) and NAT1 (B) were analyzed and subjected to predict potential c leavage sites of 2 A p r o and 3 C p r 0 using N e t P i c o R N A prediction server (version 1.0). Potential c leavage sites were indicated by arrows ( •••>- for 2 A p r o ; for 3 C p r o ) ; * represents the potential c leavage site chosen for calculating the molecular weight of the resulting fragments. -77-C R E B is a nuclear transcription factor, which is expressed in the human heart and phosphorylated to mediate cAMP-dependen t transcriptional activation (Muller et al., 1995a). Severa l studies suggested that C R E B is an important regulator of gene expression in cardiomyocytes with possible relevance for the pathophysiology of congest ive heart failure, such as ischemic and idiopathic dilated cardiomyopathy, through alteration of C R E B express ion and phosphorylation (Muller et al., 1995a&b; Muller et al., 2001). A s previously mentioned, this factor has also been found to be down-regulated in C V B 3 -infected mouse hearts from our previous differential m R N A display studies (Yang et al., 1999). The data in this thesis show that 2 A p r o or 3 C p r 0 expression resulted in a down-regulation of C R E B production in the host cells . These results suggest that C V B 3 2 A p r o and 3 C p r o are responsible for modulation of C R E B expression in myocytes, which may contribute to the development of D C M . S ince the antibody to C R E B cannot detect the C R E B c leavage products, it is not clear whether the down-regulation of C R E B is the result of a reduction at the transcription or translation level or due to degradation of the protein. However , in any case , this down-regulation will negatively affect the cell survival and contribute to H e L a cell apoptosis. In summary, my data illustrate that the mechanism of apoptosis induction in C V B 3 2 A p r o - or 3C p r o - t ransfec ted H e L a cells is likely through a caspase-8-dependent pathway and the intrinsic mitochondria-mediated pathway. D o w n -regulation of host transcription factor and c leavage of translation initiation factors by 2 A p r 0 or 3 C p r o results in the inhibition of host gene expression, which further -78-enhances apoptotic cell death. More importantly, the data suggest that the c leavage of NAT1 and down-regulation of C R E B found in 2 A p r o - or 3 C p r 0 -expressing cells require further investigation. The identification of caspase(s) responsible for the c leavage of NAT1 during 3 C p r o expression will increase our understanding of the molecular mechanisms of apoptosis induced by viral proteases, and the down-regulation of C R E B by both C V B 3 proteases will provide insight into the understanding of the mechanisms by which C V B 3 causes myocarditis and progression to D C M . 5.2 Summary of results 1. Morphological alterations and M T S assay results showed that both C V B 3 proteases can induce cell death. 2. At day 3 post-transfection, approximately 44% and 68% cells were alive in 2 A p r o - and 3C p r o -ove rexpress ing H e L a cells, respectively, as compared to the control cells. 3. Transfection with either C V B 3 2 A p r o and 3 C p r 0 induced c leavage of e l F 4 G I in H e L a cells, but 2 A p r o and 3 C p r o appeared to produce c leavage at different sites on e lF4GI . 4. NAT1 was found to be c leaved in both CVB3- infec ted and 3 C p r o -transfected H e L a cells, but not in 2A p r o - t ransfected cells . The - 5 5 k D a c leavage product observed in the CVB3- infec ted cells is smaller than the reported caspase-c leaved NAT1 (86 kDa) . -79-5 . C leavage of NAT1 by 3 C p r o in the transfected cells also generated two more c leavage products (86 k D a and - 5 0 kDa) that were not observed in CVB3- infec ted cells. The - 5 5 k D a product found in CVB3- infec ted cells could be produced from the two c leavage products that are further c leaved by other proteases from the C V B 3 or the host cells, or the 86 k D a c leavage product may be further processed by 3 C p r o to yield the smaller c leavage product ( - 5 0 kDa) observed in the 3C p r 0 - t ransfec ted cells . 6. C R E B was found to be down-regulated by both C V B 3 2 A p r o and 3 C p r o . 7. Both C V B 3 2 A p r o and 3 C p r o promoted activation of caspase-3 and c leavage of its substrate, P A R P . 8. C leavage of procaspase-8 was observed in both 2 A p r 0 - and 3 C p r o -express ing cells, and further c leavage of Bid was also detected in these cells, indicating.that caspase-8 was activated upon 2 A p r 0 and 3 C p r o express ion. 9. C V B 3 3 C p r o but not 2 A p r o could significantly up-regulate Bax express ion, a pro-apoptotic member of the Bcl-2 family. However , neither altered expression of Bcl-2 , an anti-apoptotic protein. 1 0 . Re lease of cytochrome c from mitochondria was detected in both C V B 3 2 A p r o - and 3C p r o - t ransfec ted cells. However , the 3 C p r o induced release was stronger than that of 2 A p r o . 1 1 . Caspase -9 activities were found to be increased upon C V B 3 2 A p r o or 3 C p r 0 overexpression in H e L a cells. A l so , results obtained from the -80-samples after application of the inhibition with C 9 i showed that the specificity of this assay . C o n c l u s i o n s In conclusion, these results suggest that the mechanism of apoptosis induced in C V B 3 2 A p r o - or 3C p r o - t ransfected H e L a cells is likely through multiple pathways: 1. Both 2 A p r 0 and 3 C p r o can induce a caspase-8-dependent pathway through activation of caspase-8 and caspase-3 . 2. Both 2 A p r o and/or 3 C p r o can induce intrinsic mitochondria-mediated pathway through up-regulation of Bax, the presence of tBid, cytochrome c released from mitochondria, and activation of caspase-9 . 3. 3 C p r o can promote cell death by activating or up-regulating express ion of pro-apoptotic Bcl-2 family members , Bid and Bax, rather than suppressing expression of the anti-apoptotic protein, Bcl -2 . 4. Down-regulation of host transcription factor, C R E B , by 2 A p r o and 3 C p r o may contribute to C V B 3 - i n d u c e d myocarditis and its late phase, D C M . 5. C leavage of host translation initiation factors, e lF4GI and N A T 1 , by 2 A p r o or 3 C p r o results in the inhibition of host gene expression, which further enhances apoptotic cell death. -81 -5.4 Future directions Data obtained from this study indicate that C V B 3 2 A p r o and 3 C p r o induce H e L a cell apoptosis by c leavage of host transcription and translation initiation factors and activation of caspases . However, whether these c leavages (activation) occur directly or indirectly is unknown. To address this issue, in vitro cleavage assays using purified 2 A p r o and 3 C p r o need to be performed. Therefore, future work is required to clone these genes into an express ion vector, pET-42a (Novagen), and to purify the recombinant proteases after overexpression in a prokaryotic system. 5.4.1 Cloning of CVB3 protease genes 2A and 3C into a prokaryotic expression vector To determine whether C V B 3 2 A p r 0 and/or 3 C p r o c leave NAT1 directly or not, 2A, 3 C , and NAT1 genes will be recloned into pET-42a vectors by preparing 2A, 3 C , and NAT1 gene fragments through digestion of pCI-neo(2A), pCI-neo(3C), and pCRII (NATI) , respectively. The excised D N A fragments will be ligated into the corresponding sites of the pET-42a . Overexpress ion of the c loned genes will be achieved by the induction of E. coli BL21 (DE3) cells with i sopropyl - p -D-thiogalactopyranoside (IPTG). Proteins will be purified using Glutathione S-transferase (GST) G e n e Fusion and Purification Sys tem (Amersham Pharmarcia) . -82-5.4.2 In vitro direct cleavage assay In vitro c leavage assay will be performed to determine the c leavage of NAT1 by C V B 3 2 A p r o and/or 3 C p r o . Equa l molar ratio of substrate to protease will be incubated at 2 5 ° C in a c leavage buffer overnight. Then , the protein will be analyzed by S D S - P A G E and stained with C o o m a s s i e blue. The proteins will also be transferred to polyvinylidene difluoride membranes for N-terminal sequencing to determine the c leavage site. 5.4.3 Work Completed 5.4.3.1 Construction of vectors expressing CVB3 2 A p r o or 3 C p r o I have recloned the 2A, 3 C , and NAT1 genes by excis ing the respective D N A fragments containing the entire open reading frame of the gene from the pClneo vector and pCRII vector and then ligating the exc ised fragments into the pET-42a vector. The new constructs are named as pET(2A) , pET(3C) , and pET(NAT1) , respectively. 5.4.3.2 Overexpression and purification of proteins C V B 3 3 C p r o has been successfully overexpressed and purified (Figure 23). However, the activity of 3 C p r o was difficult to detect. Perhaps , a eukaryotic expression system may be required to increase the protease activity. Overexpress ion and purification of C V B 3 2 A p r o and NAT1 are also required to be conducted before proceeding with further investigations. -83-B a c t e r i a l l y s a t e s Pur i f i ed p ro te in + + + \" - \" = N o n - i n d u c e d \"+\" = I P T G - i n d u c e d Figure 23. O v e r e x p r e s s i o n a n d pur i f i ca t ion of the r e c o m b i n a n t CVB3 3CPRO u s i n g a p r o k a r y o t i c e x p r e s s i o n s y s t e m . G S T - 3 C p r o fusion protein is overexpressed after induction with I P T G (\"+\") for 24 h at 3 7 ° C . This fusion protein is then either eluted with buffer containing glutathione (lane 3) or treated with thrombin protease to separate the G S T tag and the 3 C p r 0 (lane 4). N o n -induced bacterial cell lysate is used as a negative control in lane 1. - 8 4 -B a c t e r i a l l y sa t e s Pur i f i ed p ro t e in + + + L a n e 1 2 3 4 \" - \" = N o n - i n d u c e d \"+\" = I P T G - i n d u c e d Figure 23 . O v e r e x p r e s s i o n a n d pur i f i ca t ion o f the r e c o m b i n a n t C V B 3 3 C p r o u s i n g a p r o k a r y o t i c e x p r e s s i o n s y s t e m . G S T - 3 C p r o fusion protein is overexpressed after induction with I P T G (\"+\") for 24 h at 3 7 ° C . This fusion protein is then either eluted with buffer containing glutathione (lane 3) or treated with thrombin protease to separate the G S T tag and the 3 C p r o (lane 4). 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Virology; 213: 549-557. - 1 0 6 -"@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2004-11"@en ; edm:isShownAt "10.14288/1.0091638"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Pathology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Coxsackievirus B3 proteases 2A and 3C induce apoptotic cell death through a mitochondria-mediated pathway and cleavage of host factors for transcription and translation initiation"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/15553"@en .