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Coxsackievirus B3 proteases 2A and 3C induce apoptotic cell death through a mitochondria-mediated pathway… Chau, David Hau Wing 2004

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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 DAVID H A U WING C H A U B . S c , S i m o n F r a s e r University, 2 0 0 0  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 THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of P a t h o l o g y a n d Laboratory M e d i c i n e ; F a c u l t y of M e d i c i n e W e a c c e p t this thesis a s c o n f o r m i n g to the required s t a n d a r d  T H E UNIVERSITYOF BRITISH C O L U M B I A © David Hau Wing C h a u , 2004  THE UNIVERSITY OF BRITISH  COLUMBIA  FACULTY OF G R A D U A T E STUDIES  '  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)  ntie of Thesis^  Cot^tbtfilj)rus>  Date (dd/mm/yyyy)  &3 firtitates  Twi^us  2A  /•wfr Asb^ryj Degree:  /W,Jc-  Department of  PATIIX I o & y i A ^ i  L^o^^hr^)  The University of British Columbia Vancouver, B C Canada  grad.ubc.ca/forms/?formlD=THS  if  Year:  U^>(?cfL^__  /  U  page 1 of 1  last updated: 2Q-M-04  ABSTRACT Coxsackievirus  B3 (CVB3)-induced  d i s e a s e in children a n d y o u n g adults.  myocarditis is  a  common  T h i s viral infection c a n c a u s e  heart severe  c a r d i a c injury a n d patients m a y e x p e r i e n c e progressive heart failure, which m a y ultimately lead to dilated c a r d i o m y o p a t h y ( D C M ) . S i n c e the d e v e l o p m e n t of viral myocarditis involves c o m p l e x interactions between host cells a n d the virus, the m e c h a n i s m s by w h i c h C V B 3 c a u s e s myocarditis a n d p r o g r e s s i o n to D C M are still not well understood. T h e small C V B 3 g e n o m e contains 11 g e n e s , of w h i c h the proteases 2 A (2A  pro  ) and 3 C ( 3 C  p r o  ) are k n o w n to play critical roles in the virus life c y c l e by  p r o c e s s i n g the viral polyprotein a n d in viral p a t h o g e n e s i s by c l e a v i n g the host proteins.  R e c e n t studies h a v e demonstrated that o v e r e x p r e s s i o n of picornaviral  protease 2 A  p r o  or 3 C  p r 0  leads to c a s p a s e - 3 activation a n d apoptosis.  However,  the m o l e c u l a r m e c h a n i s m s that link t h e s e viral p r o t e a s e s to the induction of host cell death remain unclear.  M y dissertation a d d r e s s e s this issue by identifying  cellular proteins that are c l e a v e d and/or activated by these C V B 3 p r o t e a s e s a n d the s u b s e q u e n t apoptotic pathways i n d u c e d by t h e s e c l e a v a g e s .  In order to  a c h i e v e this goal, full length C V B 3 protease g e n e s 2 A a n d 3 C w e r e c l o n e d into a eukaryotic e x p r e s s i o n vector  using a  PCR-based  p l a s m i d s w e r e then transiently transfected  strategy.  into H e L a cells.  The The  resulting  transfected  s a m p l e s w e r e subjected to W e s t e r n blot a n a l y s e s to detect c l e a v a g e (activation) of v a r i o u s cellular proteins.  U p o n protease e x p r e s s i o n , cell morphological alterations a n d reduction in cell viability are o b s e r v e d in both 2 A evaluation of 2 A  or 3 C  p r o  p r o  p r 0  - a n d 3 C - t r a n s f e c t e d cells. pro  Functional  e x p r e s s i o n has a l s o s h o w n that c a s p a s e - 3 a n d -8 are  activated a n d their substrates, p o l y ( A D P - r i b o s e ) p o l y m e r a s e ( P A R P ) a n d B i d , are c l e a v e d in t h e s e transfected cells. T h e s e results indicate that the e x p r e s s i o n of C V B 3 2 A  p r o  or 3 C  p r o  in m a m m a l i a n cells is sufficient to induce cell apoptosis  through a c a s p a s e - d e p e n d e n t pathway.  W h e n other apoptotic pathways w e r e  explored, W e s t e r n blot a n a l y s e s of Bcl-2 family m e m b e r s demonstrated that only 3C  p r o  but not 2 A  p r o  c a n up-regulate e x p r e s s i o n of B a x , a pro-apoptotic protein.  H o w e v e r , e x p r e s s i o n of Bcl-2 protein, a n anti-apoptotic m e m b e r of Bcl-2 family protein, remains u n c h a n g e d  in both 2 A  p r o  - and 3C -transfected pro  cells.  Up-  regulation of B a x a n d the p r e s e n c e of truncated B i d (tBid) further contribute to the release of c y t o c h r o m e c from the mitochondria a n d activation of c a s p a s e - 9 . Finally, c l e a v a g e of host transcription factor, cyclic A M P r e s p o n s i v e  element  binding protein ( C R E B ) a n d translation initiation factors, eukaryotic translation initiation factor 4 G I (elF4GI) a n d N A T 1 , by 2 A  or 3 C  p r o  p r o  are a l s o o b s e r v e d .  T h e s e proteolytic activities are a c c o m p a n i e d by a s e v e r e inhibition of host m R N A a n d protein synthesis, w h i c h also contributes to the viral protease-induced  cell  death. In c o n c l u s i o n , the data s u g g e s t that the m e c h a n i s m of apoptosis i n d u c e d in  CVB3 2A  p r o  -  or  3C -transfected  c o n v e r g i n g pathways.  pro  HeLa  Firstly, both 2 A  p r 0  cells and  3C  is likely through p r o  can  multiple  induce H e L a  apoptosis through a c a s p a s e - d e p e n d e n t pathway. S e c o n d l y , 2 A  p r o  and 3 C  p r o  cell can  a l s o induce intrinsic mitochondria-mediated pathway through up-regulation of B a x a n d release of c y t o c h r o m e c from mitochondria. transcription a n d translation initiation factors by 2 A  Finally, c l e a v a g e of host p r o  or 3 C  p r o  results in the  inhibition of host protein e x p r e s s i o n , w h i c h further e n h a n c e s 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  Chapter One:  1.1  Introduction  General information 1.1.1 Family of  Picornaviridae  1 and genus  Enterovirus  1.1.2 Coxsackievirus: Role of Coxsackievirus B3 in myocarditis 1.2  Molecular genetics of CVB3  1 5 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 posttranslational processes 1.3  10  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  - V -  15  1.3.2.2 Effector caspases, cell surface receptor activation and mitochondrial signaling in regulation of cell death 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 i) .  Viral 2A  ii) .  Viral 3 C  20 20  pro  24  pro  1.4.2.2 Functional significance of viral 2A  pro  and 3 C  pro  in inducing  apoptosis 1.5  34  Hypothesis and Specific A i m s  Hypothesis and specific aims  Chapter Three:  3.1  32  Research focus and project rationale  Chapter Two:  2.1  15  41  Experimental Design, Materials and Methods  Cloning of CVB3 protease genes 2A and 3C into an eukaryotic expression vector  42  3.1.1 Cloning of protease genes 2A and 3C 3.1.2 Transformation of E. coli (DH5oc) and sequence analysis  42 ....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  C e l l viability a s s a y  45  3.6  C e l l culture a n d W e s t e r n blot detection of c y t o c h r o m e c r e l e a s e  45  3.7  C a s p a s e - 9 activity a s s a y  46  3.8  Statistical a n a l y s i s of M T S a s s a y  47  3.9  Limitations  47  Chapter Four: 4.1  Results and Discussion  C e l l death i n d u c e d by C V B 3 2 A  p r o  and 3 C  50  p r o  4.1.1  O v e r e x p r e s s i o n of 2 A  p r o  or 3 C  p r o  4.1.2  O v e r e x p r e s s i o n of 2 A  p r o  or 3 C  p r a  4.1.3  C l e a v a g e of translation initiation factors i n d u c e d by C V B 3 2A  p r a  and 3 C  r e d u c e s cell viability  53  O v e r e x p r e s s i o n of 2 A  p r o  4.1.3.2  O v e r e x p r e s s i o n of 3 C  p r o  or 3 C  p r o  i n d u c e s c l e a v a g e of e l F 4 G I . . . . 5 3  induces c l e a v a g e of N A T 1  C l e a v a g e of transcription factor induced by C V B 3 2 A  4.1.4.1  50  p r o  4.1.3.1  4.1.4  i n d u c e s morphological alterations...50  O v e r e x p r e s s i o n of 2 A  p r o  or 3 C  p r o  p r o  and 3 C  O v e r e x p r e s s i o n of 2 A  p r o  or 3 C  p r o  O v e r e x p r e s s i o n of C V B 3 2 A  p r o  or 3 C  of c a s p a s e - 8 a n d c l e a v a g e of B i d  - vii -  59  i n d u c e s activation  of c a s p a s e - 3 and c l e a v a g e of P A R P 4.1.5.2  57  57  C a s p a s e activation a n d c l e a v a g e of their respective substrates  4.1.5.1  p r o  i n d u c e s down-regulation  of C R E B 4.1.5  53  59 p r o  i n d u c e s activation 62  4.1.6  A l t e r a t i o n o f 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  4.1.6.1  O v e r e x p r e s s i o n of 3 C  p r 0  65  up-regulates expression  of B a x , but not B c l - 2 4.1.7  O v e r e x p r e s s i o n of 2 A  p r o  65  or 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  from mitochondria 4.1.8  O v e r e x p r e s s i o n of 2 A  Chapter Five:  68 p r o  or 3 C  p r o  induces c a s p a s e - 9 activation  70  Discussion, C o n c l u s i o n s , and Future Directions  5.1  Discussion  72  5.2  S u m m a r y of r e s u l t s  79  5.3  Conclusions  81  5.4  Future directions  82  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 expression vector  82  5.4.2  In vitro d i r e c t c l e a v a g e a s s a y  83  5.4.3  Work Completed  83  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  5.4.3.2  O v e r e x p r e s s i o n a n d purification of proteins  References  p r o  or 3 C  p r o  83 83  86  - viii -  LIST OF TABLES AND FIGURES Tables: Tablel.  A m i n o a c i d s e q u e n c e h o m o l o g y a m o n g the proteins of c o x s a c k i e B v i r u s e s ( C V B ) a n d two other picornaviruses, poliovirus (PV) a n d  T a b l e 2.  h u m a n rhinovirus ( H R V )  31  List of primary antibodies u s e d for W e s t e r n blot a n a l y s e s  49  Figures: Figure 1.  S c h e m a t i c d i a g r a m of the picornavirus c a p s i d  4  Figure 2.  T h e rate of c a r d i o v a s c u l a r d i a g n o s e s per 1,000 viral infections  6  Figure 3.  O v e r v i e w of the c o x s a c k i e v i r u s replication c y c l e  Figure 4.  G e n e organization a n d post-translational c l e a v a g e of C V B 3 polyproteins by 2 A  Figure 5.  p r o  and 3 C  12  13  p r o  Simplified s c h e m a t i c d i a g r a m s u m m a r i z i n g cell surface signaling a n d mitochondrial signaling pathways leading to apoptosis  Figure 6.  S u m m a r y of major host protein c l e a v a g e s a n d d o w n s t r e a m effects i n d u c e d by viral 2 A  Figure 7.  18  p r o  and 3 C  30  p r 0  Structural h o m o l o g y between e l F 4 G I a n d N A T 1 , a n d function of the truncated N A T 1 w h e n cells undergo apoptosis  Figure 8.  37  A model s c h e m e illustrating the contribution of N A T 1 in the p r e s e n c e of an apoptotic stimulus  Figure 9.  M o r p h o l o g i c a l alterations of H e L a cells e x p r e s s i n g C V B 3 2 A or 3 C  p r o  38 p r o  51  Figure 10.  M T S cell viability a s s a y  F i g u r e l 1.  A n a l y s i s of e l F 4 G I in H e L a cells transfected with C V B 3 2 A or 3 C  p r o  52 p r o  gene  55  Figure 12.  Immunoblot analysis of N A T 1 c l e a v a g e  Figure 13.  Down-regulation of C R E B in both C V B 3 2 A 3C -transfected pro  56 p r o  - and  H e L a cells  58  Figure 14.  C l e a v a g e of p r o c a s p a s e - 3 in gene-transfected H e L a cells  Figure 15.  C l e a v a g e of P A R P in gene-transfected H e L a cells  Figure 16.  CVB3 3C  p r o  e x p r e s s i o n i n d u c e s activation of c a s p a s e - 8  63  Figure 17.  CVB3 2A  p r o  and 3 C  64  Figure 18.  L e v e l s of B a x e x p r e s s i o n in C V B 3 2 A  p r o  60 ......61  induce c l e a v a g e of Bid p r o  - or  3C -transfected pr0  H e L a cells Figure 19.  CVB3 2A  p r o  66 and 3 C  3C -transfected pro  Figure 20.  CVB3 2A  p r o  or 3 C  p r o  do not alter Bcl-2 e x p r e s s i o n in 2 A  p r o  - and  H e L a cells p r o  67  i n d u c e s cytochrome c r e l e a s e from  mitochondria Figure 2 1 .  69  C a s p a s e - 9 activity a s s a y in C V B 3 2 A  p r o  - or 3 C  pro  -expressing  cells Figure 22.  Prediction of c l e a v a g e sites of 2 A  71 p r o  and 3 C  p r o  on e l F 4 G I a n d  NAT1 Figure 2 3 .  O v e r e x p r e s s i o n a n d purification of the recombinant C V B 3 3 C using a prokaryotic e x p r e s s i o n s y s t e m  77 p r o  84  LIST OF ABBREVIATIONS 2 A  pro  Protease 2 A  3 C  pro  Protease 3 C  3CD 3 D  p r o  pol  Protease 3 C D Polymerase 3D  Apaf-1  Apoptotic protease activating factor-1  C9i  C a s p a s e - 9 inhibitor, L E D H - C H O  CAR  C o x s a c k i e v i r u s a n d a d e n o v i r u s receptor  CREB  cyclic A M P - r e s p o n s i v e element-binding protein  CVB3  Coxsackievirus B3  DAF  D e c a y - a c c e l e r a t i n g factor  DAP5  D e a t h - a s s o c i a t e d protein 5  DCM  Dilated c a r d i o m y o p a t h y  DFF  D N A fragmentation factor  DTT  Dithiothreitol  ECL  Enhanced chemiluminescence  elF4A  Eukaryotic translation initiation factor 4 A  elF4E  Eukaryotic translation initiation factor 4 E  elF4F  Eukaryotic translation initiation factor 4 F  elF4GI  Eukaryotic translation initiation factor 4 G I  elF4GII  Eukaryotic translation initiation factor 4GII  FAK  F o c a l a d h e s i o n kinase  GST  Glutathione S-transferase  - xi -  IPTG  Isopropyl-p-D-thiogalactopyranoside  IRES  Internal r i b o s o m e entry site  moi  Multiplicity of infection  mRNA  Messenger R N A  MTS  3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium  Oct-1  O c t a m e r binding transcription factor-1  PABP  Poly(A) binding protein  PAGE  P o l y a c r y l a m i d e gel electrophoresis  PARP  Poly(ADP-ribose) polymerase  PCR  P o l y m e r a s e c h a i n reaction  pi  Post-infection  pt  Post-transfection  PTB  Poly-pyrimidine tract binding protein  SD  S t a n d a r d deviation.  SDS  S o d i u m d o d e c y l sulphate  tBid  Truncated Bid  TBP  T A T A - b i n d i n g protein  TNF-a  T u m o r n e c r o s i s factor-a  TRAIL  T u m o r n e c r o s i s factor-related a p o p t o s i s - i n d u c i n g ligand  tRNA  Transfer R N A  UTR  Untranslated region  XIAP  X - l i n k e d 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. W a n 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  encouragement I needed to follow my dreams to the end.  - xiii -  success  gave  me  Chapter One:  Introduction  1.1  General information  1.1.1  Family of Picornaviridae and genus Enterovirus T h e w o r d Picornaviridae  d e s c r i b e s the small s i z e (pico) of this group of  virus a n d the type of nucleic a c i d ( R N A ) that constitutes the viral g e n o m e .  The  picornaviruses are n o n - e n v e l o p e d v i r u s e s with a single-stranded R N A g e n o m e of positive polarity (from w h i c h R N A acts directly a s the template for translation of viral proteins). T h e R N A g e n o m e s v a r y in length from 7 2 0 9 to 8 4 5 0 nucleotides a n d contain both 5' a n d 3' untranslated  regions ( U T R s ) that are involved in  regulation a n d initiation of viral transcription a n d translation.  T h e 5' U T R s of  picornaviruses are long (vary from 6 2 4 to 1199 nucleotides) a n d highly structured, w h e r e a s the 3' U T R s are short (vary from 47 to 125 nucleotides) a n d contain secondary  structure  responsible for viral  R N A synthesis.  The genome  of  picornaviruses is infectious b e c a u s e it c a n be translated upon entry into the host cell in order to p r o d u c e all the viral proteins required for viral replication. Picornavirus virions are spherical in s h a p e with a diameter of about 30 n m . T h e virus particles lack a lipid e n v e l o p e but contain a protein shell, called c a p s i d , which  surrounds  the  naked  R N A genome  and  renders  the  viral  particle  insensitivity to organic solvents. T h e c a p s i d s of picornaviruses are c o m p o s e d of four structural proteins: V P 1 , V P 2 , V P 3 , a n d V P 4 , a n d a combination of 60 of t h e s e structural proteins are a r r a n g e d in a n i c o s a h e d r a l lattice (Rueckert et al., 1969) (Figure 1). T h e g e n o m e is covalently linked at the 5'-uridylylate moiety to a protein called V P g (virion protein, g e n o m e linked) through a n 04-(5'-uridylyl)-  tyrosine linkage ( F l a n e g a n et al., 1977; L e e et al., 1977).  A l l picornavirus  genomes  are linked to V P g , w h i c h varies in s i z e from 22 to 24 a m i n o a c i d  residues.  V P g is e n c o d e d by a single viral g e n e in all p i c o r n a v i r u s e s except the  g e n o m e of foot-and-mouth d i s e a s e virus, w h i c h e n c o d e s three V P g g e n e s ( F o r s s et al., 1982).  E v e n though V P g is e x p r e s s e d  required for infectivity of the viral R N A .  in e v e r y picornavirus, it is not  In the c a s e of poliovirus, the r e m o v a l of  V P g from the viral R N A by proteinase treatment d o e s not lead to a reduction in specific infectivity of the viral R N A ( R a c a n i e l l o , 2001).  V P g is r e m o v e d from  virion R N A by a host protein c a l l e d unlinking e n z y m e ( A m b r o s et al., 1978) a n d is present on n a s c e n t R N A c h a i n s of the replicative intermediate R N A a n d on the negative-stranded  RNAs  (Pettersson  et al.,  1978),  which  has  led  to  the  s u g g e s t i o n that viral R N A synthesis u s e s V P g a s a "primer" (Nomoto etal., 1977; P e t t e r s s o n et al., 1978). H o w e v e r , the role that V P g plays in viral R N A s y n t h e s i s during picornaviral infection is still unclear. S i n c e 1898 h u n d r e d s of picornavirus serotypes T h e family Picornaviridae acid  stability a n d  aphthovirus,  is c o m p r i s e d of six g e n e r a that are classified b a s e d on  buoyant  cardiovirus,  have been discovered.  density  enterovirus,  (Stanway, hepatovirus,  w h i c h all contain v i r u s e s that infect vertebrates.  1990).  These  parechovirus,  and  include  the  rhinovirus,  T h e g e n u s enterovirus  was  initially n a m e d for their resistance to low p H a n d their replication in the alimentary tract.  E n t e r o v i r u s e s are v e r y s m a l l , approximately 20-30 nm in s i z e , c a p a b l e of  withstanding low gastric p H , a n d are transmitted by the fecal-oral route.  Many  structures are c o n s e r v e d within this genus, n a m e l y the e x i s t e n c e of the p - s t r a n d  anti-parallel (3-sandwich of V P 1 - V P 4 ; however, the viral c a p s i d proteins  and  loops connecting the (3-strands are different ( M u c k e l b a u e r et al., 1997). This g e n u s includes poliovirus (3 serotypes), c o x s a c k i e v i r u s groups A a n d B (23 serotypes), e c h o v i r u s (28 serotypes), and  many  non-human  enteric  viruses.  h u m a n enterovirus (4 Many  serotypes),  similarities exist within  the  enteroviruses a n d classification of this group of v i r u s e s is b a s e d o n their ability to c a u s e similar d i s e a s e in murine m o d e l s and their similar growth in cell cultures. M o s t of the history of enteroviruses  is on poliovirus a s this virus w a s the first  enterovirus to be d i s c o v e r e d (Landsteiner et al., 1909).  In 1948, c o x s a c k i e v i r u s  group A w a s first isolated from the feces of paralytic children in C o x s a c k i e , N e w York, during a poliomyelitis outbreak (Dalldorf et al., 1948). In the following year, c o x s a c k i e v i r u s group B w a s isolated from c a s e s of aseptic meningitis (Melnick et al., 1949).  T h i s group of c o x s a k i e v i r u s e s p r o d u c e d a g e n e r a l i z e d infection in  n e w b o r n mice, presenting with myositis a s well a s involvement of the brain, p a n c r e a s , heart, a n d brown fat.  Figure 1.  S c h e m a t i c diagram of the picornavirus c a p s i d .  Diagrammatic  representation of the organization s h o w i n g the p s e u d o e q u i v a l e n t packing a r r a n g e m e n t of the c a p s i d proteins, V P 1 , V P 2 , a n d V P 3 d o m a i n s in a 60-subunit shell, of picornaviruses in a n i c o s a h e d r o n . V P 4 is o n the interior of the c a p s i d a n d is not visible from the external face. (Adapted from R a c a n i e l l o , 2 0 0 1 ; A r n o l d 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  infections (Figure 2).  1,000  documented virus  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 (2A ), which leads to functional impairment and morphological disruption in pro  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.  Figure 2. The  The rate of cardiovascular diagnoses per  1,000  viral infections.  r e s u l t of t h e g l o b a l s u r v e i l l a n c e o n v i r a l i n f e c t i o n s r e l a t e d 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 b y t h e W o r l d H e a l t h O r g a n i z a t i o n . c o x s a c k i e v i r u s B g r o u p is t h e 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 . era/., 1993).  The (Grist  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  CVB3  initiates  infection of cells by first attaching  to  the  host  cell  m e m b r a n e through a host cell receptor, c a l l e d c o x s a c k i e v i r u s a n d a d e n o v i r u s receptor ( C A R ) .  It is a 4 6 k D a protein w h i c h not only binds to C V B 3 , but a l s o  mediates viral R N A entry into cells for viral replication. Other than C A R , C V B 3 a l s o binds to another cell surface protein c a l l e d d e c a y - a c c e l e r a t i n g factor ( D A F ) or C D 5 5 , w h i c h  is a  m e m b e r of the c o m p l e m e n t c a s c a d e .  H o w e v e r , the  interaction between C V B 3 a n d D A F is not sufficient for infection, it may require cx p6-integrin a s a c o r e c e p t o r (Shafren et al., 1995). O n c e C V B 3 has attached to v  the cellular receptor, the viral R N A enters into the host cells a n d s e r v e s a s 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 g e n o m e is u n c o a t e d , a p r o c e s s that involves structural c h a n g e s  in the c a p s i d .  O n c e the positive-  stranded viral R N A enters the c y t o p l a s m , it is immediately translated to p r o d u c e viral proteins that are e s s e n t i a l for viral R N A replication, a s s e m b l y a n d (Figure 3).  Efficient translation initiation of most eukaryotic m e s s e n g e r  release RNAs  ( m R N A s ) involves R N A - p r o t e i n a n d protein-protein interactions at both the 5' a n d 3' e n d s of the transcript through a c a p - d e p e n d e n t m e c h a n i s m .  T h e recruitment  of a 4 0 S r i b o s o m a l subunit a n d the recognition of the 5' 7-methyl g u a n o s i n e  ( m G p p p N ) c a p p e d e n d of the m R N A require the recruitment of the eukaryotic 7  initiation factor 4 F ( e l F 4 F ) c o m p l e x , c o m p r i s e d of e l F 4 E , e l F 4 A a n d  elF4G,  w h i c h a s s o c i a t e s with the multi-subunit e l F 3 c o m p l e x . T h e poly(A) tail s e r v e s a s a translational e n h a n c e r a n d interacts with the 5' c a p to initiate translation. T h i s a s s o c i a t i o n between the c a p a n d 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  guanosine  component  c o x s a c k i e v i r a l R N A is not c a p p e d a s found  with  in all eukaryotic transcripts.  a  7-methyl  Instead,  it is  covalently linked to the v i r u s - e n c o d e d protein V P g , a s previously mentioned. D u e to its u n c a p p e d but polyadenylated g e n o m e , the viral R N A e m p l o y s a n alternative m o d e of translation initiation through a novel m e c h a n i s m .  This cap-  independent translation m e c h a n i s m requires interaction between the r i b o s o m e a n d the specific s e q u e n c e element I R E S within the 5' U T R . T h e I R E S directs the landing of the small ribosomal 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  intermediate using the input positive strand R N A a s a template. polyprotein  products,  polymerase  3D  (3D  po1  ) , is  an  negative-stranded O n e of the viral  RNA-dependent R N A  p o l y m e r a s e w h i c h is essential for this viral transcription p r o c e s s .  T h e negative  strand intermediate then s e r v e s a s a template for multiple rounds of transcription to p r o d u c e n u m e r o u s progeny g e n o m e s .  T h e n the progeny R N A is p a c k a g e d  into a mature c a p s i d a n d is r e l e a s e d from the cell (Figure 3). H o w e v e r , the e x a c t m e c h a n i s m s of viral release are u n k n o w n . M a n y picornaviruses are released a s  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 2 A 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  pro  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 intermolecular mechanism). Viral 2A  pro  (an  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-  a n d is the most significant event in the separation of the structural from the n o n structural polypeptide d o m a i n s . In addition, 2 A  p r o  a l s o r e c o g n i z e s a s e c o n d c l e a v a g e site on the 3 C D  precursor protein that g i v e s rise to the products 3 C a n d 3 D ' instead of the viral protease 3 C ( 3 C 3CD (3CD  p r o  p r o  ) and polymerase 3 D  p o 1  ; however, this c l e a v a g e of protease  ) a p p e a r s to be strain specific.  O n l y s o m e polioviruses utilize this  pathway but not all, a n d the c l e a v a g e products are replication  (Ryan  et al., 1997; K r a u s s l i c h  not e s s e n t i a l for viral  et al., 1988).  This  "secondary"  c l e a v a g e in the formation of 3 C a n d 3 D ' m a y permit the virus to modulate e x p r e s s i o n of its c a p s i d proteins a n d replication e n z y m e s (Krausslich et al., 1988).  M o s t of the remaining c l e a v a g e s of the picornaviral polyprotein are  mediated by the viral e n z y m e 3 C  p r 0  .  This protease c a t a l y z e s eight or nine  c l e a v a g e s within the polyprotein in order to r e l e a s e other viral proteins (Figure 4).  Figure 3. Overview of the coxsackievirus replication cycle. V i r u s binds to the C A R a n d D A F receptors (1) a n d the g e n o m e is u n c o a t e d (2). V P g is r e m o v e d from the viral R N A a n d the resulting R N A is then translated (3). T h e polyprotein is c l e a v e d nascently to p r o d u c e individual viral proteins (4). R N A synthesis o c c u r s in v e s i c l e s . Viral (+) strand R N A is transcribed by the viral R N A p o l y m e r a s e to form full-length (-) strand R N A (5), w h i c h is then c o p i e d to p r o d u c e additional (+) strand R N A (6). During infection, newly s y n t h e s i z e d (+) strand R N A is translated to p r o d u c e additional viral proteins (7). Later in infection, the (+) strands enter the morphogenetic pathway (8). N e w l y s y n t h e s i z e d virus particles are r e l e a s e d from the cell by lysis (9). (Adapted from R a c a n i e l l o , 2 0 0 1 ) .  -12-  <  Q CO  S*  O CO  HDO  (0  >  CO  (0  r  u  < CO  O  a  CM  CM  a> "(A  151 | C R < CM  ^_  a.  CL  ro > ro  o o  0  >  CM CL  CM  >  > C L >  o  a. >  o  IDL  ro J_  O 3 i_ <-» (A • C  o  ^ CO CM - ~ CO  CO  _  > o n ** o CO  3  "5 2  CD  CO  o  0  CM  c ca O  __r  Q."c5  r.  —  Q. >.  CO  -*—' r-  CO  .  0  II § 1 CD  CO CQ  0  CO c= CO O C c  0  E "5 O) C N CO (0 CL c c 0 .E 1 P Q- 0  CM  <  ro  2  0 CJ x:-*—> +-*  S t "  l l  ro £g C0O V) Mo to co c _  k  c ro ^  Q.  o  0  I)  4  CO CL >  C L >  Tt  C  O '53 *->  o _ i  a  >. o Q.  CO o (A C  "3 2  a. ro  CM  CL >  0  CD  ro o  CL >  co  (A i_ 3 O  CO 3  <D  o  > c  o  CQ  CL  "55  "53  CM  CO CL  CM  g  O c  >  o  k  a. >  CM  CM  o CQ CM  O  a>  * J  DO  CO  (A  'co  CM  <  0  CO CO  co  CA C  a  CO  a  o  O J C L >  0 0 C5 CT 0  o  co  O  < CO  o i_  C L >  _0  CD  ^  o CO CL  CO  CO > CO  -4—.  Q CO  C u >  D5  CO > c _ CO  Q CO  CO CQ >  0  O co o  Q.  CO  CO  £ ~  o  <l Z or  o  CL ^ 0) CD  3 •4-1  o  3 i_ *-> (0  TJ  CO  ro  0  c £  0  o  g  0  .2 CL  | -3I I' C  c  0  ro 2 to Q> 2 o =5 o co Q. ® CJ  >< E 0 §.8> _ CO £ Mo w co 0 T3 O CN 0 O . C o 2  0 C 0  rn vJ < = Tj-  0  c CO P; 3 O C O CD O) E 0 0  E 2  0  sz  CO  1.3  Overview of apoptotic cell death Apoptosis,  also  referred  to  programmed  cell  death,  is a genetically  controlled p r o c e s s w h i c h is required to maintain the integrity a n d h o m e o s t a s i s of a multicellular o r g a n i s m ( J a c o b s o n et al., 1997; N a g a t a , 1997).  It is an intrinsic  self-eliminating m e c h a n i s m w h i c h eliminates cells during d e v e l o p m e n t or during the continuous generation  of the i m m u n e repertoire.  It is a l s o required in  balancing cell division, maintaining the c o n s t a n c y of tissue m a s s , removing cells injured  by genetic defects,  aging, d i s e a s e ,  or e x p o s u r e  to noxious a g e n t s  (Golstein et al., 1991; S c h w a r t z , 1998; S a i k u m a r et al., 1999).  1.3.1  Apoptosis: Cellular and molecular characteristics Morphologically, cells undergoing a p o p t o s i s demonstrate cell shrinkage,  nuclear/cytoplasmic  condensation,  membrane  blebbing,  fragmentation  into  m e m b r a n e b o u n d apoptotic bodies, a n d m e m b r a n e alterations that eventually lead to phagocytosis of the affected cells. characterized  by a reduction in the  Biochemically, apoptotic cells are  mitochondrial t r a n s m e m b r a n e potential,  production of reactive o x y g e n s p e c i e s , externalization of residues  in m e m b r a n e  phosphatidylserine  bilayers, selective proteolysis of a s u b s e t of cellular  proteins, a n d the degradation of D N A into internucleosomal fragments (Wyllie et al., 1984; H o c k e n b e r y et al., 1993; L a z e b n i k 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  C a s p a s e s and caspase activation  A p o p t o s i s is regulated by a series of b i o c h e m i c a l e v e n t s ( S a i k u m a r et al., 1999). It is now clear that c a s p a s e s , a family of aspartic acid-directed proteases, are key effector m o l e c u l e s in apoptotic cell death.  C a s p a s e s are s y n t h e s i z e d in  the cytosol of m a m m a l i a n cells a s inactive z y m o g e n s , w h i c h b e c o m e through  intracellular c a s p a s e  cascades  (Cohen,  1997).  c a s p a s e s c l e a v e a large s u b s e t of specific d o w n s t r e a m poly(ADP-ribose)  polymerase  (PARP)  Once  active  activated,  substrates, including  ( L a z e b n i k et al.,  1994),  inhibitor of  c a s p a s e - a c t i v a t e d D N a s e (ICAD) (Liu et al., 1997) , focal a d h e s i o n kinase ( F A K ) ( W e n et al., 1997), gelsolin (Kothakota et al., 1997), lamin A ( T a k a h a s h i et al., 1996a), a n d m a n y others.  S u c h c l e a v a g e s result in disruptions of normal cellular  p r o c e s s e s and c a u s e cellular morphological a n d importantly,  caspase-dependent  cleavage  a b a t e m e n t of survival pathways,  such as  of the  structural c h a n g e s .  specific  proteins  More  induces  extracellular signal  the  regulated  kinase, w h i c h c a n interfere with the apoptotic r e s p o n s e ( W i d m a n n et al., 1998).  1.3.2.2  Effector  caspases,  cell  surface  receptor  activation  and  mitochondrial signaling in regulation of cell death A p o p t o s i s c a n be triggered by two distinct pathways, o n e initiated by extracellular  signals  and  independent  pathways  the  other  converge  caspases, such as caspase-3,  at  by the  -6, a n d -7.  -15-  intracellular activation  signals.  These  of d o w n s t r e a m  two  effector  T h e first major pathway involves  ligation of the death receptors by their ligands, s u c h a s ligation of F a s L a n d tumor n e c r o s i s factor-a ( T N F - a ) with their respective receptors, resulting in the recruitment of adaptor proteins a n d p r o c a s p a s e - 8 m o l e c u l e s to the receptors to transactivate  caspase-8.  T h e activated  caspase-8  will  either  activate  the  d o w n s t r e a m c a s p a s e s directly or induce direct c l e a v a g e of Bid, a m e m b e r of the Bcl-2 family, to yield a truncated B i d (tBid).  U p o n c l e a v a g e , tBid c a n then  translocate from the cytosol to the mitochondrial m e m b r a n e , release  of c y t o c h r o m e c, w h i c h amplifies the death  resulting in the  signals.  Mitochondrial  release of c y t o c h r o m e c c a n also be triggered by v a r i o u s cellular s t r e s s e s , s u c h a s D N A d a m a g e , toxins, a n d A T P depletion. T h e r e l e a s e d c y t o c h r o m e c, w h i c h binds to apoptotic protease activating factor-1 (Apaf-1), activates  procaspase-9  to  activate  active c a s p a s e - 9 .  downstream  This  initiator c a s p a s e  effector c a s p a s e s ,  such as  will  caspase-3,  cleave  and  inducing cell  the  apoptosis  (Figure 5). The  s e c o n d major pathway, the intercellular/intrinsic pathway, c o u l d be  directly stimulated w h e n cells e x p o s e to v a r i o u s apoptotic stimuli or s t r e s s a s mentioned a b o v e .  T h i s pathway is regulated by the Bcl-2 family proteins.  The  Bcl-2 family m e m b e r s consist of both anti-apoptotic proteins (such a s Bcl-2, B c l xL, a n d Bcl-w) w h i c h inhibit c a s p a s e activation, a s well a s pro-apoptotic proteins (such as B a x , B a k , a n d Bid) w h i c h promote c a s p a s e activation. Anti-apoptotic m e m b e r s act not only by direct binding to Apaf-1 to inactivate it, but a l s o by stabilizing mitochondrial m e m b r a n e s to inhibit the r e l e a s e of c y t o c h r o m e c.  On  the other hand, a pro-apoptotic m e m b e r like B a x translocates to mitochondria  - 16-  a n d r e l e a s e s c y t o c h r o m e c by selectively permeabilizing the outer mitochondrial m e m b r a n e ( E s k e s e r a / . , 1998). Unlike B a x , B i d requires proteolysis by c a s p a s e 8 for its pro-apoptotic function ( m e c h a n i s m a s d e s c r i b e d a b o v e ) . S u c h c a s p a s e 8-mediated  cleavage  of  B i d may  be  a  mechanism  designed  to  amplify  d o w n s t r e a m effector events during death-receptor-mediated apoptosis ( S a i k u m a r e r a / . , 1999) (Figure 5).  - 17-  S? o O ro = "co < 3 CJ  .SP «- "O CO (A b C Ja CO co c re te CL  " Vi  TJ C  1 3  o  i_  Q.  £  CL  CO o  JD CO  ^  •D  o  £ sz CO  CD CO  o  > co co  ~c o  CL  c CD W <0 CD  S  •I  O)  CO  = <? ao> ® i - aS  ro  CL  tf) CO  O CO 0 CCB o CD o O O CD 0 5 m CO CD *- 0 5 2 CO .h= C o  t  JC o  >  T  c  CO  tf) tf) = ® o  ~  -  .2 75  a> co >,.>  CD  O  ;  0  c o> N m  JD " K  CO  CJ CO  2 £2 0 o r  *-> ± 3  Q. CO i_ O u O u CO o  E 2o  1CL  1  co - Q r—  3 St CJ tf)  E ^  0  C  C5  O  T _  75  CO tH 0  .ii  *1  CO CO CO  CD  o  .2 Q CO  S -J2  1  o  CO  "D J= c  ro  g co o S.8 to 0  €8 id E co E . 2 >  O CO O CoO a 0.4= (0 co  co o  CJ CO -g CO CD.  —  0 E .E 2- SPro^ co§.  ro §  — LO  c  tf) E o o co >» L. / \ ro w to SZ ~  SI EP t o  LL  o  CO 0  a  CO o  o Q. c ro >• E CL  CO - t i  CJ O CO  00  1.4  Pathogenesis of C V B 3  1.4.1  Cytopathic effects and apoptotic cell death induced by C V B 3 A p o p t o s i s c a n also be c o n s i d e r e d as a host defence m e c h a n i s m against  virus infections to limit viral replication a n d to prevent the virus from spreading to surrounding uninfected  cells.  A n increasing n u m b e r of v i r u s e s is k n o w n to  induce apoptosis at the late s t a g e s of infection (Cuff et al., 1996; T s u n o d a et al., 1997a&b; Carthy et al., 1998; G i r a r d ef al., 1998). including c o x s a c k i e v i r u s a n d  Several  picornaviruses,  poliovirus, h a v e b e e n d e m o n s t r a t e d to  induce  apoptotic cell death ( T o l s k a y a et al., 1995; C a r t h y et al., 1998; G h a d g e et al., 1998).  P r e v i o u s studies from our laboratory o b s e r v e d C V B 3 - i n d u c e d cytopathic  effects  in an in vitro H e L a cell m o d e l (Carthy et al., 1998) a n d virus-induced  direct injuries  in multiple susceptible  infected mice (Carthy, 2002).  organs,  including the  m y o c a r d i u m , in  At d a y three post-infection (pi) of m o u s e hearts,  coagulation necrosis a n d cytopathic effects were o b s e r v e d , followed by immune cell infiltration a n d c a r d i a c remodeling at d a y nine a n d d a y 30 pi, respectively (Carthy, 2002).  B i o c h e m i c a l l y , the 32 k D a p r o c a s p a s e - 3 is found to be c l e a v e d  a n d activated following degenerative morphological c h a n g e s o b s e r v e d in infected H e L a cells. T h i s c l e a v e d c a s p a s e - 3 is proteolytically active a n d further c l e a v e s a number of substrates, including P A R P and I C A D (Carthy ef al., 1998). proteolytic activities result in alterations  These  of normal cellular h o m e o s t a s i s  and  cellular morphological c h a n g e s . U p o n virus infection, apoptosis of infected cells c a n a l s o be induced by cytotoxic T cells through the F a s / F a s L pathway a n d by g r a n z y m e  - 19-  B through  caspase  activation ( S a i k u m a r et al., 1999).  In previous studies, w e  have  demonstrated that c a s p a s e - 3 is activated in C V B 3 - i n f e c t e d H e L a cells eight h pi (Carthy et al., 1998). infection.  T h i s result s u g g e s t s that c a s p a s e activation o c c u r s after  Furthermore, recent studies h a v e s h o w n that o v e r e x p r e s s i o n of the  polioviral 2 A  p r o  or 3 C  p r 0  alone leads to c a s p a s e - 3 activation a n d apoptosis ( B a r c o  et al., 2 0 0 0 ; G o l d s t a u b et al., 2000). H o w e v e r , further studies are required s i n c e it is still u n k n o w n whether c a s p a s e - 3 is directly activated by t h e s e viral p r o t e a s e s or indirectly activated through the different death c a s c a d e s , s u c h a s 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 2A  p r o  p r o  is a small cysteine protease, approximately 17 k D a , w h i c h s h a r e s  h o m o l o g y with the bacterial trypsin-like serine protease ( R y a n et al., 1997; Y u et al., 1991). T h e frans-cleavage activity of 2 A  p r 0  involves p r o c e s s i n g an increasing  list of host cellular proteins, including e l F 4 G ( B o v e e et al., 1 9 9 8 a & b ; N o v o a et al., 1999) a n d other translation initiation factors (Figure 6). extensive studies have b e e n performed on the 2 A Investigations  have  demonstrated  that  p r o  purified  O v e r the past d e c a d e ,  frans-cleavage of e l F 4 G . recombinant  2A  p r 0  of  c o x s a c k i e v i r u s B 4 a n d h u m a n rhinovirus type 2, a s well a s poliovirus, c a n c l e a v e e l F 4 G at A r g 4 8 5 - G l y 4 8 6 directly in vitro ( L a m p h e a r et al., 1993; B o v e e et al., 1998a&b).  C l e a v a g e of e l F 4 G dysregulates host cell m e t a b o l i s m by abruptly  -20-  halting host protein synthesis. During viral infection, complete c l e a v a g e of e l F 4 G o c c u r s v e r y rapidly, approximately two h pi. T h i s c l e a v a g e event a n d s u b s e q u e n t a b a t e m e n t of host protein synthesis l e a d to the a b o l i s h m e n t of cellular m R N A translation a n d the translation.  initiation of c a p - i n d e p e n d e n t  cap-dependent viral  mRNA  Interestingly, earlier studies have s h o w n that host protein synthesis  is s u p p r e s s e d by 5 0 % or less after poliovirus infection, despite complete e l F 4 G c l e a v a g e (Lloyd et al., 1987; P e r e z et al., 1992). T h e s e findings s u g g e s t e d that c o m p l e t e inhibition of host protein synthesis may require proteolysis of other factors for translation initiation. Recently, a novel h u m a n h o m o l o g u e of e l F 4 G , called elF4GII (the original e l F 4 G w a s r e n a m e d e l F 4 G I ) , has b e e n reported a s the s e c o n d c l e a v a g e target required for complete inhibition of host protein synthesis (Gradi et al., 1998a&b). elF4GII a p p e a r s to be functionally equivalent to e l F 4 G I , but is only 4 6 % identical at the a m i n o acid level a n d has a 5 6 % overall similarity to e l F 4 G I (Gradi et al., 1998a).  Both poliovirus a n d  human  elF4GII, with s l o w e r kinetics a s  rhinovirus 2 A  p r 0  w e r e found to  cleave  c o m p a r e d to c l e a v a g e of e l F 4 G I , a n d  the  c l e a v a g e s result in the complete shutoff of host protein synthesis (Svitkin et al., 1999; G o l d s t a u b et al., 2000).  T h u s , c l e a v a g e of both e l F 4 G I a n d elF4GII  a p p e a r s to be required for the abolishment of host protein synthesis after virus infection.  T h e s e requirements m a y explain s e v e r a l earlier reports d o c u m e n t i n g  the lack of correlation between e l F 4 G I c l e a v a g e a n d the inhibition of cellular m R N A translation after enterovirus infection.  M o s t significantly, e l F 4 G I  and  elF4GII c l e a v a g e s m a y trigger host cell a p o p t o s i s either by inhibiting the c a p -  -21 -  d e p e n d e n t translation of cellular m R N A s that e n c o d e  proteins  required for  maintaining cell viability or by e n h a n c i n g c a p - i n d e p e n d e n t translation of cellular m R N A s , s u c h a s c - m y c a n d Apaf-1 ( J o h a n n e s et al., 1998), w h i c h contain I R E S a n d e n c o d e a pro-apoptotic (death-associated) A n o t h e r host protein target of 2 A Kerekatte et al., 1999) (Figure 6).  p r o  protein.  is the P A B P ( J o a c h i m s et al., 1999;  T h e c l e a v a g e of P A B P o c c u r s at the C -  terminal d o m a i n ( M e t 4 9 0 - G l y 4 9 1 ) of h u m a n ,  separating  motifs) a n d C-terminus (homodimerization domain). mediated by both poliovirus 2 A  p r o  the  N - (recognition  This cleavage  a n d c o x s a c k i e v i r u s (B3 a n d B4) 2 A  can p r o  .  be The  resulting N-terminal c l e a v a g e product of P A B P w a s found to be l e s s efficient in promoting translation than the full-length P A B P .  Furthermore, P A B P c l e a v a g e  d o e s not o c c u r during infection in the p r e s e n c e of g u a n i d i n e - H C I , a n inhibitor w h i c h prevents host translation obstruction. T h i s data s u g g e s t s that c l e a v a g e of P A B P m a y play a role in altering host protein synthesis upon virus infection. It is a l s o important to note here that P A B P is involved in translation initiation in lower eukaryotes v i a its interaction with the poly(A) tail of m R N A s .  Poly(A) tail s e r v e s  a s a translational e n h a n c e r a n d interacts with the 5' c a p to initiate translation in yeast (Preiss et al., 1998).  T h i s a s s o c i a t i o n between the c a p a n d poly(A) tail  requires the binding of P A B P to the N-terminus of e l F 4 G I (Imataka et al., 1998). B e s i d e translation initiation factors, host proteins involved in transcription are also proteolytic targets of 2 A  p r o  , s u c h a s T A T A - b i n d i n g protein ( T B P ) ( D a s et  al., 1993; Y a l a m a n c h i l i et al., 1997a) (Figure 6). T B P plays a n essential role in promoting R N A p o l y m e r a s e II- a n d Ill-mediated transcriptions.  -22-  T B P has  been  demonstrated to be c l e a v e d directly at the N-terminal d o m a i n (Tyr35-Gly36) by poliovirus  2A  p r o  .  Surprisingly, this  cleavage  of  T B P does  not  alter  its  transcriptional activity a n d the R N A p o l y m e r a s e ll-mediated transcription is not inhibited.  T h i s s u g g e s t s that T B P c l e a v a g e 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 p a t h o g e n e s i s  through  c l e a v a g e of cytoskeletal proteins h a v e b e e n d o c u m e n t e d (Figure 6). Dystrophin is a cytoskeletal protein w h i c h c o n n e c t s the internal F a c t i n - b a s e d cytoskeleton to the p l a s m a m e m b r a n e w h e r e it binds to the p-dystroglycan c o m p o n e n t of the dystrophin-glycoprotein c o m p l e x (Badorff et al., 2000).  Mutations in dystrophin  that result from premature termination of translation c a n c a u s e h u m a n X - l i n k e d DCM.  C V B 3 has b e e n postulated to c a u s e D C M ; however, the pathological  m e c h a n i s m remains u n k n o w n . CVB3 2A  p r o  Dystrophin has b e e n reported to be c l e a v e d by  directly in vitro a n d in vivo (Badorff et al., 1999; Badorff er al., 2000).  Dystrophin contains four hinge s e g m e n t s that are a c c e s s i b l e for proteolytic c l e a v a g e and C V B 3 2 A  pro  - m e d i a t e d c l e a v a g e w a s found to be at the site (human:  amino a c i d 2434; m o u s e : amino a c i d 2427) in the hinge 3 region.  Cleaved  dystrophin c a n lead to functional impairment a n d morphological disruption of heart m u s c l e cells.  H o w e v e r , the detailed pathogenic m e c h a n i s m by w h i c h  enterovirus infection begets D C M n e e d s to be studied further. Cytokeratin 8 is another structural protein c l e a v e d by 2 A  p r o  (Seipelt et al.,  2000) (Figure 6). Cytokeratin 8 is a m e m b e r of the intermediate filament family that forms the cytoskeleton together with actin filaments  -23-  and  microtubules.  C o l l a p s e of the  intermediate  filament network leads to the  deterioration of  m e c h a n i c a l support a n d the stability of cells. C l e a v a g e of cytokeratin 8 by 2 A  p r o  of h u m a n rhinovirus type 2 w a s found to be at S e r 1 4 - G l y 1 5 a n d results in the removal of 14 a m i n o acids from the N-terminal d o m a i n . T h i s c l e a v a g e is highly specific s i n c e other intermediate filament proteins, s u c h a s cytokeratin 18 a n d vimentin, are not c l e a v e d by 2 A  p r o  .  C l e a v a g e of cytokeratin 8 is s p e c u l a t e d to  destabilize the host cell a n d thus promote the s p r e a d of the virus. H o w e v e r , the molecular  basis  of  cytokeratin  8  cleavage  in  contributing  to  the  virus  p a t h o g e n e s i s has not b e e n elucidated.  ii). Viral 3C  PRO  A n o t h e r cysteine protease found in c o x s a c k i e v i r u s is the 3 C to p r o c e s s i n g the viral polyprotein, 3 C  p r o  p r o  .  In addition  (20 kDa) also specifically c l e a v e s a  n u m b e r of host proteins, including factors in both translation a n d transcription a n d proteins that regulate host cell survival a n d death. A major host protein c l e a v e d by viral 3 C 6).  p r o  is the L a autoantigen (Figure  L a is a cellular protein that has b e e n demonstrated  to stimulate internal  ribosomal entry, a m e c h a n i s m by w h i c h picornaviruses initiate translation of its genomic R N A .  Other physiological functions of L a protein include transfer R N A  (tRNA) p r o c e s s i n g , synthesis of p o l y m e r a s e  II transcripts,  a n d formation of  s n R N P c o m p l e x e s a s well a s ribosomal binding (Gottlieb et al., 1989a&b; P e e k et al., 1996; Pellizzoni er al., 1996).  C l e a v a g e of L a by the poliovirus 3 C  p r o  o c c u r s in the C-terminal region (between G l n 3 5 8 a n d G l y 3 5 9 ) (Shiroki et al.,  -24-  1999), a n d s u c h modification c a u s e s L a to relocate into the c y t o p l a s m from the nucleus.  T h i s c l e a v a g e is believed to help improve the translation efficiency of  the virus. Recently, L a autoantigen h a s b e e n demonstrated to bind to the 5' U T R of C V B 3 R N A g e n o m e ( C h e u n g et al., 2002; R a y et al., 2002). interaction between  the  L a autoantigen  a n d the  S u c h a specific  C V B 3 R N A is believed to  promote viral translation. A n o t h e r major host protein c l e a v e d by viral 3 C tract binding protein  ( P T B ) (Figure 6).  p r o  is the poly-pyrimidine  P T B is found  to be  pertinent  picornaviral translation initiation ( K a m i n s k i et al., 1995; Hunt et al., 1999).  It has  b e e n s h o w n to bind viral 5' U T R a n d c o o p e r a t e with the L a autoantigen optimal a n d a c c u r a t e translation initiation of the viral g e n o m e 1994). C l e a v a g e of the P T B by poliovirus 3 C which  generate  multiple  peptides  translation (Back et al., 2002). promotes  that  p r o  o c c u r s in three different locations  appear  to  inhibit  IRES-dependent  It has b e e n s u g g e s t e d that the c l e a v a g e event genome  H o w e v e r , the molecular m e c h a n i s m s that signal  the appropriate time of transition v i a the 3 C  p r o  remains u n k n o w n .  C l e a v a g e of e l F 4 G I a n d P A B P by viral 2 A  p r o  has b e e n p r o p o s e  d to c a u s e s e v e r e translation inhibition in virus-infected c e l l s . these cleavages  in  (Toyoda et al.,  a molecular switch from viral protein e x p r e s s i o n to viral  replication (Back et al., 2002).  to  have b e e n s h o w n to be insufficient for complete  However, translation  inhibition, a n d only lead to a partial translation shutoff ( B o n n e a u et al., 1987; P e r e z et al., 1992).  A recent study has demonstrated  that polioviral  3C  p r 0  c l e a v e s P A B P a n d r e m o v e s the C-terminal d o m a i n ( K u y u m c u - M a r t i n e z et al.,  -25-  2004), w h i c h interacts with s e v e r a l translation initiation factors, s u c h a s translation initiation factor e l F 4 B . of e n d o g e n o u s  mRNA  and  C l e a v a g e of P A B P by 3 C  reporter  inhibition is poly(A)-dependent a n d e l F 4 G I a n d e l F 4 G I I by 2 A  p r 0  .  RNA.  This 3C  pro  p r o  inhibits translation  -mediated  translation  is a s effective a s complete c l e a v a g e of  T h i s c l e a v a g e event has raised the functional  significance of P A B P in the regulation of translation a n d has s u g g e s t e d enteroviruses  use a dual strategy for host translation shutoff,  c l e a v a g e of P A B P by 3 C 3C  p r o  p r o  that  requiring both  a n d c l e a v a g e of e l F 4 G I a n d e l F 4 G I I by 2 A  p r 0  inhibits host protein e x p r e s s i o n not only at the translational level, but  also at the transcriptional level. elucidate  the  the  mechanism  of  M a n y early studies h a v e b e e n c o n d u c t e d to transcriptional  inhibition  and  a  number  of  transcription factors have b e e n demonstrated to be c l e a v e d directly by viral 3 C  p r o  .  O n e of these key proteins is T B P (Berk, 1999; P u g h , 2 0 0 0 ; G e i d u s c h e k et al., 2001) (Figure 6).  T B P has b e e n s h o w n to be directly c l e a v e d by 3 C  poliovirus infection, resulting in a n inhibition of R N A p o l y m e r a s e transcription ( Y a l a m a n c h i l i et al., 1996). Unlike 2 A T B P c l e a v a g e by 3 C  p r o  than proteolysis by 2 A  p r o  p r o  p r o  during  ll-mediated  , this inhibition indicates that  is more important in the regulation of host transcription .  A n o t h e r pivotal player in 3 C  pro  - m e d i a t e d transcription inhibition is the  cyclic A M P ( c A M P ) - r e s p o n s i v e element ( C R E ) - b i n d i n g protein, C R E B , (Figure 6). It is c l e a v e d by 3 C  p r o  between a m i n o a c i d residues 172 a n d 173 (Yalamanchili et  al., 1997b), resulting in the separation  of its D N A binding d o m a i n from  the  transcription activation d o m a i n . Recently, C R E B has a l s o b e e n demonstrated to  -26-  h a v e an anti-apoptotic effect upon induction of phosphorylation at serine residue 133  by  insulin-like growth  factor-l  in  a  phosphatidylinositol-3-OH-kinase-  d e p e n d e n t and mitogen-activated protein k i n a s e - d e p e n d e n t m a n n e r (Mehrhof et al., 2001).  A variety of stimuli, including those regulating the intracellular levels  of c A M P and C a , growth factors, a n d cellular stress, induce C R E B activation 2 +  leading to the up-regulation of B c l - 2 (Riccio et al., 1999; M e h r h o f et al., 2001). A l s o , there have b e e n reports of c a s p a s e - m e d i a t e d degradation of C R E B during neural cell apoptosis (Francois et al., 2000). 3C  p r 0  T h u s , the c l e a v a g e of C R E B by  may disrupt not only the R N A p o l y m e r a s e ll-mediated transcription but a l s o  a variety of cellular p r o c e s s e s that are involved in cell survival a n d death. A s i d e from the transcription factor C R E B , 3 C  p r 0  also cleaves  Octamer  binding transcription factor-1 (Oct-1) (Figure 6), a host protein involved in both R N A p o l y m e r a s e II- a n d Ill-mediated transcription (Yalamanchili et al., 1997c; G e i d u s c h e k et al., 2001).  Oct-1 has been implicated in the up-regulation of  inducible nitric oxide s y n t h a s e , an e n z y m e that c a t a l y z e s the synthesis of nitric oxide ( S a w a d a et al., 1997; L e e er al., 2001).  T h u s , c l e a v a g e of Oct-1 by 3 C  p r o  may alter the production of N O a n d h e n c e protect the virus against early i m m u n e responses.  H o w e v e r , further investigation is n e e d e d to verify the link a n d the  functional significance of the 3 C  p r 0  c l e a v a g e of Oct-1 in the overall p a t h o g e n e s i s  of the virus. Other than  the  R N A polymerase  inhibits R N A p o l y m e r a s e  ll-mediated transcription,  Ill-mediated transcription  through  3C  cleavage  p r o  of  also the  transcription factor IMC (TFIIIC) (Figure 6). TFIIIC is a large c o m p l e x , containing  -27-  five subunits of 2 4 0 , 110, 100, 8 0 , a n d 6 0 k D a , a n d binds the B - b o x internal promoter element of t R N A g e n e s .  TFIIIC is c l e a v e d a n d inactivated by 3 C  during poliovirus infection ( S h e n et al., 1996).  p r o  S h e n et al. demonstrated in vitro  that a n N-terminal 8 3 k D a d o m a i n of the largest subunit (240 k D a ) a s s o c i a t e s with the 110 k D a subunit to generate the TFIIIC D N A binding d o m a i n . A l t h o u g h the  D N A binding activities of the  cleavage  products  are  unchanged,  3C  p r o  c l e a v a g e of TFIIIC inactivates its transcriptional activity. S i n c e the c l e a v e d forms of TFIIIC that bind to the B - b o x promoter element a p p e a r to lack the 100, 80, a n d 60 k D a subunits a n d are defective for transcription, it s u g g e s t s that at least o n e of these subunits is required for TFIIIC's function in transcription. All of t h e s e c l e a v a g e s by both picornaviral 2 A  p r o  and 3 C  p r o  lead to a  d e c r e a s e in cellular transcription a n d translation, resulting in the down-regulation of the e x p r e s s i o n of m a n y host g e n e s a n d proteins. S u c h c l e a v a g e s also l e a d to a n i m b a l a n c e in cellular h o m e o s t a s i s , which ultimately m a y lead to host cell death a n d myocardial remodeling.  H o w e v e r , m a n y of the c l e a v a g e s d e s c r i b e d  a b o v e w e r e studied in other picornaviral models, s u c h as poliovirus a n d h u m a n rhinovirus, a n d there is currently no d o c u m e n t e d e v i d e n c e s h o w i n g that C V B 3 will perform the s a m e c l e a v a g e s or p r o c e s s the s a m e set of translation a n d transcription factors.  M o r e importantly, the a m i n o acid s e q u e n c e h o m o l o g i e s of  the c o x s a c k i e v i r a l proteins are highly c o n s e r v e d a m o n g c o x s a c k i e B viruses but not a s highly c o n s e r v e d to the respective proteins of other picornaviruses (Table 1). T a b l e 1 s h o w s 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 c o x s a c k i e B v i r u s e s ; however, they s h a r e only  -28-  6 0 % a n d 5 0 % h o m o l o g i e s with poliovirus (Enterovirus) (Rhinovirus), 2A  p r o  and 3 C  a n d h u m a n rhinovirus  respectively. Therefore, it remains to be elucidated whether C V B 3 p r o  will a l s o affect cellular h o m e o s t a s i s by p r o c e s s i n g similar target  proteins that are involved in viral pathogenesis.  -29-  C_  I s '  D) £  ro > CD jy _ LL  W O  j _  _ i  w  O  +—<  a  _  O ^ c CO CD  _2  Q-< O  <  CD CM CO  03  T a b l e 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 J e n k i n s et al., 1 9 8 7 ; K l u m p et al.,  1990).  Homology between* Protein CVB3:CVB1  CVB3:CVB4  CVB3:PV1  CVB4:PV3  CVB4:HRV14  VP4  97  94  65  71  61  VP2  83  80  51  56  57  VP3  81  79  56  56  47  VP1  77  69  39  45  36  p r o  94  92  58  59  44  2B  98  99  50  51  56  2C  98  98  62  63  59  3A  98  93  52  51  48  3B/VPg  91  91  77  73  41  99  98  55  61  53  96  97  80  74  66  2  A  3C 3  D  p r o  p o l  S e q u e n c e identities are e x p r e s s e d a s p e r c e n t a g e s .  -31 -  1.4.2.2  Functional significance of viral  2A  p r o  and  3C  p r o  in inducing  apoptosis Death a n d lysis of cells during virus infection facilitates the r e l e a s e of the virus progeny ( C a r r a s c o , 1995).  H o w e v e r , premature induction of cell death  upon virus infection will severely limit virus production, reducing the s p r e a d of the progeny in the host. Therefore, the d e g r e e of host cell lysis a n d death s h o u l d be controlled in a timely manner.  A n increasing n u m b e r of v i r u s e s is k n o w n to  induce apoptosis in a n active fashion at late s t a g e s of infection.  This process  may provide an efficient w a y to help the virus s p r e a d to neighboring cells, to protect  progeny  viruses  from  host  immune  defenses,  and  to  avoid  an  inflammatory r e s p o n s e (Teodoro et al., 1997; O ' B r i e n , 1998). A p o p t o s i s i n d u c e d by the picornaviruses remains an interesting a n d important observation b e c a u s e the persistence of picornaviral infection may be linked to the ability of the virus to propagate in cells with minimal elicitation of immune  responses.  investigations into the molecular m e c h a n i s m s by w h i c h 2 A  p r o  and 3 C  Further p r o  lead to  cellular apoptosis in a time-specific m a n n e r c a n yield insights potentially relevant to the treatment a n d understanding of persistent picornaviral infections.  A t the  present time, very little is k n o w n c o n c e r n i n g specific c o x s a c k i e v i r a l 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 b e e n strongly implicated.  Within the Picornaviridae e x p r e s s i o n of the polioviral 2 A  p r o  family, recent studies h a v e d e m o n s t r a t e d that or 3 C  p r o  alone l e a d s to c a s p a s e - 3 activation a n d  apoptosis (Barco et al., 2000; G o l d s t a u b et al., 2000).  -32-  M o r e o v e r , recent studies  h a v e s h o w n that transient e x p r e s s i o n of 2 A  p r a  or 3 C  p r o  of enterovirus 71 results in  the c l e a v a g e of the D N A repair e n z y m e P A R P (Li et al., 2002), indicating that viral proteases m a y directly or indirectly activate c a s p a s e s , w h i c h in turn leads to apoptotic r e s p o n s e s . A s previously mentioned, viral 2 A death  through c l e a v a g e s  p r 0  and 3 C  p r o  m a y i n d u c e apoptotic cell  of a n u m b e r of host cellular proteins involved in  transcription, translation initiation, a n d cytoskeletal organization. T h e r e is a l s o a s u g g e s t i o n that t h e s e viral p r o t e a s e s m a y mediate d e l a y e d apoptotic r e s p o n s e s through a n u m b e r of u n k n o w n host protein c l e a v a g e s , s u c h a s the degradation of p53 ( W e i d m a n et al., 2001).  A recent report demonstrated that p53 c a n also  induce apoptosis by perturbations of the mitochondria, resulting in c h a n g e s in the trans-membrane potential, c y t o c h r o m e c release, a n d c a s p a s e activation ( R e g u l a et al., 2001).  T h u s , the c l e a v a g e of p53 by the viral protease(s)  another strategy that v i r u s e s u s e to modulate host r e s p o n s e s  m a y be yet  a n d , thereby,  e n h a n c e viral propagation ( W e i d m a n et al., 2 0 0 1 ) . In c o n c l u s i o n , investigation of the m e c h a n i s m by w h i c h C V B 3 i n d u c e s apoptotic  cell  death  by  viral  protease  expression  will  provide  a  better  understanding of the p a t h o g e n e s i s in the induction a n d p r o g r e s s i o n of C V B 3 i n d u c e d myocarditis.  -33-  1.5  Research focus and project rationale CVB3-induced  myocarditis  and  its  sequela,  major  heart  1980; Grist et al.,  1993;  M o l e c u l a r t e c h n i q u e s from our laboratory a n d  many  d i s e a s e s in children a n d y o u n g adults (Woodruff, H o s e n p u d et al., 1994).  D C M , are  others h a v e identified a n d localized viral R N A in the infected m o u s e heart a n d other o r g a n s using in situ hybridization, all of w h i c h indicates that C V B 3 plays a n important role in t h e s e d i s e a s e s . H o w e v e r , the molecular m e c h a n i s m s , by w h i c h C V B 3 c a u s e s initiation of myocarditis and progression to D C M , are not well understood.  As a  result,  there are  still  therapeutic p r o c e d u r e s available at the  no  virus-specific  preventive  present time for protecting  and  humans  against s u c h virus-induced heart m u s c l e d i s e a s e s . T h e d e v e l o p m e n t of viral myocarditis is a c o m p l i c a t e d interaction between host a n d virus.  Earlier studies have s u g g e s t e d that the m e c h a n i s m s  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) a n d i m m u n e - or autoimmune-mediated  damage  of the heart i n d u c e d by viral infection, typically with inflammation.  Previous  studies carried out on mice with s e v e r e c o m b i n e d immunodeficiency h a v e s h o w n that C V B 3 is c a p a b l e of lytic d a m a g e to myocytes ( C h o w et al., 1992; M c M a n u s et al., 1993).  A l s o , our previous data on the activation of various c a s p a s e s by  C V B 3 - i n f e c t i o n a n d s u b s e q u e n t induction of cytopathic effect at nine h pi on H e L a cells s u g g e s t that C V B 3 induces cell death directly (Carthy et al., 1998). In  previous  work  using  a  differential  mRNA  display technique,  our  laboratory had identified 28 g e n e s that were either up- or down-regulated in the  -34-  C V B 3 - i n f e c t e d m o u s e heart ( Y a n g et al., 1999). T h e g e n e e x p r e s s i o n patterns of 15 of the 28 g e n e s w e r e further confirmed by either Northern hybridization or R T PCR.  T h u s , these g e n e s m a y h a v e potential functions in contributing to the  d e v e l o p m e n t of viral myocarditis. A m o n g these g e n e s , N A T 1 , also called deatha s s o c i a t e d protein 5 ( D A P 5 ) , is o n e of the most interesting c a n d i d a t e s involved in d i s e a s e induction. N A T 1 is a 97 k D a protein w h i c h is found to be down-regulated in the CVB3-infected characteristics.  mouse  heart a n d p o s s e s s e s unique structural a n d functional  It is predominantly e x p r e s s e d in atrial a n d ventricular myocytes  a n d plays important roles in the postnatal d e v e l o p m e n t of the heart (Pak et al., 1999).  More importantly, it is a structural h o m o l o g u e of e l F 4 G I (Figure 7).  The  N-terminal part of N A T 1 has 3 9 % identity a n d 6 3 % similarity to the central region of e l F 4 G I , while its C-terminal part is less h o m o l o g o u s to the c o r r e s p o n d i n g region of e l F 4 G I , s u g g e s t i n g that it m a y p o s s e s s unique functional properties (Imataka et al., 1997; L e v y - S t r u m p f et al., 1997; K i m c h i , 1998).  NAT1 m R N A  has no A U G initiation c o d o n a n d translation starts at G U G c o d o n to produce a 97 k D a protein.  T h i s protein l a c k s the  N-terminal region of e l F 4 G I , w h i c h is  responsible for a s s o c i a t i o n with cap-binding protein e l F 4 E .  A s a result, N A T 1  performs a s a translation repressor in a normal cell environment.  In a recent  study, c a s p a s e w a s found to c l e a v e N A T 1 at a c o n s e r v e d site to yield a novel C terminal truncated 8 6 k D a protein that promotes c a p - i n d e p e n d e n t  translation.  A n o t h e r recent study has s h o w n that translation rate of N A T 1 in apoptotic cells w a s selectively maintained, while the translation rate of other cellular proteins in  -35-  apoptotic cells w a s r e d u c e d by 6 0 to 7 0 % with the degradation of the c a p d e p e n d e n t translation mediators, e l F 4 G I a n d elF4GII. that N A T 1 is a  caspase-activated  T h e s e results s u g g e s t e d  translation factor,  which  mediates  cap-  independent translation, at least for its o w n R N A , in cells undergoing apoptosis (Figure 7).  T h e r e is a n increasing n u m b e r of reports indicating that m a n y  eukaryotic m R N A s mechanism.  c a n also initiate their translation v i a a  cap-independent  R e c e n t transfection-based experiments in cell cultures indicated  that e x p r e s s i o n of the truncated  86 k D a protein in cells stimulated protein  translation from the I R E S s of d e a t h - a s s o c i a t e d  proteins, s u c h a s c - M Y C a n d  A P A F - 1 , a n d anti-apoptotic proteins, s u c h a s X - l i n k e d inhibitor of apoptosis protein (XIAP) w h i c h is a n effective m a m m a l i a n inhibitor-of-apoptosis protein w h o s e m e c h a n i s m of action involves direct inhibition of c a s p a e - 3 a n d -7. (Holick e r a / . , 1999; Henis-Korenblit et al., 2 0 0 0 ; Henis-Korenblit et al., 2002) (Figure 8). A s previously mentioned, e l F 4 G I c a n be c l e a v e d by C V B 3 2 A s h a r e s structural h o m o l o g y with N A T L  p r 0  and elF4GI  This raises the possibility that N A T 1 m a y  be c l e a v e d by C V B 3 p r o t e a s e s , a n d the c l e a v a g e products m a y promote the translation of a specific unidentified s u b s e t of m R N A s in a m a n n e r a n d thus contribute to the apoptotic p r o c e s s (Figure 8).  -36-  cap-independent  2A PABP  elF4E  p r o  elF4A  elF3/4A Tt  9Wffl  elF4GI  63%  T  NAT1/DAP5/p97  Caspase Cells undergo apoptosis  JWiiiifi  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. T h e binding d o m a i n s of various proteins are s h o w n a n d the s a m e d o m a i n found on both e l F 4 G I a n d N A T 1 is indicated by the s a m e pattern. T h e similarity of the central region of e l F 4 G I to those of N A T 1 is indicated. T h e 2 A a n d c a s p a s e c l e a v a g e sites are indicated by arrows. W h e n cells undergo apoptosis, the truncated N A T 1 g e n e r a t e d through c a s p a s e c l e a v a g e is found to promote cap-independent translation v i a the I R E S . (Holcik era/., 2000; Henis-Korenblit etal., 2002). p r o  -37-  Apoptotic Stimulus  *\ -(^)  |  C a s p a s e activation  NAT1/p97  NAT1/p86 IRES  AH.  AUG  Stimulation of IRESmediated translation  DAP5 m R N A  {  MYC APAF-1  XIAP  " <-  <  <-  c-myc m R N A Apaf-1 m R N A  XIAP m R N A  Pro-apoptotic IRESs  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-  A n o t h e r interesting g e n e that w a s found to be down-regulated  in the  C V B 3 - i n f e c t e d m o u s e heart is C R E B ( Y a n g et al., 1999). T h i s g e n e e n c o d e s a 4 3 k D a basic leucine z i p p e r transcription factor w h i c h plays a critical role in regulating g e n e e x p r e s s i o n in r e s p o n s e to a variety of extracellular signals, including nerve growth factor in neurons a n d antigen receptor cross-linking in T lymphocytes (Arias et al., 1994; K w o k era/., 1994). T h e transcriptional activity of C R E B is d e p e n d e n t upon phosphorylation of a critical s e r i n e 1 3 3 residue protein kinase A .  T h e transcriptionally active C R E B  by  is present in c a r d i a c  m y o c y t e s . E x p e r i m e n t s using rat hearts have s h o w n that chronic stimulation with the p-adrenergic agonist isoproterenol, a m o d e l that m i m i c s the  hyperadrenergic  state, a p p e a r s to play a role in the d e v e l o p m e n t of h u m a n heart failure a s a result of a reduction of the level of C R E B m R N A (Muller et al., 1995a&b). T h u s , it is s u g g e s t e d that C R E B m a y be a n important regulator of c a r d i a c myocyte g e n e e x p r e s s i o n a n d plays a role in regulating c a r d i a c myocyte function or s u r v i v a l . Furthermore,  transgenic  phosphorylatable  mice with heart-specific  dominant-negative  overexpression  C R E B - m u t a n t (serine133  of a  replaced  nonby  alanine) d e v e l o p e d four-chamber dilation a n d s e v e r e heart failure within a few w e e k s after birth (Fentzke et al., 1998). T h e s e results s u g g e s t that a n inhibition of the C R E B - m e d i a t e d g e n e transcription is a s s o c i a t e d with the phenotype of the final s t a g e s of various c a r d i a c d i s e a s e s , s u c h a s i s c h e m i c or idiopathic D C M a n d eventually leads to altered g e n e regulation in h u m a n heart failure. H o w e v e r , little is k n o w n about the transcriptional activation mediated by C R E B a n d the c A M P d e p e n d e n t signaling pathway  in c a r d i o m y o c y t e s a n d about the  -39-  mechanisms  leading to alterations of t h e s e regulations. dystrophin by C V B 3 2 A  p r o  Interestingly, proteolytically c l e a v e d  c a n lead to functional impairment a n d morphological  disruption of heart m u s c l e cells, w h i c h c a n lead to the d e v e l o p m e n t of D C M . T h i s raises the possibility that viral proteases may a l s o p r o c e s s C R E B during infection.  S u c h c l e a v a g e c o u p l e s with C R E B m R N A down-regulation in the  C V B 3 - i n f e c t e d m o u s e heart m a y contribute to or further e n h a n c e the initiation a n d progression of viral myocarditis into D C M . A m o n g the m e m b e r s  of the Picornaviridae  family, recent studies  demonstrated that e x p r e s s i o n of the viral p r o t e a s e s 2 A  p r o  or 3 C  p r o  have  alone l e a d s to  c a s p a s e - 3 activation a n d apoptosis (Barco et al., 2 0 0 0 ; G o l d s t a u b et al., 2000). A s mentioned in chapter one, a wide s u b s e t of factors involved in transcription, translation, a n d cytoskeleton organization are proteases, w h i c h m a y further promote apoptosis. or 3 C  p r o  also p r o c e s s e d  by these two  O v e r e x p r e s s i o n of viral 2 A  p r o  in H e L a cells m a y induce cell death by i) c l e a v a g e of translation a n d  transcription  initiation factors,  expression  of  anti-  relationship  between  or the  ii) activation of c a s p a s e s ,  pro-apoptotic  molecules.  viral p r o t e a s e s a n d  and/or  Clearly,  apoptosis  iii) altered  elucidating  the  will highlight novel  m e c h a n i s m s linking translational control to cell survival/death pathways in C V B 3 infected heart d i s e a s e s .  -40-  Chapter Two: 2.1  Hypothesis and Specific Aims  Hypothesis and specific aims In this project, I c h o s e to study the role of C V B 3 protease g e n e s 2 A a n d  3 C a n d their potential substrates in promoting cell death. T h e central hypothesis of my thesis work is that coxsackieviral proteases 2A and 3C directly and/or indirectly induces apoptotic cell death via activation of c a s p a s e s and proteolysis of host transcription and translation factors.  T h i s hypothesis  w a s tested in H e L a cell cultures ( A T C C , Rockville, M D , U S A ) . T h e specific a i m s are as follows: 1. T o clone  CVB3  2A  p r o  or 3 C  p r 0  gene  into  pCI-neo  vector  and  then  transiently transfect H e L a cells. 2. T o m e a s u r e H e L a cell viability by M T S a s s a y . 3. T o detect c l e a v a g e  of e n d o g e n o u s  transcription  factor,  CREB,  and  translation initiation factors, e l F 4 G I a n d N A T L 4. T o  detect  cleavage  of  procaspases-3  and  -8  and  their  respective  substrates, P A R P a n d B i d . 5. T o detect altered e x p r e s s i o n of the B c l - 2 family m e m b e r s , s u c h a s Bcl-2 and Bax. 6. T o detect the r e l e a s e of cytochrome c from mitochondria into the cytosol a n d the activity of activated c a s p a s e - 9 .  -41 -  Chapter Three: Experimental Design, Material and Methods 3.1  Cloning of C V B 3  protease genes 2A and 3C into an eukaryotic  expression vector 3.1.1  Cloning of protease genes 2A and 3 C G e n e s encoding C V B 3 2 A  p r o  and 3 C  p r o  w e r e first prepared by p o l y m e r a s e  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) a s template.  T h e primer s e q u e n c e s u s e d for amplifying 2 A g e n e w e r e forward 5'-  CAATGGGACAACAATCAGGG-3' AGTCCTGCAGTCACTGTTCCATT-3',  and while  the  reverse primer  sequences  5'used  for  amplifying 3 C g e n e w e r e 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' a n d reverse  5'-TAAAGTCGACTTAACCTTTCTC-3'.  T h e amplified  P C R fragments  w e r e ligated into the T A v e c t o r (Invitrogen). T h e resulting T A p l a s m i d s containing the protease g e n e s were then digested with E c o R I a n d the r e l e a s e d fragments w e r e ligated into a pCI-neo v e c t o r ( P r o m e g a ) that h a d b e e n treated with E c o R I and d e p h o s p h o r y l a t e d with 2 units of calf intestinal p h o s p h a t a s e ( P r o m e g a ) .  The  new constructs were n a m e d a s pCI-neo(2A) a n d p C I - n e o ( 3 C ) , 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) a n d p C I -  n e o ( 3 C ) , w e r e a d d e d to 50 u L of competent cells (DH5oc) a n d iced for 15 min. T h e n the s a m p l e s w e r e h e a t - s h o c k e d at 4 2 ° C for 2 min a n d immediately iced for another 5 min. After incubation on ice, 1 m L of L B m e d i u m w a s a d d e d to e a c h of  -42 -  the s a m p l e s a n d then incubated at 3 7 ° C for 1 h with s h a k i n g . V a r i o u s v o l u m e s of s a m p l e s w e r e then plated on a g a r plates containing ampicillin a n d incubated overnight at 3 7 ° C . Individual c o l o n i e s w e r e s e l e c t e d a n d grown in L B m e d i u m with ampicillin.  D N A w a s extracted a n d the s e q u e n c e s of the inserts w e r e determined  by D N A s e q u e n c i n g performed at the U B C B i o t e c h n o l o g y 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 ) w e r e g r o w n in 3 5 - m m plates to 6 0 %  confluence  and  then  transfected  using  Lipofectamine™  transfection  reagent  (Invitrogen), with 1.5 pg of plasmid D N A [pCI-neo(2A) or pCI-neo(3C)] a c c o r d i n g to the manufacturer's protocol. V e c t o r (pCI-neo)-transfected H e L a cells w e r e u s e d a s a parallel control. All the s a m p l e s w e r e incubated at 3 7 ° C in a humidified 5 % C O 2 a t m o s p h e r e a n d harvested at different timepoints post-transfection (pt) to obtain h o m o g e n i z e d cell lysates, w h i c h w e r e a s s e s s e d by W e s t e r n blot a n a l y s e s .  3.3  Analysis of proteins by polyacrylamide gel electrophoresis C e l l cultures w e r e w a s h e d twice with 1x P B S a n d harvested at v a r i o u s  timepoints pt.  W h o l e cell lysates w e r e p r e p a r e d in lysis buffer [20 m M T r i s - H C L  (pH8.0); 150 m M N a C l ; 1% N a n i o d e t - P 4 0 ; 1 0 % glycerol] at 4 ° C for 20 min a n d centrifuged at 14,000 rpm at 4 ° C for 20 min. T h e supernatants w e r e heated at 9 0 ° C for 10 min in 6 x L a e m m l i s a m p l e buffer [50 m M Tris-HCI (pH 6.8), 100 m M dithiothreitol, 2 % s o d i u m d o d e c y l sulphate ( S D S ) , 0 . 1 % b r o m o p h e n o l blue, 1 0 %  -43-  glycerol] to obtain r e d u c e d conditions.  A l l s a m p l e s w e r e then a p p l i e d to a n d  s e p a r a t e d by 9-15% S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( P A G E ) .  Gels were  run at 80 V for 20 min a n d then at 100 V for 90 min, a n d the proteins transferred  onto  nitrocellulose  membranes  (Hybond™ECL™;  were  Amersham  P h a r m a c i a Biotech) at 30 V overnight or at 100 V for 2 h. T h e n m e m b r a n e s  were  b l o c k e d in blocking buffer (1x P B S containing 0.1% T w e e n 20 a n d 5% s k i m milk powder) at 4 ° C overnight.  3.4  Immunoblot analysis After blocking overnight, the m e m b r a n e s w e r e incubated with the respective  primary antibody (Table 2) for 2 h at room temperature.  The membranes  were  w a s h e d 6 times with w a s h i n g buffer (1x P B S containing 0.1% T w e e n 20) a n d then incubated with h o r s e r a d i s h  p e r o x i d a s e conjugated  goat s e c o n d a r y antibody to  mouse/rabbit immunoglobulin G ( B D B i o s c i e n c e s ) for 1 h at room temperature. T h e m e m b r a n e s w e r e a g a i n w a s h e d 6 times with w a s h i n g buffer. T h e n blots w e r e v i s u a l i z e d by e n h a n c e d c h e m i l u m i n e s c e n c e ( E C L ; A m e r s h a m P h a r m a c i a Biotech). T h e l u m i n e s c e n c e reaction w a s performed using e q u a l v o l u m e s of solution A (100 m M Tris-HCI [pH 8], 5 m M H 0 ) a n d solution B (2.5 m M luminal, 78 m M luceferin). 2  2  T h e m e m b r a n e s w e r e incubated in the a b o v e m i x e d solution for 2 min at room temperature a n d w e r e then e x p o s e d to X - r a y film ( A m e r s h a m ) . a n a l y s i s results w e r e obtained from triplicate experiments.  -44 -  A l l W e s t e r n blot  3.5  Cell viability assay T o determine the effects of C V B 3 2 A  p r o  and 3 C  p r o  e x p r e s s i o n s on H e L a cell  viability, M T S cell viability a s s a y w a s performed using a n a s s a y kit from P r o m e g a following the manufacturer's instructions. C e l l lines a n d the parallel control culture (vector-alone-transfected  cells) w e r e 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, a n d submitted for a b s o r b a n c y m e a s u r e m e n t at 4 9 0 n m . T h e number of live cells of the transfected cells a n d the control cells w e r e determined a c c o r d i n g to the a b s o r b a n c i e s corrected with the b a c k g r o u n d reading. T h e corrected a b s o r b a n c e  was expressed  relative to that of the  vector-alone-  transfected cells at e a c h respective timepoint pt (to reflect the percent viable cells of e a c h of the transfected  s a m p l e s relative to the number of viable cells of the  control at e a c h of the timepoint pt).  3.6  Cell culture and Western blot detection of cytochrome c release For total  cell  lysates,  cell w e r e  washed  twice  in cold  1x P B S  and  r e s u s p e n d e d in 1 m L of cold lysis buffer [20 m M Tris (pH 8), 137 m M N a C l , 1 0 % glycerol, 1% Nonidet P - 4 0 , 1 m M phenylmethylsulfonyl fluoride, 10 ug/ml aprotinin] per 5 6 - c m  2  culture area.  After 20 min o n ice, the supernatant w a s collected  following centrifugation at 10,000 x g. F o r cytosolic extracts,  cells w e r e w a s h e d  twice in cold  1x P B S a n d  r e s u s p e n d e d in 1 m L of ice cold buffer [250 m M s u c r o s e , 20 m M H E P E S (pH 7.4),  -45-  10 m M K C I , 1.5 m M M g C I , 1 m M E G T A , 1 m M E D T A , 1 m M Dithiothreitol (DTT), 2  s u p p l e m e n t e d with 1 m M phenylmethylsulfonyl fluoride a n d 10 ug/ml aprotinin] per 5 6 - c m culture a r e a . C e l l s w e r e gently disrupted by 20 strokes with the B pestle of 2  a  Kontes  dounce  homogenizer.  The  supernatant  was  collected  following  centrifugation at 10,000 x g a n d further centrifuged at 100,000 x g for 1 h at 4 ° C in a B e c k m a n O p t i m a ultracentrifuge using a TI-100 rotor. C e l l lysate protein concentration was* determined by the B C A method (BioRad). a  S a m p l e s w e r e separated by S D S - P A G E a n d proteins w e r e transferred onto  nitrocellulose m e m b r a n e  blocking  and  horseradish  incubation  (Hybond E C L , A m e r s h a m Biosciences). with  primary a n d  p e r o x i d a s e conjugated  secondary  secondary  antibodies  Following (Table  immunoglobulins w e r e  2),  detected  using the e n h a n c e d c h e m i l u m i n e s c e n c e ( E C L ) method ( A m e r s h a m B i o s c i e n c e s ) a n d e x p o s e d to Hyperfilm ( A m e r s h a m B i o s c i e n c e s ) .  3.7  Caspase-9 activity assay T h e activity of c a s p a s e - 9 w a s m e a s u r e d using the B D A p o A l e r t C a s p a s e -  9/6 F l u o r e s c e n t Kit (Clontech) following the manufacturer's  instructions.  Equal  n u m b e r of cells ( 1 x 1 0 cells) w e r e l y s e d with lysis buffer provided from kit a n d 6  centrifuged at 14,000 x g for 10 min.  T h e cellular extracts w e r e collected a n d  mixed with reaction buffer containing D T T (10 m M ) a n d then incubated with specific fluorescent substrates L E H D - A M C of c a s p a s e - 9  in the p r e s e n c e  a b s e n c e of its specific inhibitor, L E D H - C H O , for 1 h at 3 7 ° C .  - 4 6 -  and  H e L a cells treated  with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for 3 h at a final concentration of 150 ng/mL w e r e u s e d a s a positive control for the c a s p a s e - 9 activity a s s a y kit, while vector-transfected specific c a s p a s e - 9 inhibitor w e r e  used  H e L a cells a n d s a m p l e s treated with as  negative  controls.  A b s o r b a n c e of  s a m p l e s w a s m e a s u r e d on a fluoromicroplate reader with a 360-nm excitation filter and 465-nm  e m i s s i o n filter using cell lysis buffer for b a c k g r o u n d  calibration.  Quantification a n d normalization of c a s p a s e - 9 activities w e r e calculated following the  manufacturer's instructions a n d various publications, a n d fold increase  of  c a s p a s e - 9 activity for e a c h of the s a m p l e s w a s p r e s e n t e d ( B a l a c h a n d r a n et al., 2000; Z h a n g et al., 2002; F u k a z a w a et al., 2003; K a n d a s a m y et al., 2003).  3.8  Statistical analysis of M T S a s s a y All v a l u e s are p r e s e n t e d a s m e a n ± standard deviation ( S D ) .  Statistical  significance w a s evaluated using the Student's t test for paired c o m p a r i s o n s , with p<0.05 c o n s i d e r e d statistically significant.  3.9  Limitations In our previous studies, infection of cell cultures with the entire virus w o u l d  likely complicate the interpretation regarding the contribution of the individual viral protease to cellular effects.  Therefore, in an attempt to understanding the roles of  t h e s e viral p r o t e a s e s in inducing cell death, transient e x p r e s s i o n of the individual viral protease in H e L a cells will be u s e d to investigate a n d identify the cellular  -47  -  effects of these p r o t e a s e s o n the host.  H o w e v e r , a s e x p r e s s i o n of 2 A  p r o  and 3 C  p r o  are likely to be toxic to cells, all attempts to e x p r e s s these two p r o t e a s e s by m e a n s of transient transfection are subjected to variations in the proportion of successfully transfected cells a n d m a y result in a mixed cell population with different levels of transfection efficiency.  -48-  Table 2.  List of primary antibodies used for Western blot analyses. (Information taken from product c a t a l o g u e s of S a n t a C r u z Biotechnology, Inc, B D B i o s c i e n c e s , a n d I M G E N E X ) .  Primary antibodies  *  Protein  Species crossreactivity*  Molecular weight (kDa) detected  Source*  Company  Bax  H, M , R  22  M, m o n o c l o n a l  Santa C r u z Biotechnology  Bcl-2  H, M , R  28  M, monoclonal  Santa C r u z Biotechnology  Bid  H  15, 22  Rt, polyclonal  Santa C r u z Biotechnology  Caspase-3  H, M  17, 32  M, monoclonal  Santa C r u z Biotechnology  Caspase-8  H, M , R  20  M, monoclonal  Santa C r u z Biotechnology  CREB  H  43  M, monoclonal  Santa C r u z Biotechnology  Cytochrome c  H, M , R  15  M, monoclonal  B D Biosciences  elF4GI  H  220  M, monoclonal  B D Biosciences  NAT1  H, M  97  M , polyclonal  IMGENEX  PARP  H  89, 113  M, monoclonal  Santa C r u z Biotechnology  H = Human M = Mouse R = Rat Rt = Rabbit  -49-  Chapter Four:  Results  4.1  Cell death induced by C V B 3 2 A  4.1.1  Overexpression of 2 A  p r o  or 3 C  p r o  p r o  and 3 C  p r o  induces morphological alterations  C e l l morphological c h a n g e s w e r e o b s e r v e d at different timepoints pt with o v e r e x p r e s s i o n of C V B 3 2 A contrast m i c r o s c o p e .  p r o  or 3 C  in H e L a cells a s o b s e r v e d u n d e r a p h a s e -  p r o  A s s h o w n in Figure 9, the a p p e a r a n c e of cell s h r i n k a g e  a n d loss of cell a d h e r a n c e b e g a n at 24 h pt a n d 4 8 h pt in 2 A transfected cells, respectively.  p r 0  - and  3C  p r o  -  B y 72 h pt, most cells h a v e d i e d a s e v i d e n c e d by  u n a d h e r e d , floating particles in the cell culture media, particularly in the c a s e of 2 A  pro  4.1.2  Overexpression of 2 A  p r o  or 3 C  p r o  reduces cell viability  T o determine the p e r c e n t a g e of cell death induced by C V B 3 protease e x p r e s s i o n , cell viability w a s m e a s u r e d using the M T S a s s a y kit (Promega).  As  s h o w n in Figure 10, the percent viable cells rapidly d e c r e a s e d upon transient e x p r e s s i o n of 2 A 3C  pro  -expressing  p r o  or 3 C  cells  p r 0  was  .  A t 72 h pt, the percent cell viability of 2 A approximately  c o m p a r e d to control cells.  -50-  44%  and  68%,  p r o  respectively,  - or as  CD  CL  LO  D  CO  LL  CO  Figure 10. M T S c e l l v i a b i l i t y a s s a y . P e r c e n t of v i a b l e cells of 2 A - or 3 C transfected cells w a s plotted against indicated timepoints pt. H e L a cells w e r e transfected a n d cultured in a 6-well plate. A t 72 h pt, approximately 4 4 % a n d 6 8 % cells a p p e a r v i a b l e in the 2 A - e x p r e s s i n g a n d 3 C - e x p r e s s i n g cells, respectively, relative to the vector-transfected control cells. B a r s s h o w n are ± S D of the m e a n from triplicate experiments; * indicates P<0.05. p r o  pro  -52-  pr0  p r o  4.1.3  Cleavage of translation factors induced by C V B 3 2 A  4.1.3.1 elF4GI  Overexpression of 2 A is a  key factor  determine whether C V B 3 2 A  p r o  p r o  or 3 C  p r o  p r o  and 3 C  p r o  induces cleavage of elF4GI  in c a p - d e p e n d e n t and 3 C  p r o  translation  initiation.  To  c a n affect e l F 4 G I integrity a n d thus  influence translation initiation of the host cells, W e s t e r n blot w a s performed using h o m o g e n i z e d cell lysates of H e L a cells transfected with either 2 A  p r 0  - or 3 C  p r o  -  e x p r e s s i n g p l a s m i d s a n d the s i g n a l s w e r e a n a l y z e d with m o u s e a n t i - e l F 4 G I antibody. Figure 11 d e m o n s t r a t e s that 220 k D a e l F 4 G I w a s c l e a v e d by 2 A 3C  p r o  2A  p r o  at 72 h pt.  p r o  and  H o w e v e r , different c l e a v a g e products of e l F 4 G I a p p e a r e d in  - a n d 3 C - t r a n s f e c t e d cells. pro  2 A - t r a n f e c t e d cells p r o d u c e d a 100 k D a pro  product which is a n a l o g o u s to the c l e a v a g e product found in the C V B 3 - i n f e c t e d cells, while 3 C - t r a n s f e c t e d cells a p p e a r e d to p r o d u c e more than o n e proteolytic pro  product of various m o l e c u l a r weights, ranging from 100 k D a to 120 k D a .  4.1.3.2  Overexpression of 3 C  p r o  induces cleavage of NAT1  N A T 1 , a l s o c a l l e d p97 or d e a t h - a s s o c i a t e d protein 5, is a newly identified protein that s h a r e s structural h o m o l o g y with e l F 4 G I (Levy-Strumpf et al., 1997). T h e 86 k D a c l e a v a g e product of N A T 1 by c a s p a s e w a s found to promote c a p independent translation of m a n y pro-apoptotic proteins, s u c h a s c - M y c a n d A p f - 1 , a n d anti-apoptotic proteins, s u c h a s X I A P (Henis-Korenblit et al., 2002).  To  determine whether C V B 3 infection c a n induce c l e a v a g e of N A T 1 , H e L a cells w e r e infected with C V B 3 at a moi of 10 a n d harvested at different timepoints pi. T h e C V B 3 - i n f e c t e d H e L a cell lysates w e r e a s s e s s e d  -53-  by W e s t e r n blot a n a l y s e s  using anti-NAT1 antibody.  Figure 1 2 A d e m o n s t r a t e s that the c l e a v a g e of full-  length N A T 1 (97 k D a ) w a s o b s e r v e d a s early a s 5 h pi to give a - 5 5 k D a product. T h e n w e further investigated whether the c l e a v a g e of N A T 1 c o u l d be attributed to the activity of C V B 3 3 C with 3 C  pro  p r o  .  T o this e n d , H e L a cells w e r e transiently transfected  - e x p r e s s i n g p l a s m i d , p C I - n e o ( 3 C ) . Immunoblot a n a l y s i s revealed that  N A T 1 w a s c l e a v e d a n d yielded two products ranging from 50 k D a to 5 5 k D a in the transfected cells (Figure 12B). T h e p r e s e n c e of a n 86 k D a protein w a s a l s o o b s e r v e d in Figure 1 2 B , w h i c h indicated that c a s p a s e ( s ) w a s / w e r e activated in the transfected cells. T h i s observation strongly s u g g e s t s that C V B 3 3 C a n important role in apoptotic cell death.  -54-  p r o  plays  CVB3infected  Cleaved elF4GI  Vector  2A  p r o  3C  p r o k  D  a  ' ^~~*^ ^0  •^ggMMwp 72  72  72  H o u r s pt  Figure 11. A n a l y s i s of elF4GI in H e L a cells transfected with C V B 3 2 A or 3C g e n e . C e l l s h a r v e s t e d at different timepoints pt w e r e a n a l y z e d by W e s t e r n blotting using a m o u s e m o n o c l o n a l antibody against h u m a n e l F 4 G I . CVB3infected H e L a cells (8 h pi) w e r e u s e d a s positive control a n d vector-transfected cells w e r e u s e d a s negative control. p r o  p r o  -55-  ^  CVB3 Jurkat Sham  B Jurkat  30'  1  3  CVB3infected  5  Vector  7  9  3C  p r o  h pi  kDa  kDa  NAT1-*  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 o f NAT1 c l e a v a g e . H e L a cells w e r e infected with C V B 3 (A) or transfected with pCI-neo(3C) (B). Total proteins w e r e prepared from the infected or transfected cells harvested at the indicated timepoints pi/pt a n d subjected to W e s t e r n blot a n a l y s i s of NAT1. CVB3-infected H e L a cells (9 h pi) w e r e u s e d a s positive control, while Jurkat cell lysate, s h a m infected H e L a cells, a n d empty vector-transfected cells w e r e u s e d a s negative controls.  -56-  4.1.4  Cleavage of transcription factor induced by C V B 3 2 A  4.1.4.1  Overexpression of 2 A  p r o  or 3 C  p r o  p r o  and 3 C  p r o  induces down-regulation of  CREB C R E B is a 4 3 k D a transcription factor w h i c h is c o n s i d e r e d to be  an  important regulator of c a r d i a c myocyte g e n e e x p r e s s i o n a n d plays a role in regulating c a r d i a c myocyte function a n d survival. T o determine whether C V B 3 2A  p r o  and 3 C  p r 0  c a n influence host g e n e e x p r e s s i o n by affecting C R E B protein  regulation, W e s t e r n blot w a s performed using H e L a cells transfected with either 2A  p r o  - or 3 C  pro  - e x p r e s s i n g plasmids a n d the signals w e r e a n a l y z e d with m o u s e  a n t i - C R E B antibody.  Figure 13 d e m o n s t r a t e s that e x p r e s s i o n of 2 A  p r 0  or 3 C  p r o  i n d u c e d down-regulation of C R E B a s early a s 24 h pt, a s c o m p a r e d to the nontransfected a n d vector-transfected both C V B 3 2 A  p r o  and 3 C  p r o  H e L a cell lysates. T h e s e results indicate that  c a n influence cellular m R N A transcription by d o w n -  regulation of the transcription factor C R E B v i a proteolytic c l e a v a g e , w h i c h in turn c a n affect the translation of host cellular proteins, leading to the e n h a n c e m e n t of apoptotic r e s p o n s e s .  -57-  2A  p r o  H o u r s pt HeLa  Vector  24  48  72  kDa  GAPDH  3 C  pro  H o u r s pt HeLa  Vector  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 - and 3 C t r a n s f e c t e d H e L a c e l l s . Cell extracts were prepared a n d detected for C R E B e x p r e s s i o n levels at the indicated timepoints pt. L e v e l s of C R E B protein w e r e found to be down-regulated in both transfected cells beginning at 24 h pt. H o w e v e r , C R E B e x p r e s s i o n returned to basal level at 72 h pt in 3 C - t r n a s f e c t e d cells. G A P D H w a s u s e d a s a n e q u a l loading control. Non-transfected H e L a cells a n d empty vector-transfected H e L a cells (72 h pt) w e r e u s e d a s negative controls. p r o  p r o  pro  -58-  4.1.5  C a s p a s e activation and cleavage of their respective substrates  4.1.5.1  Overexpression of  2A  p r o  or  3C  induces  p r o  activation  of  caspase-3 and cleavage of P A R P C a s p a s e s , a family of cysteine proteases,  play a n e s s e n t i a l role in the  p r o c e s s of apoptosis ( S a l v e s e n et al., 1997; Hengartner, 2000).  It has  been  s h o w n that t h e s e c a s p a s e s p r o c e s s s e v e r a l key structural proteins w h i c h result in the systematic a n d orderly d i s a s s e m b l y of the cells. the transient e x p r e s s i o n of C V B 3 2 A  p r o  or 3 C  p r 0  A s s h o w n in Figure 14,  could induce p r o c a s p a s e - 3 (32  kDa) c l e a v a g e a s e v i d e n c e d by a n increase of the c l e a v a g e product (17 k D a ) . Activation of c a s p a s e - 3 w a s further verified by the c l e a v a g e of its substrate, P A R P , a nuclear protein involved in D N A repair.  T h e 113 k D a P A R P  was  c l e a v e d 14 h pi with C V B 3 into 24 k D a (not s h o w n ) a n d 89 k D a fragments (Figure 15), a p r o c e s s w h i c h represents a specific marker for c a s p a s e activation. PARP  cleavage  was  detected  in both  2A  p r o  -  and  suggesting that c a s p a s e - 3 w a s activated by both 2 A (Figure 15).  -59-  3C p r o  p r 0  -expressing  and 3 C  p r o  cells,  expression  CVB3infected  Vector  2A  p r o  2A  p r o  Vector  3C  p r o  Procaspase-3 « - 17  Caspase-3 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-transfected H e L a cells. C e l l s w e r e transfected with C V B 3 2 A or 3 C g e n e a n d harvested at the indicated timepoints pt. C l e a v a g e of p r o c a s p a s e - 3 w a s 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 using m o u s e m o n o c l o n a l c a s p a s e - 3 antibody. C V B 3 - i n f e c t e d H e L a cells (8 h pi) a n d vector-transfected cells w e r e u s e d a s positive a n d negative controls, respectively. p r o  -60-  p r 0  2 A  pro  Hours pt  CVB3infected  Vector  24  48  72  PARP-*  kDa 113  Cleaved — • PARP  89  3 C  pro  Hours pt  CVB3infected  Vector  24  PARP-*  48  72  kDa 113  Cleaved — • PARP  89  Figure 15. Cleavage of P A R P in gene-transfected HeLa cells. C e l l s w e r e transfected with C V B 3 2 A or 3 C g e n e a n d harvested at the indicated timepoints pt. C l e a v a g e of P A R P w a s determined by W e s t e r n blot analysis u s i n g m o u s e m o n o c l o n a l P A R P antibody. C V B 3 - i n f e c t e d H e L a c e l l s (14 h pi) a n d vector-transfected cells (72 h pt) w e r e used as positive a n d negative controls, respectively. p r o  p r o  -61 -  4.1.5.2  Overexpression of  2A  p r o  or  3C  p r o  induces  activation  of  caspase-8 and cleavage of Bid T o determine the activity of other c a s p a s e s that c o u l d activate c a s p a s e - 3 , cleavage expressing  of p r o c a s p a s e - 8 cells.  Results  was  detected  obtained  using the  from  CVB3 2A  p r o  -  immunoblot a n a l y s i s  or  3C  showed  p r 0  -  a  significant prolonged increase in the 20 k D a p r o c a s p a s e - 8 c l e a v a g e product in 3 C - t r a n s f e c t e d cells. pro  In 2 A - t r a n s f e c t e d cells, the p r o c a s p a s e - 8 pro  product p e a k e d at 24 h pt a n d returned to b a s a l level thereafter Activation of c a s p a s e - 8 w a s then a s s e s s e d in 2 A  p r o  - or 3 C  pro  cleavage  (Figure 16).  - e x p r e s s i n g cells by  the c l e a v a g e of its substrate B i d . Figure 17 s h o w s that B i d (22 kDa) w a s c l e a v e d a n d yielded the tBid (15 kDa) in both 2 A  p r o  - and 3C  p o r  - e x p r s s i n g cells.  p r e s e n c e of tBid indicated that c a s p a s e - 8 w a s activated upon both 2 A 3C  p r o  e x p r e s s i o n in H e L a cells.  -62-  p r o  The and  2 A  Pro  Hours pt CVB3-  3 C  pro  Hours pt CVB3-  Figure 16.  CVB3 3C  PRO  expression induces activation of caspase-8.  HeLa  cells w e r e transfected with 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 at 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 protein levels w e r e  determined  b y 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 h e 2 0 k D a c l e a v a g e N o t e that c l e a v a g e i n d u c e d b y 2 A  p r o  a n d r e t u r n e d to b a s a l l e v e l t h e r e a f t e r .  product.  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 v e l 2 4 h pt C V B 3 - i n f e c t e d c e l l e x t r a c t ( 1 4 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 c e l l e x t r a c t ( 7 2 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 controls, respectively.  -63-  2 A  CVB3infected  Hours pt Vector  24  Bid -* Cleaved —• Bid  i  48 .  —  —  3 C  Vector  24 1  Bid-* Cleaved-*— Bid  CVB3 2A  Figure 17. harvested  pro  and 3C  pro  blot.  k  D  ^- 22 15  —  pro  p r a  48  72  induce cleavage of Bid.  at t h e i n d i c a t e d t i m e p o i n t s  d e t e c t i o n o f B i d c l e a v a g e in 2 A Western  72  Hours pt  CVB3infected  Pro  pt.  - or 3C  p r o  Cell  extracts  kD «• 22 • 15  H e L a cells were  were  prepared a n d  - e x p r e s s i n g cells w a s performed by  C V B 3 - i n f e c t e d ( 1 2 h pi) H e L a  cell extract a n d empty  vector-  t r a n s f e c t e d c e l l e x t r a c t ( 7 2 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 , respectively.  -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 T h e Bcl-2 family proteins include both anti-apoptotic, s u c h a s Bcl-2 a n d Bcl-xL, and  pro-apoptotic, s u c h a s  Bax and  Bid, members.  To  determine  whether C V B 3 protease e x p r e s s i o n c a n induce altered e x p r e s s i o n of Bcl-2 a n d Bax, W e s t e r n blot a n a l y s e s w e r e c o n d u c t e d using 2 A H e L a cell lysates.  p r o  - or 3 C - t r a n s f e c t e d  Figure 18 s h o w s that e x p r e s s i o n of B a x in 3 C  cells w a s significantly i n c r e a s e d but not in 2 A Figure 19 d e m o n s t r a t e s that both C V B 3 2 A  p r o  pro  pro  pro  -expressing  - e x p r e s s i n g cells.  However,  and 3 C  p r o  did not induce c h a n g e s  in the e x p r e s s i o n of B c l - 2 , indicating that apoptotic cell death w a s triggered by CVB3 3C  p r 0  through activation of the pro-apoptotic Bcl-2 family protein B a x rather  than s u p p r e s s i o n of the anti-apoptotic protein B c l - 2 .  -65-  Figure 18. Levels of Bax expression in CVB3 2A - or 3C -transfected HeLa cells. C e l l extracts w e r e prepared a n d detected for B a x protein levels by W e s t e r n blot at the indicated timepoints pt. B a x up-regulation started 24 h pt a n d significantly i n c r e a s e d by 72 h pt in the transfected cells. C V B 3 - i n f e c t e d H e L a cells (14 h pi) a n d vector-transfected cells (72 h pt) w e r e u s e d a s positive a n d negative controls, respectively. PRO  -66-  pro  2A  Hours pt  CVB3infected  p r o  Vector  Bcl-2  GAPDH—•  Figure 19 CVB3 2 A and 3 C do not alter Bcl-2 expression in 2 A and 3C -transfectd HeLa cells. Total protein extracts w e r e prepared at indicated timepoints pt a n d the Bcl-2 e x p r e s s i o n w a s a n a l y z e d by W e s t e r n blotting. C V B 3 - i n f e c t e d (14 h pi) a n d empty vector-transfected H e L a cells (72 h pt) w e r e u s e d a s controls. p r o  p r o  p r o  pro  -67-  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. overexpression of 2 A  pr0  Thus, my next experiment was to test whether the or 3 C  pr0  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 2 A - and 3 C pr0  transfected HeLa cells. This indicates that CVB3 2 A  pro  and 3 C  lead to cytochrome c redistribution in the transfected cells.  -68-  pro  pro  expression can  CVB3infected  Vector  2A  72  p r o  72  3C  HeLa  p r o  kDa  72  H o u r s pt  Figure 2 0 . CVB3 2A or 3 C induces c y t o c h r o m e c release from mitochondria. H e L a cells w e r e transfected with 2 A or 3 C gene. Cell extracts were p r e p a r e d from cytosolic a n d w h o l e cell l y s e s at 72 h pt. C y t o c h r o m e c w a s found in the cytosolic fraction of 2 A - or 3 C - e x p r e s s i n g cells. C V B 3 - i n f e c t e d (14 h pi) H e L a cells w e r e u s e d a s a positive control, while vector-transfected cells a s well a s non-transfected cells w e r e u s e d a s negative controls. p r o  p r o  p r o  p r o  -69-  p r 0  pro  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 - and 3C -transfected cells activates pro  pro  procaspase-9, caspase-9 activity assay were performed. Figure 21 shows that caspase-9 activities were increased upon 2A as compared to the controls.  pr0  or 3 C  pro  expression in HeLa cells  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  pro  or 3 C  pr0  expression, in addition to a  caspase-dependent pathway through activation of caspase-8 and -3.  -70-  Figure 2 1 . Caspase-9 activity a s s a y in CVB3 2A - or 3 C - e x p r e s s i n g cells. Activity of c a s p a s e - 9 w a s m e a s u r e d using the A p o A l e r t Kit following the manufacture's instructions. C e l l extracts (72 h pt) w e r e p r e p a r e d a n d incubated with specific c a s p a s e - 9 substrate ( L E H D - A M C ) in the p r e s e n c e a n d a b s e n c e of specific inhibitor of c a s p a s e - 9 ( L E H D - C H O , C 9 i ) . T R A I L - t r e a t e d H e L a cells (3 h post induction) were u s e d a s positive control, while empty vector-transfected H e L a cells (72 h pt) a n d protease-transfected s a m p l e s treated with C 9 i w e r e u s e d a s negative controls. C a s p a s e - 9 activities w e r e m e a s u r e d a n d c a l c u l a t e d a s per the manufacturer's instructions (Clontech); bars represents ± S D of the m e a n from triplicate experiments; * indicates P<0.05. pro  -71 -  pro  Chapter Five: 5.1  Discussion, Conclusions, and Future Directions  Discussion •  It has  been  k n o w n that m a n y viral  infections  can  induce  host  cell  apoptosis. T h i s p r o c e s s is c o n s i d e r e d a s a host defence m e c h a n i s m to limit viral replication and prevent virus s p r e a d to re-infect surrounding cells ( Z h a n g et al., 2 0 0 2 ; Z h a n g et al., 2003). T h u s , this r e s p o n s e is obviously beneficial to the host against viral infection. cannot  be  regenerated  H o w e v e r , the overall cost will be huge if the dying c e l l s or  regenerated  at  a  very  low rate,  such  as  the  c a r d i o m y o c y t e s ( A n v e r s a et al., 2002). T h u s , study of the molecular m e c h a n i s m of CVF33-induced c a r d i o m y o c y t e apoptosis  and development  of strategies to  prevent cardiomyocyte d a m a g e are important in c a r d i o v a s c u l a r r e s e a r c h . Picornaviral  proteases,  such  as  those  found  in  polioviruses  and  enteroviruses, have b e e n reported to play a n important role in inducing host cell apoptosis (Barco et al., 2000; G o l d s t a u b et al., 2 0 0 0 ; K u o et al., 2002; L i et al., 2002).  H o w e v e r , the molecular m e c h a n i s m s of how t h e s e proteases induce  apoptosis  are  still unclear.  M o r e importantly, the  2A  p r o  and  3C  p r o  of  the  cardiovirulant virus, C V B 3 , has not been studied in the context of apoptosis. Here, experiments w e r e c o n d u c t e d to elucidate the death signal transduction c a s c a d e induced by the C V B 3 2 A  p r o  and 3 C  p r o  expression.  In C V B 3 2 A  p r o  - or  3 C - t r a n s f e c t e d cells, protease e x p r e s s i o n w a s found to induce reduction of cell pro  viability a n d induction of apoptotic cell death. 2A  p r o  and 3 C  p r 0  c a n promote  apoptosis  I a l s o demonstrated  that C V B 3  by activation of c a s p a s e - 8 a n d  up-  regulation of pro-apoptotic m e m b e r s of Bcl-2 family, B a x . A l s o , down-regulation  -72 -  of  protein e x p r e s s i o n of host transcription factor,  translation  initiation factors,  elF4GI and  CREB,  and  NAT1, w o u l d further  c l e a v a g e of enhance  the  apoptotic cell death. A s mentioned in chapter one, the induction/initiation of apoptotic pathways can  be either c a s p a s e - d e p e n d e n t  or c a s p a s e - i n d e p e n d e n t .  In the  caspase-  d e p e n d e n t pathway, upstream c a s p a s e s , s u c h a s c a s p a s e - 8 , are activated v i a ligation of the cell m e m b r a n e death receptors by their ligands. O n c e this initiator c a s p a s e is activated, it will either activate the d o w n s t r e a m effector c a s p a s e - 3 or induce c l e a v a g e of its substrate B i d . In the present study, I h a v e s h o w n that c a s p a s e - 3 w a s activated a n d further c l e a v e d its substrate P A R P in both 2 A 3 C - t r a n s f e c t e d cells through activation of c a s p a s e - 8 .  and  p r o  -  Moreover, I also  pro  demonstrated that the activation of c a s p a s e - 8 w a s a s s o c i a t e d with c l e a v a g e of Bid in both 2 A and 3 C  p r o  p r 0  - and 3C  pro  - e x p r e s s i n g cells.  A l l t h e s e data s u g g e s t that 2 A  c a n induce apoptosis through a c a s p a s e - 8 - d e p e n d e n t  p r o  pathway, w h i c h  can subsequently activate effector c a s p a s e - 3 most likely v i a direct c l e a v a g e by caspase-8. Apoptosis  can  also  mediated  pathway.  The  mediated  apoptotic event  be  triggered  are the  and 3 C  p r o  of B a x a n d 3 C  intrinsic mitochondriain this mitochondria-  Bcl-2 family proteins. p r 0  and 3 C  B a x e x p r e s s i o n w a s found only in the 3 C p r 0  an  key regulatory c o m p o n e n t s  evaluation of the c o n s e q u e n c e s of 2 A  2A  through  pro  p r o  In the functional  transfection, up-regulation of  - e x p r e s s i n g cells, but both C V B 3  did not induce c h a n g e s of Bcl-2 e x p r e s s i o n . S u c h up-regulation  p r o  - i n d u c e d c l e a v a g e of B i d could further contribute to the r e l e a s e  -73-  of c y t o c h r o m e c from mitochondria. In this study, I also s h o w e d the release of c y t o c h r o m e c from  mitochondria a n d this release  c o u l d further activate  c a s p a s e - 9 , w h i c h in turn could activate d o w n s t r e a m c a s p a s e - 3 . s u g g e s t that apoptotic cell death w a s triggered by C V B 3 3 C  p r o  the  T h e s e results  through activation  of pro-apoptotic m e m b e r s of Bcl-2 family rather than s u p p r e s s i n g e x p r e s s i o n of anti-apoptotic m e m b e r s . In the investigation of the detailed molecular m e c h a n i s m of C V B 3 2 A and 3 C  p r o  - i n d u c e d apoptosis, w e must take into a c c o u n t that t h e s e p r o t e a s e s  also c l e a v e a variety of host translation factors. initiating eukaryotic cellular protein synthesis. binding  complex  translation. and  p r o  (elF4F),  which  leads  to  e l F 4 G I is a key player in  It is a c o m p o n e n t of the c a p the  initiation  of  cap-dependent  C l e a v a g e of e l F 4 G I results in the disruption of the e l F 4 F c o m p l e x  inhibition of c a p - d e p e n d e n t  recombinant 2 A  p r 0  translation.  It w a s  demonstrated  that  the  of poliovirus a n d h u m a n rhinovirus c a n c l e a v e e l F 4 G I directly  ( S o m m e r g r u b e r e r a / . , 1994; B o v e e e r a / . , 1998a&b; G r a d i et al., 1998; N o v o a et al., 1999). It has b e e n p r o p o s e d that the inhibition of c a p - d e p e n d e n t translation is a major m e c h a n i s m of the e x e c u t i o n p h a s e of apoptosis, w h i c h leads to rapid cell death ( C l e m e n s e r a / . , 1998; M a r i s s e n e r a / . , 1998). In this report, c l e a v a g e of e l F 4 G I w a s found in both C V B 3 2 A 3 C - t r a n s f e c t e d cells. pr0  However, 2 A  p r o  and 3 C  p r o  pro  - e x p r e s s i n g cells.  for this divergent p h e n o m e n o n .  - and  a p p e a r e d to h a v e different  c l e a v a g e sites o n e l F 4 G I , w h i c h w a s e v i d e n c e d by the products obtained in 3 C  p r 0  different  cleavage  T h e r e are a few potential r e a s o n s  O n e of the possibilities is that e l F 4 G I c a n s e r v e  -74-  a s a substrate for c a s p a s e - 3 a s c a s p a s e - 3 is activated upon 3 C  expression.  p r o  However, I did not o b s e r v e similar c l e a v a g e products in 2 A - t r a n s f e c t e d cells, pro  although c a s p a s e - 3  w a s also activated by 2 A  p r 0  expression.  Therefore, this  possibility is unlikely. T h e other possibility is that e l F 4 G I is directly p r o c e s s e d by 3C  p r o  to produce the c l e a v a g e product of approximately 120 k D a . F r o m  prediction  (NetPicoRNA  V 1 . 0 , Prediction of  http://www.cbs.dtu.dk/services/NetPicoRNA),  Picornavirus  it has  contains only o n e potential c l e a v a g e site for 2 A  p r 0  been  in silico  Protease  Sites,  s h o w n that  elF4GI  but eight for 3 C  T h e molecular weight of the predicted c l e a v a g e product by 2 A  p r 0  p r 0  (Figure 2 2 A ) .  is approximately  100 k D a , while two of the potential c l e a v a g e sites, G l n 4 2 4 a n d G l n 4 6 5 , by 3 C  p r 0  yield two c l e a v a g e products of approximately 120 a n d 125 k D a , respectively (Figure 2 2 A ) .  Therefore, the s e c o n d possibility is a more s o u n d  hypothesis  b e c a u s e the predicted c l e a v a g e products are similar in s i z e to the o n e s o b s e r v e d in the W e s t e r n blot a n a l y s i s .  A l t h o u g h both p r o t e a s e s w e r e found to c l e a v e  e l F 4 G I , only the 100 k D a c l e a v a g e product w a s found in C V B 3 - i n f e c t e d cells.  HeLa  T h i s m a y be b e c a u s e the 120 k D a c l e a v a g e product g e n e r a t e d by 3 C  w a s further p r o c e s s e d by 2 A  p r o  p r 0  to yield the 100 k D a product w h i c h w a s then  detected by the antibody against e l F 4 G I at the C-terminus, or the activity of 3 C towards e l F 4 G I is m u c h lower than that of 2 A  p r o  in the viral infected cells.  p r 0  Also,  previous studies h a v e demonstrated that c o m p l e t e c l e a v a g e of e l F 4 G I by viral 2A  p r 0  takes  an  average  of two h after  infection ( B o v e e et  al., 1 9 9 8 a & b ) .  Therefore, the 100 k D a w o u l d be e x p e c t e d to be the only c l e a v a g e product found in C V B 3 - i n f e c t e d H e L a cells.  -75-  A n o t h e r factor involved in regulating host translation is N A T L  In a recent  study, N A T 1 w a s reported to be c l e a v e d by c a s p a s e ( s ) at a c o n s e r v e d site to yield a novel C-terminal truncated 86 k D a protein that promoted c a p - i n d e p e n d e n t translation of death a s s o c i a t e d proteins including itself, s u g g e s t i n g that N A T 1 may play a role in apoptosis modulation (Henis-Korenblit et al., 2000; H e n i s Korenblit et al., 2002). Here, I demonstrated that the c l e a v a g e of N A T 1 o c c u r r e d in both C V B 3 - i n f e c t e d a n d C V B 3 3 C - t r a n s f e c t e d pr0  cells.  H o w e v e r , the two  c l e a v a g e products (ranging from 50 to 5 5 k D a ) of N A T 1 w e r e found to be s m a l l e r than the c l e a v a g e product (86 kDa) by c a s p a s e ( s ) (Figure 2 2 B ) . T h i s m a y be b e c a u s e N A T 1 c a n be c l e a v e d directly by 3 C activity of 3 C cells.  p r o  in the transfected cells, or the  p r o  in C V B 3 - i n f e c t e d cells is higher than that in 3 C - t r a n s f e c t e d  F r o m in silico  pro  prediction, it h a s b e e n s h o w n that N A T 1 contains three  potential c l e a v a g e sites for 3 C  p r 0  but none for 2 A  the predicted c l e a v a g e products by 3 C  p r o  p r o  , a n d the m o l e c u l a r weight of  are approximately 5 5 k D a . Therefore, it  s u g g e s t s that N A T 1 may be a target protein directly c l e a v e d by 3 C  p r 0  in 3 C  p r o  -  e x p r e s s i n g cells to give the 50 to 5 5 k D a products a n d the larger 86 k D a c l e a v a g e product obtained by W e s t e r n blot a n a l y s i s in Figure 12 m a y be residual products from c a s p a s e activation a s a result of 3 C  p r o  transfection. T h i s truncated  86 k D a protein m a y be further p r o c e s s e d by 3 C  p r 0  to yield the s m a l l c l e a v a g e  product, a s s h o w n in the W e s t e r n blot. T h e s e observations strongly s u g g e s t that CVB3 3C  p r 0  plays a n important role in promoting cell death.  Whether these  c l e a v a g e products perform the s a m e function a s the truncated 86 k D a protein in promoting the c a p - i n d e p e n d e n t translation remains to be elucidated.  -76-  ^ Potential c l e a v a g e T sites for 2 A  Potential c l e a v a g e sites f o r 3 C  p r o  elF4GI (1404a.a  *  w\ •• •  *  *  p r o  *  \ •  • • + • • Gln424 Glu465  GInlOO Gln218 Gln108  Arg490  Gln547 Glu631  NAT1 (907a.a.)  220  Gln1318  kDa  -100  -54  B  kDa  -50  -120  kDa  -45  -125  kDa  -60  -95  kDa  caspase cleavage site  * **  1  • ••  •  # •  Gln419 Gln433 Gln444  Glu152  D  -50  E  T  D  7  9  0  kDa 97  Gln889  kDa  -55  kDa  -45  kDa  86  Figure 2 2 . Prediction of cleavage sites of 2A and 3C on e l F 4 G I and NATL Full length of protein s e q u e n c e of e l F 4 G I (A) a n d N A T 1 (B) w e r e a n a l y z e d a n d subjected to predict potential c l e a v a g e sites of 2 A and 3 C using N e t P i c o R N A prediction server (version 1.0). Potential c l e a v a g e sites w e r e indicated by arrows ( •••>- for 2 A ; for 3 C ) ; * represents the potential c l e a v a g e site c h o s e n for calculating the molecular weight of the resulting fragments. PRO  PRO  p r o  p r o  p r o  -77-  p r 0  C R E B is a nuclear transcription factor, w h i c h is e x p r e s s e d in the h u m a n heart a n d phosphorylated to mediate c A M P - d e p e n d e n t transcriptional activation (Muller et al., 1995a).  S e v e r a l studies s u g g e s t e d that C R E B is a n important  regulator of g e n e e x p r e s s i o n in c a r d i o m y o c y t e s with possible relevance for the pathophysiology of c o n g e s t i v e heart failure, s u c h a s i s c h e m i c a n d idiopathic dilated  cardiomyopathy,  through  alteration  of  CREB  expression  phosphorylation (Muller et al., 1995a&b; Muller et al., 2001).  and  A s previously  mentioned, this factor has also b e e n found to be down-regulated in C V B 3 infected m o u s e hearts from our previous differential m R N A display studies ( Y a n g et al., 1999). T h e data in this thesis s h o w that 2 A  p r o  or 3 C  p r 0  e x p r e s s i o n resulted  in a down-regulation of C R E B production in the host cells. T h e s e results s u g g e s t 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 e x p r e s s i o n in  myocytes, w h i c h m a y contribute to the d e v e l o p m e n t of D C M . S i n c e the antibody to C R E B cannot detect the C R E B c l e a v a g e products, it is not c l e a r 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. H o w e v e r , in a n y c a s e , this down-regulation will negatively affect the cell survival a n d contribute to H e L a cell apoptosis. In s u m m a r y , my data illustrate that the m e c h a n i s m of a p o p t o s i s induction in C V B 3 2 A  p r o  - or 3 C - t r a n s f e c t e d H e L a cells is likely through a pro  caspase-8-  d e p e n d e n t pathway a n d the intrinsic mitochondria-mediated pathway.  Down-  regulation of host transcription factor a n d c l e a v a g e of translation initiation factors by 2 A  p r 0  or 3 C  p r o  results in the inhibition of host g e n e e x p r e s s i o n , which further  -78-  e n h a n c e s apoptotic cell death.  M o r e importantly, the data s u g g e s t that the  c l e a v a g e of N A T 1 a n d down-regulation of C R E B found in 2 A e x p r e s s i n g cells require further investigation.  of the  - or  3C  p r 0  -  T h e identification of c a s p a s e ( s )  responsible for the c l e a v a g e of N A T 1 during 3 C understanding  p r o  molecular mechanisms  p r o  e x p r e s s i o n will increase our  of apoptosis  p r o t e a s e s , a n d the down-regulation of C R E B  i n d u c e d by viral  by both C V B 3 p r o t e a s e s will  provide insight into the understanding of the m e c h a n i s m s by w h i c h C V B 3 c a u s e s myocarditis a n d p r o g r e s s i o n to D C M .  5.2  Summary of results 1.  M o r p h o l o g i c a l alterations a n d M T S a s s a y results s h o w e d that both C V B 3 p r o t e a s e s c a n induce cell death.  2.  A t day 3 post-transfection, approximately 4 4 % a n d 6 8 % cells w e r e alive in 2 A  p r o  - and 3C  pro  - o v e r e x p r e s s i n g H e L a cells, respectively, a s  c o m p a r e d to the control cells. 3.  Transfection with either C V B 3 2 A e l F 4 G I in H e L a cells, but 2 A  p r o  p r o  and 3 C  and 3 C  p r o  p r 0  i n d u c e d c l e a v a g e of  appeared  to  produce  c l e a v a g e at different sites on e l F 4 G I . 4.  N A T 1 w a s found to be c l e a v e d in both C V B 3 - i n f e c t e d a n d transfected H e L a cells, but not in 2 A - t r a n s f e c t e d cells. pro  3C  p r o  -  The - 5 5  k D a c l e a v a g e product o b s e r v e d in the C V B 3 - i n f e c t e d cells is s m a l l e r than the reported c a s p a s e - c l e a v e d N A T 1 (86 k D a ) .  -79-  5.  C l e a v a g e of N A T 1 by 3 C  p r o  in the transfected cells a l s o generated  two more c l e a v a g e products (86 k D a a n d - 5 0 kDa) that w e r e not o b s e r v e d in C V B 3 - i n f e c t e d cells. CVB3-infected  cells c o u l d  be  T h e - 5 5 k D a product found in  p r o d u c e d from  the  two  cleavage  products that are further c l e a v e d by other p r o t e a s e s from the C V B 3 or the host cells, or the 86 k D a c l e a v a g e product m a y be further p r o c e s s e d by 3 C  to yield the s m a l l e r c l e a v a g e product ( - 5 0 k D a )  p r o  o b s e r v e d in the 3 C - t r a n s f e c t e d cells. pr0  6.  C R E B w a s found to be down-regulated by both C V B 3 2 A  7.  Both C V B 3 2 A  p r o  and 3 C  p r o  p r o  and 3 C  p r o  .  promoted activation of c a s p a s e - 3 a n d  c l e a v a g e of its substrate, P A R P . 8.  C l e a v a g e of p r o c a s p a s e - 8 w a s o b s e r v e d in both 2 A  p r 0  - and  3C  p r o  -  e x p r e s s i n g cells, a n d further c l e a v a g e of B i d w a s also detected in t h e s e cells, indicating.that c a s p a s e - 8 w a s activated upon 2 A 3C 9.  p r o  CVB3  p r 0  and  expression. 3C  p r o  but  not  2A  p r o  could  significantly  up-regulate  e x p r e s s i o n , a pro-apoptotic m e m b e r of the Bcl-2 family.  Bax  However,  neither altered e x p r e s s i o n of Bcl-2, a n anti-apoptotic protein. 10.  R e l e a s e of c y t o c h r o m e c from mitochondria w a s detected in both CVB3 2A  p r o  - a n d 3 C - t r a n s f e c t e d cells. H o w e v e r , the 3 C pro  r e l e a s e w a s stronger than that of 2 A 11.  p r o  p r o  induced  .  C a s p a s e - 9 activities w e r e found to be i n c r e a s e d upon C V B 3 2 A 3C  p r 0  o v e r e x p r e s s i o n in H e L a cells.  -80-  p r o  or  A l s o , results obtained from the  s a m p l e s after application of the inhibition with C 9 i s h o w e d that the specificity of this a s s a y .  Conclusions In c o n c l u s i o n , these results s u g g e s t that the m e c h a n i s m of a p o p t o s i s i n d u c e d in C V B 3 2 A  p r o  - or 3 C - t r a n s f e c t e d H e L a cells is likely through multiple pro  pathways: 1. Both 2 A  p r 0  and 3 C  p r o  c a n induce a c a s p a s e - 8 - d e p e n d e n t  pathway  through activation of c a s p a s e - 8 a n d c a s p a s e - 3 . 2. Both 2 A pathway  and/or 3 C  p r o  through  p r o  c a n induce intrinsic mitochondria-mediated  up-regulation  of  Bax,  the  presence  of  tBid,  c y t o c h r o m e c r e l e a s e d from mitochondria, a n d activation of c a s p a s e - 9 . 3. 3 C  p r o  c a n promote cell death by activating or up-regulating e x p r e s s i o n  of pro-apoptotic Bcl-2 family m e m b e r s ,  Bid and  B a x , rather  than  s u p p r e s s i n g e x p r e s s i o n of the anti-apoptotic protein, B c l - 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 a n d its late phase, D C M . 5. C l e a v a g e of host translation initiation factors, e l F 4 G I a n d N A T 1 , by 2A  p r o  or 3 C  p r o  results in the inhibition of host g e n e e x p r e s s i o n , w h i c h  further e n h a n c e s 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 a p o p t o s i s by c l e a v a g e of host transcription a n d translation initiation factors  and  activation  of  caspases.  H o w e v e r , whether  these  cleavages  (activation) o c c u r directly or indirectly is u n k n o w n . T o a d d r e s s this issue, in vitro c l e a v a g e a s s a y s using purified 2 A  p r o  and 3 C  p r o  n e e d to be performed.  Therefore,  future work is required to c l o n e t h e s e g e n e s into a n e x p r e s s i o n vector, p E T - 4 2 a ( N o v a g e n ) , a n d to purify the recombinant p r o t e a s e s after o v e r e x p r e s s i o n in a prokaryotic s y s t e m .  5.4.1  Cloning of C V B 3 protease genes 2A and 3C into a prokaryotic expression vector T o determine whether C V B 3 2 A  p r 0  and/or 3 C  p r o  c l e a v e N A T 1 directly or not,  2 A , 3 C , a n d N A T 1 g e n e s will be r e c l o n e d into p E T - 4 2 a vectors by preparing 2 A , 3 C , a n d N A T 1 g e n e fragments through digestion of pCI-neo(2A), p C I - n e o ( 3 C ) , a n d p C R I I ( N A T I ) , respectively.  T h e e x c i s e d D N A fragments will be ligated into  the c o r r e s p o n d i n g sites of the p E T - 4 2 a . O v e r e x p r e s s i o n of the c l o n e d g e n e s will be a c h i e v e d by the induction of E. coli B L 2 1 ( D E 3 ) cells with i s o p r o p y l - p - D thiogalactopyranoside  (IPTG).  Proteins will be purified using Glutathione S -  transferase ( G S T ) G e n e F u s i o n a n d Purification S y s t e m ( A m e r s h a m P h a r m a r c i a ) .  -82-  5.4.2  In vitro direct cleavage assay In vitro c l e a v a g e a s s a y will be performed to determine the c l e a v a g e of  N A T 1 by C V B 3 2 A  p r o  and/or 3 C  p r o  . E q u a l molar ratio of substrate to protease will  be incubated at 2 5 ° C in a c l e a v a g e buffer overnight.  T h e n , the protein will be  a n a l y z e d by S D S - P A G E a n d stained with C o o m a s s i e blue. T h e proteins will also be transferred to polyvinylidene difluoride m e m b r a n e s for N-terminal s e q u e n c i n g to determine the c l e a v a g e site.  5.4.3 Work Completed 5.4.3.1  Construction of vectors expressing C V B 3 2 A  p r o  or 3 C  p r o  I have recloned the 2 A , 3 C , a n d N A T 1 g e n e s by e x c i s i n g the respective D N A fragments containing the entire o p e n reading frame of the g e n e from the p C l n e o vector a n d pCRII vector a n d then ligating the e x c i s e d fragments into the p E T - 4 2 a vector.  T h e new constructs are n a m e d as p E T ( 2 A ) , p E T ( 3 C ) , a n d  p E T ( N A T 1 ) , respectively.  5.4.3.2  Overexpression and purification of proteins  CVB3 3C  p r o  has b e e n successfully o v e r e x p r e s s e d a n d purified (Figure 23).  H o w e v e r , the activity of 3 C expression  system  may  p r o  be  w a s difficult to detect. required  to  O v e r e x p r e s s i o n a n d purification of C V B 3 2 A  p r o  increase  the  protease  activity.  a n d N A T 1 are a l s o required to be  c o n d u c t e d before proceeding with further investigations.  -83-  P e r h a p s , a eukaryotic  Bacterial lysates  Purified protein  +  +  +  "-" = Non-induced "+" = I P T G - i n d u c e d  Figure 2 3 . O v e r e x p r e s s i o n a n d p u r i f i c a t i o n o f t h e r e c o m b i n a n t CVB3 PRO using a prokaryotic expression system. G S T - 3 C fusion protein is o v e r e x p r e s s e d 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 s e p a r a t e the G S T tag a n d the 3 C (lane 4). N o n i n d u c e d bacterial cell lysate is u s e d a s a negative control in lane 1.  3C  p r o  p r 0  -84-  Bacterial lysates  Lane  1  Purified protein  +  +  +  2  3  4  "-" = Non-induced "+" = I P T G - i n d u c e d  Figure 2 3 . O v e r e x p r e s s i o n a n d purification of the recombinant C V B 3 3C using a prokaryotic expression system. G S T - 3 C fusion protein is o v e r e x p r e s s e d after induction with I P T G ("+") for 24 h at 3 7 ° C . T h i s fusion protein is then either eluted with buffer containing glutathione (lane 3) or treated with thrombin p r o t e a s e to separate the G S T tag a n d the 3 C (lane 4). 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