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The molecular characterization of Coxsackievirus B3 Pathogenisis Stadnick, Ellamae 1999

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The Molecular Characterization of Coxsackievirus B3 Pathogenesis by Ellamae Stadnick B.Sc. (Biochemistry), University of Alberta, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pathology and Laboratory Medicine University of British Columbia We accept the following thesis in conforming to the required standard: The University of British Columbia September, 1999 © Ellamae Stadnick In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract The group B coxsackieviruses (CVBs) cause a variety of clinical manifestations in humans and are often fatal in infants under the age of three months. In the cardiovascular field, CVBs are considered the primary etiologic agent of virus-induced myocarditis. Although often a subclinical illness in adults, myocarditis may lead to chronic dilated cardiomyopathy, arrhythmias and sudden death. Unlike other family members such as poliovirus, there is relatively little understanding of the mechanisms of pathogenesis of coxsackievirus and no vaccine is available. Therefore, the objective of this project was to map genetic regions associated with CVB3 virulence by studying antibody escape mutants which differ in their pathogenesis for heart tissue. In conjunction with previous work from the laboratory, a panel of ten antibody escape mutants was isolated: EM1-EM10. Sequence analysis of the genes encoding the capsid proteins (VP1-VP4) revealed that all but two mutants, EM7 and E M 10, contained a lysine to arginine mutation within the 'puff region' of VP2. In contrast, EM7 and EM10 contained a glutamate to glycine mutation within the 'knob region' of VP3. Both of these regions, the 'puff of VP2 and the 'knob' of VP3, have been shown to be neutralizing and/or immunodominant sites on the capsid surface of other picornaviruses. Complete sequence analysis of EMI and E M 10 revealed further mutations within the viral polymerase gene and the 5' nontranslated region which may be responsible for the phenotypes of these variants. As characterized in previous studies, EMI produces about tenfold lower titres in the hearts of infected mice as compared to the wildtype strain. This inability of EMI to replicate to wildtype levels in the hearts of infected animals does not appear to be the result of an inability to replicate in cardiomyocytes as demonstrated by in vitro analysis. The pathogenic phenotype of E M 10 in A/J mice has also been examined as part of this study. Data from these experiments iii suggest that E M 10 has a higher level of attenuation than EMI producing 100-1000 fold lower titres in the hearts of infected animals as compared to the wild type CVB3(RK) strain. In an attempt to determine if the lysine to argihine mutation in VP2 was responsible for the reduced myocarditic potential of EMI, this mutation was incorporated into an infectious clone of the wildtype strain. Despite multiple attempts the mutant clones did not produce infectious virus that could be tested for cardiovirulence. In summary this project has identified several loci within the CVB3 genome which may be responsible for the attenuated phenotypes of EMI and EM10. Correlation of such mutations with an alteration in a phenotypic property of interest, such as tropism for heart tissue, will lead to an increased understanding of CVB pathogenesis. This knowledge may be exploited in vaccine development. Table of Contents I. Abstract ii H. Table of Contents iv m. List of Figures viii IV. List of Tables x V. Abbreviations xi VI. Acknowledgements xv Chapter One: Introduction 1 1.1 Historical Background 1 1.2 Classification 1 1.3 Pathogenesis : 2 1.4 Clinical Diagnosis and Treatment 2 1.5 Coxsackievirus B and IDDM 3 1.6 Coxsackievirus and Myocarditis '..: 5 1.7 The Virion 7 1.8 The RNA Genome 10 1.9 The Coxsackievirus Cellular Receptor 18 1.10 Coxsackievirus Replication 20 1.11 Pathogenic Variants , 25 Chapter Two: Research Objective 29 Chapter Three:MateriaIs -31 3.1 Tissue Culture... 31 3.2 Fixatives, Stains and Growth Medium. 31 3.3 Virus Strains ., 33 3.4 Infectious Clone 33 3.5 Restriction Enzymes 34 3.6 PCR 34 3.7 Reverse Transcription-PGR 35 3.8 In-Situ Hybridization (ISH) Solutions 36 3.9 ISH Digoxigenin Labeled Probe 39 3.10 Large Scale Plasmid Preparation Solutions 40 3.11 Neonatal Mice Cardiomyocyte Culturing 41 3.12 In Vivo Transcription 41 3.13 Antibodies. •- 42 3.14 Animals .' 42 Chapter Four: Methods 43 4.1 Cell Maintenance 43 4.2 Virus Stock Preparation. 43 4.3 Virus Titration By Plaque Assay.: 44 4.4 Isolation of Antibody Escape Mutants 44 4.5 Antibody Neutralization Assay 45 4.6 Viral RNA Isolation 46 4.7 Reverse Transcription - Polymerase Chain Reaction 46 4.8 Temperature Sensitivity of Viral Strains 48 4.9 Viral Decay Curves 48 4.10 Viral Growth in Cultured Cardiomyocytes 49 4.11 Large Scale Plasmid Preparation 50 4.12 Preparations of DIG-Labeled RNA Probe 51 4.13 Quantification of DIG-Labeled RNA Probe : 52 4.14 In-Situ Hybridization : 53 4.15 Animal Experiments 54 4.16 Histological Analysis of Tissue Samples 56 4.17 Titration of Virus in Tissue Samples 56 4.18 DNA Sequencing 56 4.19 Construction of the pCVB3(KR) Clone 57 4.20 Isolation of Virus By In Vitro Transcription 59 Chapter Five: Experimental Results and Discussion 61 5.1 Isolation of Antibody Escape Mutants 61 5.2 Viral Sequence Analysis : 62 5.3 Monoclonal Antibody Neutralization Assay 71 5.4 EMI Replication in Cardiomyocytes 72 5.5 Temperature Sensitivity Analysis 74 Vll 5.6 Viral Decay Curve 78 5.7 Timecourse of Infection of Susceptible A/J Mice With CVB3(RK) 80 5.8 In Vivo Analysis of CVB3(T7) in Comparison with CVB3(RK) 83 5.9 Comparison of the Virulence In Vivo of EMI, EM10, and CVB3(RK) 85 5.10 Site-Directed Mutagenesis of pCVB3(T7) 91 Chapter Six: Conclusions 95 Bibliography 101 Appendix I: 113 Appendix II: 130 viii List of Figures: Figure: 1.1 The Icosahedral Capsid of Coxsackieviruses 9 1.2 The Coxsackievirus Genome 10 1.3 The Internal Ribosomal Entry Site of Picornavirues 12 1.4 The Coxsackievirus B3 5'NTR 13 1.5 Expression of the Picornaviral Genome 14 1.6 Coxsackievirus Binding To Its Cellular Receptor 18 1.7 Hypothetical Structure of the Picornaviral Infectosome 21 1.8 Two Models For RNA Synthesis 23 1.9 Replication of Picornavirus RNA 24 4.1 CVB3(T7) Infectious Clone Restriction Enzyme Map 57 5.1 PCR Primer Location and Products 62 5.2 Location of the Lysine to Arginine Mutation in the VP2 Protein of EMI 66 5.3 Location of the Glutamate to Glycine Mutation in the VP3 Protein of E M 10 67 5.4 Location of EMI and EM10 Capsid Mutations 68 5.5 Replication of CVB3(RK) and EMI in Balb/c Cardiomyocytes 73 5.6 Replication of CVB3(RK) and EMI in A/J Cardiomyocytes 73 5.7 Temperature Sensitivity Assay 76 5.8 Viral Plaque Phenotypes at 37°C and 39°C 77 5.9 Viral Decay Curves 78 5.10 Timecourse of CVB3(RK) Infection 81 5.11 Histological Assessment of A/J Mice Infected With CVB3(RK) 82 5.12 Infection of A/J Mice With CVB3(T7) 84 5.13 Serum Titres of CVB3(RK), EMI, and EM10 85 5.14 Spleen Titres of CVB3(RK), EMI, and EM10 86 5.15 Pancreas Titres of CVB3(RK), EMI and EM10 87 5.16 Histological Analysis and In-Situ Hybridization of Pancreas Infected With Either CVB3(RK) or EM10 88 5.17 Heart Titres of CVB3(RK), EMI, and EM10 89 5.18 Histological Analysis and In-Situ Hybridization of Heart Infected With Either CVB3(RK) or EM10 90 5.19 Strategy For pCVB3(KR) Construction 93 List of Tables: Tables: 3.1 PCR Primers 5.1 Neutralization By the mAb aCVB3-948 xi Abbreviations: Ab Antibody AP Alkaline Phosphatase ATP Adenosine Triphosphate bp Base Pair BCIP 5-bromo-4-chloro-3-indoly lphosphate BSA Bovine Serum Albumin CAR Coxsackievirus Adenovirus Receptor CBC Cap Binding Complex cDNA Complementary Deoxyribonucleic Acid CIAP Calf Intestinal Alkaline Phosphatase CHO Chinese Hamster Ovary CPE Cytopathic Effect CTL Cytotoxic T-Lymphocytes CV Coxsackievirus CVB Coxsackievirus Group B CVB3(RK) Coxsackievirus B3 (Reinhard Kandolf) C V B 3 ( T 7 ) Coxsackieviurs B3 (infectious clone derived) DAF Decay Accelerating Factor ddH20 Distilled Water DEPC Diethylpyrocarbonate DIG Digoxigenin DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic Acid DNase Deoxyribonuclease dNTP Deoxyribonucleosidetriphosphate DTT Dithiothreitol EDTA Ethylenediaminetetraacetic Acid eEF Eukaryotic Initiation Factor EM Escape Mutant FBS Fetal Bovine Serum HJEBS Heat Inactivated Fetal Bovine Serum HLA Human Leukocyte Antigen hrs. Hours HRV Human Rhinovirus IDDM Insulin Dependent Diabetes Mellitus IRES Internal Ribosomal Entry Site kbp Kilobase Pair La Lupus Autoantigen mAb Monoclonal Antibody mRNA Messenger Ribonucleic Acid NAPS Nucleic Acid and Protein Services at UBC NBT Nitroblue Tetrazolium NEB New England Biolabs X l l l NIm Neutralizing / Immunogenic NTP Nucleosidetriphosphate NTR Nontranslated Region PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction pCVB3(T7) CVB3 Infectious Clone pCVB3(KR) CVB3 Clone With Lysine > Arginine Mutation PF Paraformaldehyde PFU Plaque Forming Unit p.i. Post Infection PTB Polypyrimidine Tract Binding Protein PVP Polyvinylpyruvate RD Rhabdomyosarcoma RNA Ribonucleic Acid RNase Ribonuclease RPM Revolutions Per Minute RT-PCR Reverse Transcription Polymerase Chain Reaction SDS Sodium Dodecyl Sulphate TAE Tris-Acetate TE Tris-EDTA TUT Terminal Uridylyltransferase UBC University of British Columbia Uracil Triphosphate Vascular Endothelial Cells African Green Monkey Kidney Genome Viral Protein XV Acknowledgements: The author would like to acknowledge the following for their contribution to this thesis: Dr. Janet Chantler Dr. Shirley Gillam Dr. George Mackie Dr. Simon Rabkin Dr. Michael Murphy Jennifer Kong UBC NAPS Unit BCRICWH Animal Unit Neil Chomos Jaskamal Girn This research was funded by the BC Research Institute For Child and Women's Health and The Canadian Heart and Stroke Foundation. 1 Chapter One: Introduction 1.1 Historical Background In the summer of 1947 upstate New York experienced five small outbreaks of paralytic poliomyelitis. In light of this epidemic Gilbert Dalldorf and Grace Sickles attempted to isolate polioviruses from the stools of two paralyzed boys (Dalldorf et al., 1948). Newborn mice inoculated intracerebrally with 20% fecal suspensions prepared from the affected boys exhibited paralysis (Dalldorf et al., 1949b). The virus isolates recovered from these animals were considered unique because they could not be neutralized by poliovirus antisera, and poliovirus had not been shown to replicate in mice. Dalldorf suggested the new group of viruses be named coxsackieviruses after the location of their original isolation: the village of Coxsackie, New York State (Dalldorf, 1949a). 1.2 Classification The coxsackieviruses belong to the family Picornaviridae; genus Enterovirus. As the name suggests these are small, RNA-containing viruses which infect via the enteric tract. The Picornaviridae family is divided into five genera: the rhinoviruses, the enteroviruses, the apthoviruses, the cardioviruses, and the hepatoviruses. Other members of the genus Enterovirus include the polioviruses (3 serotypes) and the echoviruses (31 serotypes). The coxsackieviruses are further divided into A and B subgroups based on their pathogenicity in newborn mice, and their ability to replicate in tissue culture. Group A coxsackieviruses, of which there are 23 serotypes, induce flaccid paralysis, primarily infect both skeletal and heart muscle, and are difficult to culture. On the other hand, group B coxsackieviruses (6 serotypes) induce spastic paralysis, infect many tissues including striated muscle, the central 2 nervous system, the liver, the exocrine pancreas, brown fat, and replicate readily in cell cultures (Hyypia et al., 1993; Sickles et al., 1955; Sickles et al., 1959) . 1.3 Pathogenesis Although infection with the group B coxsackieviruses (CVBs) often manifests as a subclinical illness, these viruses have been associated with various clinical diseases including aseptic meningitis, insulin-dependent diabetes mellitus, hepatitis, and fatal systemic infections of neonates. The CVBs are considered the most prominent viral etiologic agent of acute myocarditis and pericarditis, conditions which may lead to chronic dilated cardiomyopathy, arrhythmias, or sudden death (Woodruff, 1980). The primary mode of transmission of the CVBs is fecal-oral; however, infections may also be spread by the respiratory route. The onset of disease from the time of exposure may vary from 2 to 35 days, but most often occurs within 1 to 2 weeks. Once orally ingested, the virus initially replicates in the oropharynx and small intestine (alimentary phase). This is followed by extensive infection of the tonsils, lymph nodes of the neck, and Peyer's patches of the small intestine (lymphatic phase). From here the virus may enter the blood stream (viremic phase) leading to a systemic infection of target organs throughout the body (Melnick, 1996). 1.4 Clinical Diagnosis and Treatment Coxsackieviruses may be isolated from nasal secretions, cerebral spinal fluid (CSF), blood, feces, or rectal and throat swabs of individuals with either a clinical or subclinical infection. Traditionally, identification of the virus was achieved using neutralization assays with reference antisera (Grandien et al., 1989); however, the past ten years have lead to the 3 development of cheaper more efficient techniques for virus detection and identification. These new advances include the use of RNA probes for in situ hybridization (Hohenadl et al., 1991), PCR (Shimizu et al., 1994, Severini, 1993 #82, Weiss, 1991 #80), and enzyme-linked immunosorbent assays (ELISA) for CVB-specific IgM antibodies (Goldwater, 1995). There are no antiviral drug therapies available for coxsackievirus infections, and, unlike the situation for poliovirus, a vaccine has yet to be developed. 1.5 Coxsackievirus B and Insulin-Dependent Diabetes Mellitus (IDDM) Insulin-dependent diabetes mellitus (Type 1 diabetes) results from the destruction of insulin-producing beta cells in the Islets of Langerhans within the pancreas. This leads to a reduction of serum insulin levels resulting in hyperglycemia. Multiple etiologic factors are thought to play a role in the development of this disease including a genetic predisposition and triggering environmental factors. Several studies have suggested a link between MHC haplotypes HLA-D3 and HLA-D4 and an increased genetic susceptibility for IDDM (D'Alessio et al., 1992; Schernthaner et al., 1985). Viruses have long been suspected as one of the environmental triggers associated with the development of IDDM (reviewed by Yoon et al., 1993). In 1969 Gamble suggested an association between CVB infections and the onset of diabetes (Gamble et al., 1969). Since then CVB, in particular CVB4, has been implicated in the pathogenesis of IDDM using various experimental techniques. Seroepidemiological studies have shown a positive correlation between an increase in CVB-specific IgM levels and recent onset IDDM (reviewed by Yoon et al., 1996). In further support, CVBs have been isolated from the pancreas of IDDM patients showing that these viruses are able to replicate in pancreatic islet cells (Gladisch et al., 1976; Yoon et al., 1979). Perhaps the most convincing evidence linking CVB infection with the onset of IDDM has come from animal studies in which virus isolated from patients with recent onset IDDM was found to induce diabetes in mice (Champsaur et al., 1982; Yoon et al., 1979). While these studies support an association between CVB infection and the onset of IDDM, the role of the virus in the pathogenesis of the disease remains to be determined. Several mechanisms have been postulated to explain how CVB infection induces the destruction of pancreatic beta cells. CVB may cause direct damage to the pancreas by lytic infection. Alternatively the virus may trigger an autoimmune response which in turn destroys the insulin-producing cells. This is thought to occur by either molecular mimicry or activation of bystander T-cells. The molecular mimicry theory suggests that antibodies directed against viral proteins can cross-react with host proteins present on the surface of beta cells thereby initiating an autoimmune response. Several studies have identified glutamic acid decarboxylase (GAD-65) as the autoantigen responsible for the cross-reactivity as it displays a region of homology with the P2C nonstructural protein of CVB4 (Baekkeskov et al., 1990; Ellis et al., 1996; Kaufman et al., 1992). The 'bystander' theory suggests that virus infection causes local tissue damage and inflammation thereby releasing a sequestered islet antigen. This results in the activation of autoreactive T cells which mediate beta cell destruction (Horwitz et al., 1998). As a final mechanism, persistent infection by CVB may induce cytokine and interferon production which trigger an autoimmune response against the pancreatic endocrine cells (Foulis et al., 1990). More recently, in an attempt to understand the molecular determinants of viral-induced diabetes, the ability of CVB4 to induce hyperglycemia has been mapped to the PI region of the genome (discussed further under pathogenic variants). ' 5 1.6 Coxsackievirus and Myocarditis The group B Coxsackieviruses are the primary etiologic agents of acute viral myocardits (Woodruff, 1980). Acute myocarditis is defined as myocyte necrosis, either focal or diffuse, associated with a heavy infiltrate of inflammatory cells (predominately T-lymphocytes) in the absence of an ischemic event. Under histological analysis the cardiac tissue exhibits myofilament contraction band formation while the myocytes undergo membrane proliferation and cytoplasmic vacuolation. It has been postulated that idiopathic dilated cardiomyopathy, characterized by a dilated left ventricle with impaired systolic function, and a leading cause of congestive cardiac failure, is a product of chronic myocarditis due to a persistent viral infection (Baboonian et al., 1997). The pathogenesis of viral myocarditis is a complicated, poorly understood phenomenon. Three distinct mechanisms have been proposed to explain the development of cardiac tissue damage subsequent to coxsackievirus infection. The first suggests that myocarditis is a product of an autoimmune response mediated by the cellular immune system; primarily T-lymphocytes. The second suggests the humoral immune system produces autoantibodies which lead to cardiac damage. Most recently it has been suggested that myocyte damage occurs directly as a product of CVB infection and replication. Viral Myocarditis and Cellular Immunity The role of T lymphocytes in the development of myocarditis following CVB infection was first shown by Woodruff et al. when T-cell deficient mice were unable to develop cardiac inflammation and myocardial necrosis (Woodruff et al., 1974). Since then multiple other studies have been performed which suggest myocyte damage is dependent upon cytotoxic T 6 lymphocyte (CTL) activity (Henke et al. 1995; Huber et al., 1984b). Upon CVB infection two CTL populations are generated. One is virus-specific and associated with lysis of infected myocytes while the other is autoreactive and causes lysis of uninfected cells. The autoreactive CTLs are thought to play a primary role in the development of myocarditis since they produce 3-4 times greater myocyte damage as compared to the virus specific CTLs when adoptively transfused into CVB3-infected, T lymphocyte-depleted Balb/c mice (Huber et al., 1984a). Although the exact mechanism of autoreactive CTL killing is unknown, it is evident that viral infection is a prerequisite for the generation of this CTL subpopulation. Viral Myocarditis and Humoral Immunity The second proposed mechanism for the pathogenesis of myocarditis invokes a role for autoantibodies in directing complement-mediated lysis of myocytes. Maisch et al. were the first to report the presence of heart-reactive autoantibodies in a patient with CVB myocarditis (Maisch et al., 1980; Maish et al., 1982). In further support, studies in animals have lead to the detection of autoantibodies directed against cardiac myosin in CVB3-infected mice (Alvarez et al., 1987; Wolfgram et al., 1985). It is not known how the viral infection leads to the production of self-reactive antibodies. One suggestion is that virus infection reveals self-antigens which were otherwise sequestered from the immune system. Alternatively the infection may induce the production of antibodies directed against viral epitopes which cross react with host cell antigens in a process termed molecular mimicry (reviewed by Wolfgram etal., 1989). Direct Injury to Myocytes by CVB The final mechanism that has been put forward to explain the role of CVB in myocarditis is the direct injury hypothesis. This hypothesis suggests that CVB infection of the cardiac tissue and subsequent lysis of infected myocytes accounts for the majority of necrosis. This theory suggests that the immune response plays a protective role in eliminating the virus rather than a pathological one. Evidence supporting this mechanism has been provided by experiments using in-situ hybridization to identify CVB within the myocarditic lesions in the hearts of infected mice (McManus et al., 1993). Further support for this theory is provided by experiments in which immunocompromised hosts show an increase in disease severity (Chow etal., 1992). CVB-induced myocarditis is a multifactorial disease, the pathogenesis of which is poorly understood. Undoubtedly the disease process is a combination of both viral and immunopathogenic mechanisms dependent upon host age, sex, and genetic background. As with diabetes, current research is focused on mapping the ability to induce myocarditis with specific regions of the genome. Evidence suggests that the 5' nontranslated region of the genome along with neutralizing immunogenic sites within the capsid structure may contain these virulence determinants (discussed further under pathogenic variants). 1.7 The Virion Coxsackieviruses are small, nonenveloped viruses capable of withstanding low pH. This property enables them to survive the acidic environment found in the stomach. The capsid of a mature virion is approximately 300 Angstroms in diameter and exhibits a T=l. icosahedral lattice structure (Casper et al., 1962). The capsid shell of the virus serves several 8 functions. It acts as a physical barrier protecting the virus's RNA genome from degradation by environmental nucleases! It binds to receptors on target cells, and delivers the virion's RNA into the cytosol of the host cell thereby determining host range and tissue tropism. It directs the packaging of the RNA into the capsid and provides a proteinase required for the production of mature virions. Finally the capsid determines the antigenicity of the virus particle (Rueckert et al., 1996). As with other members in the Picornaviridae family, the coxsackievirus capsid is composed of 60 protomers; each protomer containing one copy of each of the four viral proteins VP1, VP2, VP3 and VP4. The protomers are arranged as pentamers around each icosahedral fivefold axis with the VP1 proteins forming each vertex while the VP2 and VP3 subunits form a surrounding ridge. This results in each fivefold axis being surrounded by a depression referred to as the 'canyon region' which is thought to be the site of receptor binding [Figure 1.1] (Rossmann, 1989). The atomic structure of CVB3 has been determined by Muckelbauer et al. and demonstrates an eight-stranded antiparallel Beta-sandwich motif within each of VP1, VP2 and VP3 (Muckelbauer et al., 1995). This motif is conserved among other members of the Picornaviridae family with areas of dissimilarity occurring at the amino and carboxy termini and within the loops connecting the Beta-strands. These regions of dissimilarity occur predominately on the viral surface which may account for the differences in antigenicity and tissue tropism between family members (Muckelbauer et al., 1997). The VP1 Beta-sandwich has a hydrophobic pocket which contains an unidentified pocket factor predicted to be a C 1 6 fatty acid with its aliphatic chain extending into the pocket. This pocket factor resembles that found in poliovirus (Filman et al., 1989). The VP4 protein exists on 9 the interior of the capsid structure and its amino terminus is covalently linked to a myristate residue which mediates interactions with the amino terminal of VP3. Several functions of the myristate residue have been suggested. It may play a role in virus assembly by stabilizing the pentameric subunits (Ansardi et al., 1992). It may also play a role in disassembly during infection by mediating the interaction with cellular membranes (Chow et al., 1987). Figure 1.1 The Icosahedral Capsid of Coxsackievirus Figure 1.1 The icosahedral arrangement of the coxsackievirus structural proteins. The shaded circle around each fivefold axis represents the canyon region. The dark lines around VP1, VP2 and VP3 outline five protomers which compose each pentameric subunit (text and figure scanned from Muckelbauer et al., 1997). 10 1.8 The RNA Genome The coxsackievirus genome is a single stranded message sense RNA molecule approximately 7400 nucleotides in length. Its organization is the same as the poliovirus genome with a virally encoded protein VPg covalently linked to its 5'end and a polyadenylated tail at the 3' end. The first 741 nucleotides do not code for any known protein and are referred to as the 5' nontranslated (NTR) region. The single open reading frame is divided into three regions, PI, P2 and P3, which are cotranslationally cleaved to produce the proteins required for capsid assembly and RNA replication. The PI region encodes the structural proteins while the P2 and P3 regions encode the nonstructural proteins required for viral replication. The open reading frame is terminated by a stop codon which is followed by 100 noncoding nucleotides appropriately named the 3' nontranslated region (Tracy, 1988; Rueckert, 1996 ) [Figure 1.2]. Figure 1.2 The Coxsackievirus Genome 5'NTR 3'NTR I M P2 P3 AAA, (n) Figure 1.2 The coxsackievirus genome depicting VPg, the 5' and 3' NTRs, the poly-A tail, and the PI, P2 and P3 coding regions. 11 VPg: The coxsackievirus genome has a small viral-encoded protein, VPg, at the 5' end of its genome. VPg is covalently attached to the RNA by a phosphodiester linkage to the phenolic hydroxyl group of a tyrosine residue (Wimmer, 1982). This protein plays a role in the initiation of coxsackievirus RNA synthesis (discussed further under CVB3 Replication). 5' NTR: The 5' nontranslated region is unusually long and exhibits an extensive, well-conserved, complex secondary structure. When compared to typical eukaryotic messenger RNAs several differences are apparent. Most importantly, members of the Picornaviridae family do not have a 7-methylguanosine cap structure attached to the 5' end of their genome (Jang et al., 1990). Cap-independent initiation of translation occurs by binding of specific host cell proteins and ribosomes to a region within the 5'NTR (Schmid et al., 1994; Pelletier et al., 1988). This region is a cis-acting element known as the 'internal ribosomal entry site' (IRES). Based on sequence comparison and secondary structures, the IRES is present in one of two forms. Type 1 IRES elements are found in the rhinoviruses and the enteroviruses while type 2 are found in the cardiovirus, apthovirus and hepatovirus genera. (Brown et al., 1991; Pilipenko et al., 1989a; Pilipenko et al., 1989b) . The 3' border of both elements exhibits a conserved YnXmAUG motif which consists of a stretch of 8-10 pyrimidine residues (Yn) separated from an AUG triplet by 18-22 nonconserved nucleotides (Xra). In viruses with the type 2 IRES element, the AUG triplet in the YnXmAUG motif acts as the translation initiation site. However in those with the type 1 IRES, this triplet is silent and translation is initiated further downstream (Jang et al., 1990; Pilipenko et al., 1992) [Figure 1.3]. 12 Figure 1.3 The Internal Ribosomal Entry Site of Picornaviruses (A) EMCV 5' VP» Figure 1.3 Schematic representations of the predicted secondary strucutres for the E M C V (A) and poliovirus (B) IRES elements. Yn-Xm A U G motifs are indicated by shaded boxes (where the light shaded box = Yn, the dark shaded box = A U G and the space between these boxes = Xm). Stem loop structures of the E M C V IRES and poliovirus IRES elements are labeled with letters and roman numerals respectively. The borders of the IRES are indicated by dashed boxes, (test and figure scanned from Harber et al., 1993). As a member of the Enterovirus genus, coxsackieviruses have a type 1 IRES element. Analysis of various deletion mutants has shown the CVB3 IRES to span nucleotides 529-630 which are located in stem-loops G, H, and I [Figure 1.4]. In addition to the IRES element, nucleotides 1-249 are crucial for translation initiation. The function of this region is not known; however, it has been suggested that it may bind translation initiation factors (Yang et 13 al., 1997). Translation of the genome results in a polyprotein which is cotranslationally cleaved into the functional proteins [Fig. 1.5]. As the diagram depicts, the initial proteolytic cleavage events produce multiple combined proteins (2BC, 3 A B , 3CD) that may have different functional properties than their individual subcomponents (2B, 2C, 3A, 3B, 3C, and 3D). Among the C V B s the length of the 5' N T R is relatively conserved ranging from 741 to 743 nucleotides; however, there are two regions of variability. The first begins at a pyrimidine tract located around nucleotides 90-100 and extends for approximately 80 nucleotides downstream. The second begins at nucleotide 680 and continues until the initiation codon. Neither of these variable regions lie in an area of predicted secondary structure (Romero et al., 1997). O f particular interest, the 5' N T R of C V B 3 appears to contain virulence determinants required for the induction of myocarditis (Tu et al., 1995). Figure 1.4 The Coxsackievirus B3 5'NTR 10-34 40-81 104-180 188-208 209-481 484-514 519-560 581-624 625-641 646-659 688-741 ,50 l ,1 .300 350 "1(400 25ol K Q i The IRES H 450 ' F ^ o 550 100 200 LUUCAUUUU '700 B50 742 AUG g' Figure 1.4 Diagram of R N A secondary structures in the 5'NTR of CVB3 predicted by the method of Zuker (Zuker, 1989). The range of numbers indicates the nucleotides of respective stem-loop structures which are labeled A through K. The IRES, authentic initiation codon A U G , and conserved polypyrimidine/AUG tract are marked (text and figure scanned from Yang et al., 1997). 1 4 Figure 1.5 Expression of the Picornaviral Genome 5' Coat protein genes Genes for cleavage & RNA synthesis VPO VPg V P 3 VP1 a pro TRANSLATION PRODUCTS N-terminus -polyprotein-C-terminus • -PI- •P2 • - -P3-1ABCD 2ABC +3ABCD 1ABC 2A 2BC 3CD VPO 1CD JB]l 2C USA) Qj 3C j 3D ryp4i VP2 VP3 VP1 VPg 3' pol f-AAAA polyA Figure 1.5 Organization and expression of the picornaviral genome. Synthesis of the polyprotein occurs from the N-terminus to the C-terminus. Cleavage of the polyprotein occurs by the virally encoded proteinases 2A (or 2AB) and 3C (or 3CD) (text and figure scanned from Rueckert, 1996 with minor modifications). PI: The PI region encodes the viral capsid proteins VP4, VP2, VP3 and VP1 (Reuckert et al., 1984) [Figure 1.2]. The VP4 protein is the smallest at 69 amino acids and is the most highly conserved among other enteroviruses, most likely due to its internal location in the capsid thereby protecting it from selective pressure to mutate. VP1, VP2 and VP3 form the exterior of the capsid shell and define the major antigenic epitopes of the virus. Three neutralizing immunogenic sites (NIm) have been determined in Poliovirus (Mahoney) and Human Rhinovirus 14 (HRV14). These same regions are also thought to exist in the 15 coxsackieviruses. The first is in the BC loop of VP1 and consists of amino acid residues 81-85 which flank the north rim of the canyon [Figure 1.6]. The second NIm site occurs in the EF loop of VP2 (amino acid residues 129-180); also referred to as the 'puff region. The third epitope includes amino acid residues 58-69 form the surface protruding 'knob' region of VP3 which also contains a NIm site in both rhino- and polioviruses (reviewed in Muckelbauer et al., 1997). In light of this, it is not surprising that VP 1-3 exhibit the lowest levels of amino acid conservation among the various CVBs and that the regions with the most amino acid variability occur within the NIm sites or surface-exposed regions. P2: The P2 region lies in the middle of the genome and encodes for four nonstructural proteins: 2A, 2BC, 2B, and 2C. The functions of the P2 and P3 nonstructural proteins have been analyzed for poliovirus and are thought to be very similar in a coxsackievirus infection. Since these proteins are not exposed to external selective pressures, and many have enzymatic functions, the variability in this region of the genome is much less than in PI. The 2A protein is a proteinase which functions to cotranslationally cleave the PI region from the polypeptide chain, an essential event to allow efficient translation to continue (Toyoda et al., 1986) . Protein 2A is also involved in shut-off of host protein synthesis by cleaving the 220-kDa component of the initiation factor eIF-4F (Bernstein et al., 1985; Krausslich et al., 1987) . Interestingly 2A exhibits the lowest level of amino acid conservation of any of the CVB proteins at 90% (Romero et al., 1997). 16 Expression of the 2BC protein induces the formation of small membrane vesicles which are thought to be the site of viral RNA replication. In addition, expression of the 2B protein causes several biochemical alterations in the host cell. It inhibits protein secretion and increases the permeability of the plasma membrane (Doedens et al., 1995; Van Kuppeveld et al., 1997). The role of these biochemical changes in viral replication is unknown. The 2C gene is the most highly conserved region of the entire genome (Argos et al., 1984), and its encoded protein 2C functions as a small NTPase located on the surface of the membrane vesicles. Here it is thought to bind viral RNA thereby attaching it to the membranous replication complex (Barco et al., 1995; Bienz et al., 1983; Bienz et al., 1987; Bienz et al., 1990; Bienz et al., 1994; Mirzayan et al., 1994; Rodriguez et al., 1993). In addition the ability of poliovirus to replicate in the presence of the drug guanidine hydrochloride has been mapped to this region (Anderson-Sillman et al., 1984). The 2C protein has the highest level of amino acid conservation between CVBs at approximately 98%, in which there exists a 41 nucleotide tract (331-371) which is absolutely conserved among all CVBs (Romero et al., 1997). P3: The P3 region of the genome is divided into four regions, A, B, C, and D, which are expressed as six different proteins: 3AB, 3A, 3B, 3CD, 3C, and 3D. Like the P2 region, the genetic variability of the P3 region is very low with greater than 90% conservation of amino acids in the encoded proteins. The 3AB protein plays a role in viral RNA synthesis as displayed by its RNA-binding activity and its ability to upregulate the activity of 3D polymerase more than 50-fold in vitro (Lama et al., 1994). Like the 2B protein, the 3A 17 protein has membrane-permeabilizing activity (Lama et al., 1992). Recent work with poliovirus also suggests a role for the 3 A protein in the virus-induced cytopathic effect (CPE) observed in infected Vero cells (Lama et al., 1998). The 3B gene encodes the viral protein VPg (Pallansch et al., 1980). Both the 3CD and 3C gene products are proteinases which cleave the polyprotein synthesized from the viral RNA. The 3C proteinase has also been shown for poliovirus to cleave host cell transcription factor IIIC in order to shut down host cell metabolism as shown in poliovirus (Clark et al., 1991). The 3D protein is an RNA-dependent RNA polymerase required for viral replication (Van Dyke et al., 1980). In addition the polymerase has been shown to contain a RNA duplex unwinding activity which is utilized during RNA replication (Cho et al., 1993). 3'NTR: The last 100 nucleotides of the CVB3 genome occur after the stop codon of the open reading frame arid are referred to as the 3' nontranslated region. Like its 5' counterpart, the 3' NTR displays a high level of secondary structure. Although the exact functions of these RNA structures are unknown, they are thought to play a role in the initiation of negative-strand RNA synthesis (Melchers et al., 1997). Following the 3' NTR is a string of genetically encoded adenosine residues known as the poly-A tract. The length of the pbly-A tract varies greatly between family members ranging from 35 residues in the cardioviruses to 100 residues in the apthoviruses (Carroll et al., 1984; Palmenberg et al., 1984). In coxsackieviruses it comprises 50-80 residues. The function of the poly-A tract is unknown; however, it is a requirement for poliovirus infectivity (Spector ef al., 1974). It has been 1 8 suggested that it may play a role in negative strand synthesis, but this remains to be elucidated. 1.9 The Coxsackievirus Cellular Receptor The role of the cellular receptor is to mediate virus attachment and entry of the viral genome into the host cell. As a result, receptors help define the cell tropism and pathogenesis for a given viral infection. In addition the virus possesses recognition sites for the cell receptor, and strain variation occurs in the affinity of these sites for the host cell molecule. CVB3 has two potential receptor recognition sites on its surface including the canyon region about its fivefold axes and a large depression at its twofold axes (Muckelbauer et al., 1995). These sites are major targets for neutralizing antibodies [Figure 1.6]. Figure 1.6 The Hypothesized Structure of Coxsackievirus Binding to its Cellular Receptor 5—FOLD VERTEX Figure 1.6 Binding of the cellular receptor to the floor of the canyon on the C V B capsid surface. This regions also contains epitopes recognized by neutralizing antibodies (figure scanned from Rueckert, 1996). 19 The primary receptor for coxsackieviruses has been identified as a 46 kilodalton, transmembrane glycoprotein (Bergelson et al., 1997; Hsu et al., 1988; Tomko et al., 1997). It contains two extracellular immunoglobulin-like domains thereby making it a member of the immunoglobulin-like superfamily, and its gene has been mapped to human chromosome 21. Interestingly this protein also acts as the cellular receptor for several of the adenovirus subgroups leading to its name; CAR (coxsackie-adenovirus receptor) (Bergelson et al, 1997; Roelvink et al., 1998; Tomko et al., 1997). A secondary receptor has been identified for coxsackievirus strains Bl, B3 and B5 as the 'decay accelerating factor' (DAF or CD55) (Bergelson et al., 1995; Martino et al., 1998; Shafren DR, 1995). DAF was first identified as a CVB receptor when a CVB3 variant adapted to growth in human rhabdomyosarcoma cells was shown to bind to CHO cells transfected with the human DAF gene (Bergelson et al., 1995). DAF is a 70 kilodalton glycophosphatidylinositol-anchored, transmembrane protein involved in the complement regulatory system (Lublin et al., 1989). Unlike CAR, DAF is not a classical receptor because it alone cannot induce a productive, lytic infection. Instead DAF is thought to be important in the attachment phase of the infection process. Unlike the CAR receptor which binds in the canyon region about the fivefold axes, it has been suggested that the DAF receptor binds in the twofold axes depression on the viral capsid (Kuhn, 1997). The ability of cardiovirulent CVB 3 strains to bind to DAF suggests it may play a role in the pathogenesis of CVB-induced myocarditis (Martino et al., 1998). In a recent study Martino et al. suggest several mechanisms whereby DAF binding may play a role in the pathogenesis of viral heart disease. These include: (1) presenting the virus to secondary cell surface receptor(s), (2) promoting virus tropism for specific target tissues expressing DAF, (3) 20 initiating signaling cascades with pathogenic effects such as T-cell activation, commonly found in myocarditis, and (4) removing DAF from the cell surface thereby increasing the cell's susceptibility to complement-mediated lysis and release of progeny virus (Martino et al., 1998). Regardless of the roles these receptors may play in the pathogenic mechanisms of the virus it should be noted that not all cells expressing the receptor molecules are infected. This suggests there are other elements within the cell such as host factors involved in viral translation or RNA replication which determine the virus's ability to replicate in a specific tissue. 1.10 Coxsackievirus Replication The coxsackievirus lifecycle may be divided into four phases: (1) entry and uncoating (2) expression of the viral genome (3) replication of the viral genome (4) assembly and release of mature virus particles. Initially the virus binds loosely to the cell's surface by attaching to a single receptor (as discussed above). Binding allows additional receptors to be recruited resulting in the progressive formation of a tight virus-cell association. As this occurs, the cellular membrane begins to invaginate around the virion, the first step in endocytosis. Once bound, the virion undergoes an irreversible conformational change. The VP4 proteins are extruded from the capsid causing it to swell and extend the hydrophobic N-termini of its VP1 residues into the lipid bilayer of the host cell, thereby forming a transmembrane channel. Finally infection may occur during which the RNA genome is delivered through the membrane and into the cytosol. Those transient virus-membrane complexes successful in delivering the viral RNA into the cell are referred to as infectosomes [Figure 1.7] (Lee et al., 1993; Rueckert, 1,996). 21 Figure 1.7 Hypothetical Structure of Picornaviral Infectosome Figure 1.7 (A) Multiple receptor units, recruited from the membrane, position the virus particle close enough to implant into the host cell membrane. (B) Hypothetical structure of picornaviral infectosome showing how extrusion of hydrophobic capsid elements might form a channel or pore enabling the RNA genome to pass through the host cell membrane and into the cytosol ( figure scanned from Rueckert, 1996). The remaining three phases occur exclusively in the cytosol of the host cell. Host-cell protein synthesis is shut down and translation of the viral RNA begins upon entry. Inhibition of host protein synthesis is achieved by viral protein 2A which cleaves the eukaryotic initiation factor 4F (eIF4F) (formerly termed p220). Cleavage of eIF4F prevents formation of the cap-binding complex (CBC) (Etchison et al., 1982) that attaches to the 5' 7-methylguanosine cap of cellular mRNA in order to promote ribosome recognition and subsequently translation. Inhibition of host protein synthesis allows the translational machinery to be exploited for viral protein synthesis. Unlike eukaryotic mRNA, the viral genome does not have a 5' cap structure eliminating its need for eIF4F. Instead the 40S ribosomal subunits are capable of attaching directly to the viral RNA at the IRES. Multiple host proteins are required for ribosomal binding and the initiation of translation. These include the 52 kilodalton La protein (an autoantigen in Lupus) (Meerovitch et al., 1993a; Meerovitch et al , 1989), the 57 kDa 22 protein identified as the polypyrimidine tract-binding protein (PTB) (Hellen et al., 1993; Pestova et al., 1991) and eukaryotic initiation factors 4B and 4A (Meyer et al., 1995). The precise roles these proteins play in the initiation of cap-independent translation is unknown. It has been suggested that the La and PTB proteins act as RNA chaperones thereby stabilizing those RNA structures required for ribosomal binding (Meerovitch et al., 1993b) while elF 4A and 4B form an ATP-dependent helicase complex which unwinds certain regions of the IRES required for ribosomal binding (Belshamet al., 1996). Once the ribosomal complex has bound the IRES it translates the genome's single open reading frame commencing at AUG (nucleotide 742) into a polyprotein which is cotranslationally cleaved (autocatalytically) into the various viral proteins (see genome section). The first step in the replication of the picornaviral genome is the synthesis of a complementary (-) RNA strand which will serve as the template for production of progeny (+) strands. Two models have been proposed for synthesis of the negative RNA of picornaviruses. In the first model VPg-pUpU acts as a primer which is elongated by the viral 3D RNA polymerase (protein 3Dpo1) using the (+) strand as a template. In the second model a host enzyme, terminal uridylyltransferase (TUT), adds a poly-U tail to the 3' end of the genome which forms a hairpin that can self prime the synthesis of the negative RNA strand. Subsequent VPg addition and cleavage separates the heterodimeric RNA releasing both a (+) and (-) strand [Figure 1.8] (Rueckert, 1996; Young et al.,1986). 23 Figure 1.8 Two Models For RNA Synthesis Model 1 + VPg as primer J—PApAnApApApA-QH (-) -^ fgDj -{pU PU-Q-Tyr)VPg Model 2 VPg as strand separator ApApApApAl IpUpUpUpU-o^^Q VPg B (+)5'VPg-pUUAAAACAG GGAAApoly(A)..AAApA-OH 3' (-)3' HO-AAUUUUGUC -CCUUUpoly(U)UpUp-VPg 5' Figure 1.8 Two models for picornaviral (-) R N A synthesis. (A) In Model 1 the newly forming R N A strand is initiated by elongation of a VPg-pUpU primer derived from protein 3AB. In Model 2, the template strand is elongated at the 3' end by TUT. The oligo- (U) tract folds back to hybridize with the poly(A) tract of the template strand; thereby permitting the nascent strand to be elongated by the viral 3D polymerase. Cleavage of the A - U bond is thought to occur by transesterification using the tyrosyl group of VPg. (B) RNA heterodimer containing a copy of both the (+) and (-) strands (figure scanned from Rueckert, 1996 with minor modifications). In the second step of RNA replication secondary structures at the 5' end of the (+) strand of the heterodimeric duplex are recognized by the 3C region of an uncleaved 3 CD molecule along with a 36 kDa cellular protein (Andino et al., 1993). This replication complex is anchored to smooth membranes by 3AB. VPg (3B) and 3D are released from the complex by intramolecular proteolysis by 3C protease activity. Several uridylate residues are added to the tyrosine residue of the VPg molecule which serves as a primer for the synthesis of a new (+) strand by 3D polymerase. Replication continues displacing the orignal (+) strand as the polymerase adds more residues to the growing chain which in turn binds a new 3 CD 24 molecule, the first step in initiating the synthesis of another (+) RNA strand. As a result RNA structures containing one full length (-) strand and many growing (+) strands are formed. These are called replicative intermediates [Figure 1.9]. It should be noted that the majority of the work aimed at understanding the replication of picomaviruses has used poliovirus as the viral model. Figure 1.9 Figure 1.9 Three initial postulated step of picornavirus plus strand R N A synthesis modeled from poliovirus. The upper picture represents a negative strand (-) acting as a template and a resident positive strand (+) holding the initiation complex consisting of the 5'-cloverleaf RNA, 3CD, p36 and the 3AB precursor of the primer peptide (VPg) attached to the membrane. It is suggested that 3CD acts in three ways in the complex: addition of Replication of Picornavirus RNA 25 one or two U residues to VPg (3B), cleavage of 3B from 3A and cleavage of itself to yield 3C and 3D. The middle picture shows the progress of the reaction after VPg-pU-pU is used as a primer for 3D to begin the elongation reaction. The bottom picture shows that after 3D has synthesized at least the first 100 nucleotides of the genomic RNA a new cloverleaf structure will form, allowing the formation of a new complex that can then catalyze the initiation of the next plus strand RNA. (figure scanned from Andino R, 1993) The pool of (+) RNA strands may be translated to make more viral proteins, utilized as a template for more (-) strand synthesis, or encapsidated. Packaging the (+) strand RNA into virions is the final phase of the virus lifecycle, the mechanism for which is unknown. Once assembled, the mature progeny virions are thought to be released by cell lysis. 1.11 Pathogenic Variants The study of viral variants is a useful approach to associate a pathogenic phenotype with a given genotype in order to understand the molecular mechanism of disease. Every virus stock is a genotypically heterogenous population in which the dominant phenotype is expressed. This genotypic heterogeneity and ultimately phenotypic variation is a product of mutations. The mutation rate per base in single stranded RNA viruses is approximately 10"4 (Holland et al., 1982). These mutations may have phenotypic consequences such as an alteration in virulence, plaque morphology, antigenicity and host range. An analysis of the intratypic genetic variation among multiple CVB3 strains (Romero et al., 1997) revealed greatest variation within the 5'NTR which exhibited a nucleic acid indentity of 79%-99% between strains. The PI region displayed a high level of amino acid conservation of greater than 95% for all four capsid proteins while the P2 and P3 26 nonstructural genes were the most highly conserved at 98%-100% identity. Among the nonstructural proteins, the 2A protease exhibits the greatest variance while the VPg protein (3B) remained absolutely conserved in its nucleotide sequence and hence in its amino acid sequence. Among various CVB3 strains the 3'NTR varies in length from 97-99 nucleotides and exhibits a nucleic acid identity of 94% to 99%. The data suggests a high level of conservation among CVB3 strains; however, it should be noted these data were compiled from the comparison of thirteen different strains including 9 clinical isolates. If more strains were sequenced one would expect the amount of variation to increase. Multiple types of CVB variants have been isolated including temperature-sensitivity variants, receptor variants, antigenic variants and clinical isolates. In 1983 Gauntt et al. isolated 10 temperature-sensitive variants from a myocarditic parental strain that replicated equally well at both 34°C and 39°C. The mutant strains were unable to replicate efficiently at the higher temperature and exhibited an altered pathogenic phenotype in adolescent mice including the inability to induce myocarditis (Gauntt et al., 1983). In 1984 Reagan et. al isolated a CVB3 variant (CV3-RD) after serial passages in human rhabdomyosarcoma (RD) cells which could infect both RD and HeLa cells (Reagen et al., 1984). Further analysis revealed that the CB3-RD variant utilizes the DAF (Decay Accelerating Factor) (CD55) molecule as a secondary receptor, thereby increasing its host cell range (Bergelson et al., 1995). This phenotypic property has since been mapped to a threonine to serine mutation at amino acid 151 of VP2 which occurs in the EF loop otherwise referred to as the 'puff-region'(Lindberg et al., 1992). 27 Since this early work over fifteen years ago, multiple other CVB variants have been isolated and analyzed in order to correlate a unique pathogenic phenotype with a specific region or sequence within the genome. Ramsingh et al. (1993) have isolated a nonvirulent CVB4 variant which is incapable of inducing acute pancreatitis and severe, prolonged hyperglycemia unlike its parental strain. The virulent phenotype has been mapped to two locations; a threonine residue at position 129 on the DE loop of VP1 and an arginine residue at amino acid position 16 of VP4 (Caggana et al.1993; Ramsingh et al.1995; Ramsingh et al., 1990). As a point of interest, these authors suggest Thr-129 of VP1 would be positioned in the same region as Ile-143 of poliovirus which has been shown to be a major attenuation determinant (Ren et al., 1991). In 1994 Chapman et al. isolated a non-cardiovirulent CVB3 strain (Chapman et al., 1994) whose phenotype was mapped to a single nucleotide change in the 5'NTR (Tu et al., 1995). Virus containing a U at nucleotide position 234 exhibited a cardiovirulent phenotype while a C at this position resulted in an avirulent strain. Interestingly position 234 does not appear to be located in one of the stem-loop structures of the 5' NTR and transcriptional efficiency is about 10-fold lower in the non-cardiovirulent strain. In 1996 Knowlton et al. (1996) isolated a CVB3 antibody escape mutant which also displayed an amyocarditic phenotype. This reduction of myocarditic potential was mapped to a single nucleotide change at position 1442 of the genome. This corresponds to an asparagine (myocarditic phenotype) to aspartate (amyocarditic phenotype) mutation at amino acid 165 of the EF loop of VP2 otherwise referred to as the 'puff-region', the same region of the threonine to serine mutation found in the CVB-RD strain. The presence of an asparagine residue at amino acid 165 of VP2 is also associated with a high-level of production of tumour 28 necrosis factor alpha (TNFa) from infected Balb/c monocytes (Knowlton etal., 1996). Most recently a cardiovirulent revertant strain was isolated after passage of an amyocarditic strain through SCID (severe combined immunodeficient disease) mouse heart. The attenuated phenotype was mapped to an aspartate to glycine mutation at amino acid position 155 of VP1 (Cameron-Wilson et al., 1998). The study of variants has helped to define the molecular basis for CVB virulence. The determination of specific genomic regions and/or sequences responsible for a pathogenic phenotype will ultimately lead to the development of a 'library' of attenuating mutations that may be utilized in vaccine development, as has been done for poliovirus. 29 Chapter Two: Research Objective The long term objective of this research is to understand the molecular basis for CVB3 virulence. As described in the Introduction, the study of pathogenic variants is a useful method to help identify regions of the genome responsible for a given biological phenotype such as the ability to induce myocarditis or diabetes. The underlying hypothesis of this study is that differences in virulence among CVB3 strains may be mapped to specific nucleotide changes in the genome. Previous work in our laboratory has led to the isolation of an antibody escape mutant of CVB3 (EMI) which exhibits similar viral titres in the liver and pancreas as compared to the wild-type strain, but ten-fold lower titres in the hearts of infected mice (Sadeghi, 1997). This amyocarditic variant of CVB3 has been the basis of the studies reported in this thesis which had the following specific objectives: 1. To sequence the genome of EMI in order to determine the position of mutations relative to the parental CVB3(RK) wild-type strain. 2. To produce a panel of antibody-escape mutant strains by repeating the procedure used to isolate EMI in order to identify other mutations which may affect viral pathogenic phenotype. 3. To analyze the biological properties of the isolated mutants in comparison with the wild-type strain CVB3(RK) in an attempt to correlate a biological phenotype with the sequence data. 4. To analyze the various strains in vitro for temperature-sensitivity and stability. 5. To engineer an altered strain containing one of the mutations found in the EMI structural proteins. 30 In this manner it should be possible to correlate specific mutations with an alteration in a phenotypic property of interest such as tropism for heart or pancreatic tissue. Ultimately this will lead to an increased understanding of CVB pathogenesis, and identify potential mutations which may be exploited in vaccine development. Chapter Three: Materials 3.1 Tissue Culture a) Cell line: Vero ( African Green Monkey Kidney Cells) b) Medium: DMEM.F12 (Dulbecco's Modified Eagle Medium) c) Fetal Bovine Serum (FBS) d) Heat Inactivated Fetal Bovine Serum (HIFBS): Heat FBS 60min. at 60°C e) Antibiotic: Gentamicin 50mg/ml (Gibco/BRL) used at 1% (v/v) f) Trypsin (0.25%) 2.5g 1:250 Trypsin (Fisher) Dissolve in IL of Hanks balanced salt solution ( without Ca or Mg ) (Gibco/BRL) Sterilize using a 0.22um filter g) Phosphate Buffered Saline (PBS) 34.0gNaCl 4.28gNa2HP04 1.38gNaH2P04-H20 Dissolve in 4L of distilled water and autoclave to sterilize 3.2 Fixatives, Stains, and Growth Medium a) Carnoy's Fixative 750mls 95% Ethanol 250mls Glacial Acetic Acid b) 4% Paraformaldehyde (PF) 40.5mls 0.1MNa 2HPO 4 9.5 mis 0. IM NaH 2P0 4-H 20 Heat the phosphate buffer to 80°C Dissolve 2g of PF Adjust pH to 7.4 with 5N NaOH (5-10 drops) c) Coomassie Blue Stain lOOmls Glacial Acetic Acid (10% v/v) 250mls Isopropanol (25% v/v) 2.50g Coomassie Blue Stain (Sigma) Dilute to IL with distilled water d) Eosin 70mls Ethanol(70%) 25mls Glacial Acetic Acid 0.5g Eosin (0.05% w/v) e) Haematoxylin 0.2g Haematoxylin (0.2% w/v) 0.7g Al2(S04)3-15H20(12.6mM) 0.008g NalOs (0.4 mM) 25 ml 1,2-Ethanediol (25% v/v) 2 ml Glacial Acetic Acid (2% v/v) Dilute to 100ml with ddH20 f) Massons's Trichrome Bouin's Sodium bicarbonate (2% w/v) Weigert's iron Haematoxylin (a mixture of 2 solutions in equal parts): Solution A: 1% (w/v) haematoxylin in absolute ethanol Solution B: 40% (w/v) FeCl 3 in 10ml HCI per IL Ponceau 2R (1% w/v): acid fuchsin (l%w/v) mixed (70:30) in acetic acid Acetic acid (1% v/v) Phosphomolybdic acid (1% v/v) Aniline Blue (2.5%) in acetic acid (2.5%) g) LB-Ampicillin Plates lO.Og Tryptone 5.00g Yeast Extract lO.Og NaCl 20.0g Agar Dissolve in IL of distilled water. Autoclave and cool to 55°C prior to adding ampicillin to a final concentration of 150ug/ml. Pour into petri dishes and allow to harden prior to storing at 4°C. 33 h) Preparation of SOC Medium i) Prepare 2M M g 2 + stock as a IM MgCl 2 and IM MgS0 4 solution. Filter Sterilize. ii) Prepare SOB: lO.Og Bacto Tryptone 2.50g Yeast Extract 0.30g NaCl 0.09g KC1 Dissolve in 500ml of distilled water and autoclave to sterilize. Add 1/100 volume of 2M M g 2 + stock. iii) Prepare a 2M glucose stock and filter sterilize. iv) To prepare SOC add 1/100 volume of 2M glucose stock to SOB. 3.3 Virus Strains The wildtype strain; CVB3(RK) was obtained from Dr. Reinhard Kandolf. The virus was derived from an infectious clone pCVB3/T7 prepared from Nancy strain which was transcribed and transfected into HeLa cells. The progeny virus was passaged in mice and virus harvested from the heart tissue was used to make the CVB3(RK) stock. The CVB3 antibody escape mutant strain EMI was originally isolated by Assal Sadeghi as described in material and methods. 3.4 Infectious Clone The CVB3 infectious clone pCVB3/T7 was kindly provided by Dr. Reinhard Kandolf. 3.5 Restriction Enzymes The following restriction enzymes and their respective reaction buffers were obtained from New England Biolabs (NEB): Age I BlpI BsiWI HindEI Bgin Bsal BstI Sail Smal 34 3.6 PCR a) DNA polymerase Taq DNA Polymerase (NEB) Deep Vent DNA Polymerase (GIBCO/BRL) b) 25mM dNTPs (Pharmacia) c) Light Mineral Oil (Fisher) d) PCR Clean-up Kit (Qiagen) e) 50X TAE Gel Running Buffer f) 0.7% Agarose Gel Dissolve 0.7g agarose (GIBCO/BRL) in 100ml IX TAE g) PCR Primers PCR primers were synthesized by the UBC NAPS (Nucleic Acid and Protein Services) Unit. The following table [Table 3.1] lists the primer pairs used in RT-PCR to span the entire genome for sequence analysis. 35 Table 3.1 PCR Primers LP. Sequence 5' position la: 5'- GTC GTC GAC GTT TAA AAC AGC CTG TGG GTT GA-3' ' (0) lb: 5'- CGG GAT CCC AGG GTC TTG AGT GAA ATC CTG C- 3' (890) 2a: 5'- AAC TCA GCC AAT CGG CAG GAT- 3' (856) 2b: 5'- GGT CCT TCA AAC GAA ATT GGG-3' (3511) 3a: 5'-TGG ATA CCT AGA CCA CCT AGA CT-3' (3184) 3b: 5'-GCC CAG CAT GGT AAA CTC GCC-3' (5448) 4a: 5'-AAG CCC AGA GTG CCT ACC C-3' (5323) 4b: 5'- CAC GTG ATC TTG GGT GTT CTT -3' (7155) 5a: 5'- ACA CCA GCA GAT AAG GG -3' (6976) 5b: 5'- TTT TTT TTT TTT TTT CCG CAC-3' (3'-polyA) 3.7 Reverse Transcriptase RT-PCR a) Dulbecco's PBS 0.2g KC1 0.2g KH 2P0 4 8.0g NaCl 2.16g NaHP04-7H20 Dissolve in IL distilled water b) Dulbecco's PBS + 30% Sucrose 150g Sucrose (Beckman) Dissolve in 500mls of Dulbecco's PBS c) ' Trizol Reagent (Gibco/BRL) d) Reverse Transcriptase: Superscript II (Gibco/BRL) e) RNAse Inhibitor (Gibco/BRL) f) 0. IM Dithiolthreitol (DTT) g) Hexanucleotides (Pharmacia) h) 25mM dNTPs (Pharmacia) 3.8 In-Situ Hybridization (ISH) Solutions a) DEPC treated water 0.1% (v/v) diethypyrocarbonate in distilled water Incubate overnight at 37°C Autoclave b) 10% SDS lOg SDS in 100ml DEPC-ddH20 c) 4M NaCl 233.7g NaCl in IL DEPC-ddH20 d) 20XSSC 175.3g NaCl 88.20g Na Citrate Dilute in IL DEPC-ddH20 e) lMTris pH 7.4 242.2g Tris in IL DEPC-ddH20 Adjust pH to 7.4 with HCI Dilute to 2L with DEPC-ddH20 f) IM Tr is /O. lMEDTApH 7.4 60.55g Tris 18.61g EDTA Dilute in 250ml DEPC-ddH20 Adjust pH to 7.4 and dilute up to 500ml with DEPC-ddH20 g) 0.2MCaCl 2 11.lg CaCl 2 Dilute in 500ml DEPC-ddH20 h) 0.5M MgCl 2 101.7g MgCl 2 Dilute in IL DEPC-ddH20 i) Proteinase K lOmg proteinase K dissolved in 1ml DEPC-ddH20 j) 100% Formamide (Gibco/BRL) k) 1.8MNaCl (10% Dextran Sulfate) 10.5 lg NaCl lO.OOg Dextran Sulfate Dissolve in 100ml DEPC-ddH20 1) 1MDTT 250mg DTT Dissolve in 1.62ml DEPC-ddH20 m) Polyvinylpyrrolidone Ficoll BSA (PFB) (100X [2%]) 2.0g PVP 2.0g Ficoll 5.0g BSA Dissolve in 100ml DEPC-ddH20 n) Salmon Sperm DNA 1 mg/ml o) 0.2NHC1 13.9ml 32% HCI Dilute to 600ml with DEPC-ddH20 p) 20mM Tris / 2mM CaCl 2 12ml lMTris 6ml 0.2M CaCl 2 Dilute to 600ml with DEPC-ddH20 q) 2xSSC 400ml 20xSSC Dilute to 4000ml with DEPC-ddH20 r) Wash Buffer (50% Formamide, lOmM Tris /ImM EDTA, 600mM NaCl) 600ml Formamide 12ml IM Tris /0.1M EDTA 180ml 4MNaCl Dilute to 1200ml with DEPC-ddH20 s) Buffer 1 (0.15M NaCl, 0.1 M Tris-HCl) 75ml 4M NaCl 200ml IM Tris-HCl Dilute to 2000ml with DEPC-ddH20 t) Buffer 2 (2% Lamb Serum) Add 12ml lamb serum to 600ml Buffer 1 u) Buffer 3 (0. IM NaCl, 0.1 M Tris-HCl, 0.05M MgCl2) 16ml 4MNaCl 60ml IM Tris-HCl 60ml 0.5MMgCl 2 Dilute to 600ml with DEPC-ddH20; adjust to pH 9.5 v) Colour Substrate Dissolve 2 Sigma Fast NBT/BCIP tablets (Sigma) in 10ml ddH20. Dilute to 300ml with ddH20. w) Anti-Digoxigenin Antibody Dilute anti-digoxigenin antibody conjugate 1:500 with Buffer 1 containing 1% lamb serum x) 0.25% Acetic Anhydride / 0. IM TEA 0.5ml Acetic Anhydride 3.7g TEA Dilute to 200ml with DEPC-ddH20 y) Hybridization Solution 50% Formamide 33% 1.8M NaCl (10% Dextran Sulfate) 1% IM Tris / 0. IM EDTA pH 7.4 1% 1MDTT 1% 100xPFB(2%) 1% 10% SDS Add salmon sperm DNA to a final concentration of 0.04ug/ul Add dig-labelled probe to a final concentration of 20ng/ml 3.9 ISH Digoxigenin-Labeled Probe a) DIG RNA Labeling Kit (SP6/T7) (Boehringer Mannheim) b) Buffer 1 (lOOmM Maleic Acid, 150mM NaCl, pH 7.5) 5.8g Maleic Acid 4.38g NaCl Adjust pH to 7.5 and dilute to 500ml with DEPC-ddH20 c) Buffer 2 Dissolve 1% (w/v) of blocking reagent (available in kit) in Buffer 1 d) Buffer 3 (lOOmM Tris-HCl pH 9.5, lOOmM NaCl, 50mM MgCl 2 3.03g Tris 1.46g NaCl 2.54g MgCl 2 Adjust pH to 9.5 and dilute to 250ml with DEPC-ddH20 e) Hybond Membrane (Amersham) f) T7/ SP6 RNA Polymerase (NEB) 3.10 Large Scale Plasmid Prep. Solutions a) 2xYT Broth 20g Bacto-tryptone lOg Bacto-yeast extract lOg NaCl Dissolve in IL ddH20 b) TE pH 8.0 (lOmM Tris, ImM EDTA) 1.2g Tris 0.3g EDTA Dissolve in 800mls ddH20 and adjust pH to 8.0 with HCI Make up final volume to IL with ddH20 c) Solution I (50mM Glucose, 25mM Tris-HCl, lOmM EDTA) 9.0g Glucose 3.0g Tris 2.9g EDTA Dilute in IL of ddH20 and adjust pH to 8.0 with HCI d) Solution II (0.2M NaOH, 0.1% SDS) 8.0g NaOH lOg SDS Dilute in IL with ddH20 e) Solution III (5M KOAc, 11.5% Glacial Acetic Acid) 245g KOAc 57.5ml Glacial Acetic Acid Dilute to 500ml with ddH20 f) Phenol: Chloroform: Isoamylalcohol g) 5MLiCl 21.2g LiCl Dissolve in 100ml ddH20 h) 3M NaOAc 21.2g NaOAc Dissolve in 100ml ddH20 i) 1.6M NaCl containing 13% polyethylene glycol 8000 (w/v) j) Ampicillin 3.11 Culturing of Cardiomyocytes From Neonatal Mice a) DMS8 6.80g NaCl 0.40g KC1 0.06g NaH 2 P0 4 -H20 0.27g Na 2HP0 4-7H20 l.OOg Glucose Dilute to 100ml with ddH20 b) Solution I 0.01% Trypsin (v/v) 0.2% BSA (w/v) Dilute to 100ml with DMS8 c) Solution II DNAse A 1ml of a 1.1 mg/ml DNAse A stock Dilute to 100ml with DMS8 d) Collagenase 3mg Collagenase 2mg BSA lOul lOmM CaCl 2 stock solution 2ml DMEM e) DMEM containing 10% FCS and 1% antibiotic 3.12 In Vitro Transcription a) T7 RNA Polymerase (NEB) b) 25 mM NTPs c) RNAse Inhibitor d) Lipofectamine Reagent (GIBCO/BRL) 3.13 Antibodies a) Murine monoclonal antibody against coxsackievirus B3: Anti-CVB3 948 (Chemicon International Inc.) b) Anti-Digoxigenin-Alkaline Phosphatase Conjugate (Boehringer Mannheim) 3.14 Animals a) A/J mice were purchased from Jackson Laboratories. b) Balb/c mice were purchased from the UBC Animal Unit. 43 Chapter Four: Methods 4.1 Cell Maintenance Vero cells were grown in a 5% C0 2 atmosphere within a 37°C humidified incubator. Cells were passaged twice a week at a 1:6 split. The monolayer of cells was washed with phosphate buffered saline (PBS) and incubated for approximately 25 minutes at 37°C in the presence of 0.25% trypsin. Once the cells were detached from the flask they were resuspended in DMEM.F12 culture medium supplemented with 10% FBS and 1% gentamicin, and distributed to fresh tissue culture flasks and/or petri dishes. 4.2 Virus Stock Preparation Vero cells were subcultured 24 hours prior to infection such that the monolayer was 80-90%) confluent. The cells were washed with 7.5 mis PBS to remove residual medium and infected with 0.1-1.0 plaque forming unit of virus per cell. The virus was diluted in 1ml of DMEM.F12 and allowed to adsorb to the monolayer of cells for one hour at 37°C. The inoculum was removed and replaced with 7.5 mis DMEM.F12 containing 5% HIFBS and 1% gentamicin. The cells were incubated at 37°C in a humidified atmosphere of 5% C0 2 until cytopathic effect (CPE) was observed (usually within 24 hours). The flask was frozen at -70°C and subsequently thawed at 37°C to lyse the cells and release progeny virus. The flask's contents was centrifuged for 10 minutes at 2500 RPM to separate the cell debris from the virus-containing supernatant. The supernatant was removed, aliquoted, and stored at -70°C for future use. 44 4.3 Virus Titration By Plaque Assay Titration of virus was achieved by quantifying plaque formation in Vero cells. Tenfold serial dilutions of the virus sample were used to infect 85-95% confluent Vero cells in 35mm petri dishes. Serial dilutions were prepared in DMEM.F12 and each dilution was titred in duplicate. Each dish was inoculated with 0.5ml of a serial dilution and allowed to adsorb for lhour at 37°C in a 5% C0 2 , humidified incubator. Following adsorption, the inoculum was removed and replaced with an overlay of 0.5% agarose in DMEM.F12 containing 5% HIFBS and 1% gentamicin. After the cells were incubated for 48 hours at 37°C in an atmosphere containing 5% C 0 2 they were fixed with 2ml of Carnoy's fixative for 30 minutes. The overlay was removed and the monolayer stained for 10 minutes with coomassie blue dye. Once stained, the petri dishes were rinsed with tap water, allowed to dry, and the plaque forming units (PFU) were counted as circular, colorless regions within the blue-stained monolayer. 4.4 Isolation of Antibody Escape Mutants The antibody escape mutants (EM2-EM10) were isolated using a commercially available, murine, neutralizing monoclonal antibody (MAb) specific for wild type coxsackievirus B3. CVB3(RK) virus stock was pretreated for one hour with a 1:50 dilution of the antibody to neutralize the dominant wild type strain. The neutralized stock was then incubated for one hour with a goat anti-mouse IgG-bead conjugate, and the suspension centrifuged for 5 minutes at 14K rpm in a microcentrifuge to remove the virus-MAb complexes. The remaining supernatant was used to infect Vero cells. Following virus adsorption for one hour, the cells were cultured for 48 hours in medium containing MAb diluted 1:1000. The 45 medium was replaced 24 hours post-infection at which time approximately 10% of the cells exhibited CPE. By 48 hours post-infection all of the cells displayed CPE, and the flask was frozen at -70°C and subsequently thawed. The culture medium was clarified by centrifugation for 10 minutes at 2800 RPM. The clarified supernatant containing virus was serially diluted in DMEM.F12, and 0.5ml of each dilution was used to infect Vero cells in 35mm petri dishes. After a one hour incubation at 37°C, the monolayers were overlaid with DMEM.F12 medium containing 5% heat inactivated fetal bovine serum (HIFBS), 1% antibiotic, and 0.5% agarose. The plates were incubated for 48 hours at 37°C in a 5% C0 2 humidified incubator at which time they were examined under an inverted microscope to identify individual plaques. The medium under one single plaque was removed using a pasteur pipette and stored at -70°C in DMEM.F12 containing 5% HIFBS. Eleven different plaques were purified in this manner, and the samples later used to infect Vero cells in the presence of the MAb. Nine of the eleven samples induced significant CPE 24 hours post-infection. These were selected to make escape mutant virus stocks EM2-EM10. 4.5 Antibody Neutralization Assay Monoclonal antibody aCVB3 948 was incubated (in duplicate) with 15ul of either CVB3(RK), EMI or EM10 diluted in DMEM.F12 at a ratio of 1:50 for one hour on ice. In order to determine if the Ab: virus complexes were infectious half of the samples were treated for 45 minutes with 150 ul of Zysorbin resin which binds to the constant region of antibodies thereby removing the Ab-virus complexes. The Zysorbin was pelleted by centrifugation and the supernatant saved for titration. Untreated virus stock, virus stock treated with antibody 46 and virus stock treated with both antibody and Zysorbin were titrated by plaque assay as described above. 4.6 Viral RNA Isolation Five flasks of Vero cells were infected with the virus stock of interest at a low multiplicity of infection. After maximal cytopathic effect (CPE) was observed 24-48 hours post-infection, the flasks were frozen at -70°C overnight. The flasks were thawed at 37°C and the culture medium was clarified by centrifugation for 10 minutes at 2800rpm. 25mls of the virus-containing supernatant was overlayed onto 6mls of Dulbecco's phosphate saline containing 30% sucrose in Beckman Ultra-Clear centrifuge tubes. The tubes were centrifuged at 20K rpm for 3 hours at 4°C. The supernatant was decanted and the virus pellet was resuspended in lOOul of phosphate buffered saline (PBS). In order to isolate RNA from the viral suspension, 1ml of Trizol Reagent (Canadian Life Technologies) was added and the sample incubated for 5 minutes at room temperature facilitating complete dissociation of nucleoprotein complexes. Samples were extracted with 0.2ml of chloroform and RNA in the upper aqueous phase was precipitated with an equal volume of isopropanol for 10 minutes at room temperature followed by centrifugation. The RNA pellet was washed with 75% ethanol, air dried and dissolved in 40ul of diethylpyrocarbonate (DEPC) treated water. 4.7 Reverse Transcriptase - Polymerase Chain Reaction a) First Strand Synthesis Synthesis of cDNA from viral RNA was carried out using the superscript kit (Canadian Life Technologies) and random hexonucleotides (Pharmacia pd(N)6) as primers. Each 25ul reaction was composed of 13p.l DEPC water, 5ul 5X Buffer (Superscript kit), 2ul 0.1M DTT, 47 lul containing 250 pmoles of pd(N)6 lul of 25mM dNTPs, 2ul Trizol purified RNA. The mixture was denatured at 95°C for 2 minutes and allowed to cool on ice for one minute at which time lul of RNase Inhibitor and lul of Superscript II Reverse Transcriptase was added. The tube was incubated for one hour at 42°C. b) Polymerase Chain Reactions (PCR) PCR was carried out using Taq DNA polymerase. Five primer sets (lab, 2ab, 3ab, 4ab, 5ab) were designed to amplify the 5'NTR, PI, P2, P3, and 3'NTR regions of the genome respectively. Each 50ul PCR reaction mixture contained IX PCR buffer, 0.25mM of each dNTP, 0.6uM of each primer, 2pi of cDNA, and 2 units of Taq DNA polymerase. The samples were overlay ed with mineral oil and underwent 35 cycles of amplification in a minicycler. The amplification parameters for each region of the genome are listed below: 5'NTR: (PCR product is 750bp long.) 2 minutes at 95°C 30 seconds at 95°C 30 seconds at 60°C 60 seconds at 72°C PI: (PCRproduct is 2.5 kbp long.) 2 minutes at 95°C 30 seconds at 95°C 3 minutes at 60°C P2: (PCR product is 2.2 kbp long.) 2 minutes at 95°C 30 seconds at 95°C 30 seconds at 5 7°C 2min.l5sec. at 72°C 48 P3: (PCR product is 1.8 kbp long.) 2 minutes at 95°C 30 seconds at 95°C 30 seconds at 59°C 2minutes at 72°C 3'NTR: (PCR product is 450bp long.) 2 minutes at 95°C 30 seconds at 95°C 30 seconds at 50°C 40 seconds at 72°C PCR products were examined on 0.7% agarose gels to ensure that the correct size bands were produced. Samples were purified using Qiagen quick-spin purification columns. The purified DNA was sequenced at the University of British Columbia's NAPS (nucleic acid and protein services) unit using ABI's AmpliTaq Dye Terminator Cycle Sequencing chemistry. 4.8 Temperature Sensitivity of Viral Strains Plaque assays were performed on the various virus stocks at either 37°C or 39°C. The plaque assays were carried out as described previously with the following exceptions. The one hour adsorption and 48 hour post-infection incubation were performed at 39°C for one of the assays. The virus titres were calculated for each virus strain at both temperatures and compared. 4.9 Viral Decay Curves In order to determine the decay rates of the various virus strains a sample of each virus stock was incubated at either 37°C or 39°C for ten days. A sample of each strain was removed on days 1, 3, 5, 7.5, and 10 following the start of incubation. The titre of each sample was 4 9 determined by plaque assay and plotted to determine the rate of viral decay. Decay rates were compared between strains and temperatures. 4.10 Viral Growth in Cultured Cardiomyocytes a) Culturing of cardiomyocytes from neonatal mice The hearts were removed from neonatal (<24 hours old) mice (Balb/c or A/J) and placed in a petri dish containing DMEM supplemented with with 10% FCS and 1% antibiotic. The atria were removed and the ventricles transferred to a fresh dish containing a few drops of collagenase. The ventricles were minced in a total volume of 1.5ml of collagenase and were transferred to a small erlenmeyer flask containing a stir bar. The tissue was stirred for 7 minutes at 37°C at which time the liquid was discarded, replaced with 1.5ml of collagenase, and allowed to stir for an additional 7 minutes at 37°C. The digestate was transferred to a tube containing DMEM (10% FCS, 1% antibiotic) and mixed gently to inactivate the collagenase. 1.5ml of both solutions I and II (Section 3.11) were added to the mixture and allowed to stir for 7 minutes at 37°C at which time the digestion was stopped by transferring to a tube containing DMEM (10% FCS, 1% antibiotic). This treatment with solutions I and II was repeated 2-4 further times until the minced tissue was completely digested. The cells were pelleted by centrifugation at <1000g for 3-4 minutes and resuspended in fresh DMEM (10% FCS, 1% antibiotic). Approximately 2ml of medium was used per heart to resuspend the myocytes which were aliquoted into a 35mm petri dish (2ml per dish) and allowed to grow at 37°C in a 5% C0 2 humidified incubator. Neonatal hearts were kindly cultured by Jennifer Kong in Dr. Simon Rabkin's laboratory at the University of British Columbia. 50 b) Infection of Cardiomyocytes Cultured cardiomyocytes from either Balb/c or A/J neonatal mice were grown overnight in 2ml of medium. The following day 1.5 ml of medium was removed from each petri dish and stored at 37°C. Approximately 1000 pfu of either CVB3(RK) or EMI was added to each dish containing 0.5ml of medium and allowed to adsorb for 1 hour at 37°C. Following adsorption the inoculum was removed and each dish was rinsed three times with prewarmed DMEM. The inoculum was replaced with 0.5ml of fresh, prewarmed DMEM (10% FCS, 1% antibiotic) and 1ml of the old medium removed from the cells that morning. A sample dish was taken every three hours for 12 hours. The medium was removed and the cells were washed three times with cold PBS. Following the wash, the cells were scraped into 1ml of cold DMEM and stored at -70°C. The virus titre in each sample was determined by plaque assay and the results plotted in order to compare the rates of viral growth. 4.11 Large Scale Plasmid Preparation Overnight cultures of the bacterial strain carrying the plasmid of interest were grown in 200mls of 2X YT broth containing 150u,g/ml ampicillin at 37°C with shaking. The cells were collected by centrifugation for 10 minutes at 6000rpm in a GSA rotor for a Sorvall centrifuge. The cells were resuspended in 4ml of Solution I (Section 3.10) and treated with 25mg of lysozyme dissolved in 1ml of Solution I for 10 minutes to digest the bacterial cell walls. After cell lysis 10ml of solution II (Section 3.10) was added for 10 minutes (alkaline denaturation phase) followed by 7.5 ml of solution III (Section 3.10)for 20 minutes ( neutralization phase). The cellular debris was pelleted by centrifugation at 9000 rpm for 20 minutes at which point the supernatant was filtered through a cheesecloth into a fresh 51 centrifuge bottle. The nucleic acid was precipitated from the supernatant with isopropanol and pelleted by centrifugation. After being washed in 70% ethanol the pellet was gently dissolved in 3ml of TE pH 8.0. 3ml of 5M LiCl was added to precipitate high molecular weight RNA. Following centrifugation the supernantant was removed to a fresh tube and precipitated with isopropanol. After a 70% ethanol wash the pellet was resuspended in 700ul of TE and an equal volume of 1.6M NaCl containing 13% PEG 8000 was added to precipitate the DNA. The pellet was resuspended in TE and extracted with phenol/chloroform followed by chloroform alone. The DNA was precipitated from the aqueous phase with 3M NaOAc (1/10 volume) and 100% ethanol (2volumes). The pellet was washed with 70% ethanol to remove excess salt, dried and dissolved in lOOul TE. The concentration of the plasmid was determined by electrophoresing lul on a 0.7% agarose gel next to a standard quantity of X, DNA digested with Hind III. 4.12 Preparation of DIG-Labeled RNA Probe The plasmid pCVB3(Rl) (provided by Dr. Reinhard Kandolf) was used as the template for synthesis of both positive and negative strand RNA probes. PCVB3(R1) contains CVB3 cDNA cloned into the EcoRI site of the pSPT18 plasmid flanked by SP6 and T7 RNA polymerase promoters. In vitro transcription was carried out, as described below, using a digoxigenin labeled NTP mixture. Approximately lug of the purified pCVB3(Rl) plasmid was linearized with either Sal I or Sma I for two hours at either 37°C or 25°C respectively. The linear DNA was precipitated using 3M NaOAc (1/10 volume) and 100% ethanol (2 volumes), and resuspended in lOpl of 52 DEPC treated water. The concentration of the linear DNA was determined by electrophoresing a lul sample on a 0.7% agarose gel next to a standard amount of X DNA digested with Hind III. Each in vitro transcription reaction was performed in a final volume of 20ul which contained 50ng of linear DNA, 2ul of a lOx DIG-labeled NTP mixture (lOmM ATP, CTP, GTP and 3.5 mM DIG-labeled UTP), 2ul of a 1 Ox transcription buffer, lul of 0.1 M DTT, lul of RNAse Inhibitor (20 units/ul), and either T7 (used with Sal I linearized DNA) or SP6 (used with Sma I linearized DNA) RNA polymerase. The reaction mixture was incubated for 2 hours at 37°C at which time the DIG-labeled RNA transcript was ethanol precipitated and resuspended in 20ul ofTEpH8.0. 4.13 Quantification of DIG-Labeled RNA Probe In order to perform a calorimetric quantification of the RNA probes a sample of each was serially diluted ten fold five times. Similarly, a DIG-labeled control DNA was serially diluted into samples of known concentration. Each dilution of either RNA probe or control DNA was spotted onto Hybond membrane and UV crosslinked for 2 minutes. The membrane was rinsed in Buffer I (Section 3.9) and incubated for 10 minutes in Buffer II (Section 3.9) with slight agitation. Buffer II was removed and the Anti-DIG-Alkaline Phosphatase conjugate was applied for 10 minutes diluted 1:5000 in Buffer II. The antibody was removed and the membrane washed in Buffer I twice for 10 minutes. Following the Buffer I washes the membrane was equilibrated to pH 9.5 for 2 minutes in Buffer III (Section 3.9). The colour development solution was applied (45ul NBT and 35ul BCIP in 10ml Buffer III) and the membrane allowed to develop for 45 minutes in the dark. After development the membrane 53 was rinsed in distilled water and the concentration of each probe determined by comparison with the standardized control D N A samples. 4.14 In-Situ Hybridization (ISH) Tissues fixed in a 4% paraformaldehyde solution were embedded in paraffin and cut into 4pm sections. The sections were baked overnight at 60°C, deparaffinized in xylene, and rehydrated in graded alcohols of 90%, 70% and 40%. The tissues were permeablilized in the following treatments: 20 minutes in 0.2N HCI, 30 minutes in 2 x SSC at 70°C, 15 minutes in 20mM Tris/2mM C a C l 2 containing lug/ml proteinase K at 37°C, and 10 minutes in 0.25% acetic anhydride containing 0.1M triethanolamine. The slides were then dehydrated using graded alcohols. Once dried, the tissues were treated with 25 ul of hybridization solution containing 20ng/ml of DIG-labeled sense or anti-sense probe. The sections were covered with glass coverslips and incubated in a sealed humidified dish overnight at 42°C. Post-hybridization, the sections were incubated overnight in Wash Buffer at 56°C. This step is critical to remove nonspecific hybridization thereby reducing background staining. This was followed by three 5 minute washes in 2X SSC. The slides were equilibrated for 10 minutes in Buffer 1 (Section 3.8), blocked for 30 minutes in Buffer 2 (Section 3.8), and rewashed for 10 minutes in Buffer 1 (Section 3.8). Each section was incubated with lOOul of anti-DIG-AP (diluted 1:500 in buffer 1 containing 1% lamb serum) 60 minutes at room temperature. The slides were washed with Buffer 1 twice for 5 minutes, and were equilibrated to p H 9.5 in Buffer 3 (Section 3.8) for 10 minutes. The alkaline phosphatase linked anti-DIG antibody was detected by incubation with the colour substrate for 24 hours at room temperature. The slides were counterstained with haematoxylin and eosin and were examined with a light microscope for a positive reaction indicated by a blue-black colour. 54 4.15 Animal Experiments 4 week old A / J mice were purchased form Jackson Laboratories, Maine. The animals were acclimatised for one week following arrival prior to infection. a) Animal Experiment #1: CVB3(RK) Time course In the first experiment 30 A / J mice were divided into 2 groups of 27 and 3 animals (control group) and injected intraperitoneally with the following: Group 1: 105 P F U of C V B 3 ( R K ) in 0.2ml of PBS Group 2: 0.2ml of PBS (control group) Three animals in Group 1 were sacrificed each day for 6 days post infection (by day six p.i. the remaining animals in this group had died). The group 2 animals were sacrificed on day 6 post infection. The animals were anaesthetized with halothane, a blood sample was taken, and the heart, liver, spleen, and pancreas were removed. The tissues were sectioned into 2 pieces; one was fixed in 4% paraformaldehyde for histological analysis while the other was frozen on dry ice for viral titration by plaque assay. The blood samples were allowed to separate overnight at 4°C at which time the serum was removed and the virus titre determined by plaque assay. 55 b) Animal Experiment #2: Infection with CVB3 (RK) and Virus Derived from pCVB3(T7) In the second animal experiment 18 animals were divided into 2 groups of 8 and one control group of 2. The animals were injected intraperitoneally with the following: Group 1: 105 P F U of CVB3(RK) in 0.2ml PBS Group 2: 105 P F U of C V B 3 (T7) in 0.2ml PBS Group 3: 0.2ml of PBS Half of the animals in groups 1 and 2 were sacrificed on either day 3 or 6 post infection. A l l control animals were sacrificed on day 6 post infection. Blood and tissue samples were removed and analyzed as described previously. c) Animal Experiment #3: Infection with CVB3 (RK), EMI and EM10 In the third animal experiment 34 animals were divided into three groups of 10 and one group of four (control group). The animals were injected intraperitoneally with the following: Group 1: 105 P F U of CVB3(RK) in 0.2ml PBS Group 2: 105 P F U of E M I in 0.2ml PBS Group 3: 105 P F U of E M I 0 in 0.2ml PBS Group 4: 0.2ml of PBS Half of the animals from each group were sacrificed on either day 3 or 6 post infection. Blood and tissue samples were removed and analyzed as described above. A sample of the 56 tissues sent for histological analysis were sectioned and mounted on slides in an RNAse-free environment to be analyzed by in-situ hybridization. 4.16 Histological Analysis of Tissue Samples Tissue samples were fixed in 4% paraformaldehyde and sent to the histology laboratory in the Department of Pathology and Laboratory Medicine at the University of British Columbia. Samples were embedded in paraffin, cut into 3 pm sections and mounted on slides. Following mounting the tissue sections were treated with a Masson's trichrome stain which dyes regions of fibrosis, collagen and cartilage blue. 4.17 Titration of Virus in Tissue Samples Frozen tissue samples were weighed and homogenized in 1ml of D M E M in a thick-walled homogenization tube by a pestle attached to an electric drill. The amount of virus per ml was determined by plaque assay which was then converted to a standardized unit of pfu/g. 4.18 DNA Sequencing D N A sequencing was performed by the University of British Columbia's N A P S (Nucleic Ac id and Protein Services) Unit. The samples were sequenced using ABI 's AmpliTaq Dye Terminator Cycle Sequencing chemistry in which all four base reactions take place in a single tube with AmpliTaq; a mutant form of Taq D N A polymerase developed specifically for fluorescent cycle sequencing. 57 4.19 Construction of the pCVB3(KR) Clone Figure 4.1 CVB3(T7) Infectious Clone Restriction Enzyme Map H K KBtBJ ABwBg HA Ba Ba K Ba H K psa PT18 AnpR Kk CVB3 KHndm K:KmI BtrBstI Bl:BlpI BwBsiWI Bg :B^n Ba: BsaBI A: Agsl Figure 4.1 A linear representation of pCVB3(i7). Tn restriction enzyme cleavags sites are labeled The lysine to argjnine rnutation found in EMI i denoted by KR a) Synthesis of the K R P C R Fragment The C V B 3 (T7) infectious clone was digested with K p n I and B s i W I to generate a 1.6 kbp fragment which was used as the template for the P C R reaction in order to reduce mispriming. A 1.2 kbp P C R product was synthesized which contained the A > G mutation responsible for the lysine to arginine mutation found in the V P 2 protein of E M I . The mutation was incorporated into the 3 ' primer as an A>T mutation. The primers and the P C R reaction conditions are listed below: 58 5* Primer (5' nucleotide at position 244): 5'- A C T A C T T C G A A A A A C C T A G T A A C A C - 3' 3' Primer (5' nucleotide at position 1434): 5' - G G A T G C G A C C G G T C T G T C C G C - 3' PCR Reaction Parameters (35 cycles of steps 1-3) 2min. at 95°C 30sec. at 95°C (Step 1: Denaturation) 30sec. a t64°C (Step 2:Annealing) 80sec. at 72°C (Step 3: Extension) The P C R reaction was performed in a 50ul volume containing 5ul of lOx reaction buffer, 250uM dNTPs, lOng of template D N A , 0.6uM of each primer, and 2.5 Units of Deep Vent D N A Polymerase. The 1.2kbp product was purified with a Qiagen P C R Clean-up Kit and digested with Agel and Blpl . b) Construction of the HIII Subcloning Vector The infectious clone pCVB3(T7) was digested with Hind III and Bsa B L The 5.2kbp Hind III fragment was gel purified , religated with T4 D N A ligase, and electroporated into WM1100 electrocompetent E. coli cells (BIO-RAD). The Gene Pulser apparatus (BIO-RAD) was set to the 25uP capacitor, 1.80 kV and the Pulse Controller unit was set to 200Q. After a one minute incubation on ice in the presence of the plasmid D N A , the cells were transferred to a 0.1cm electroporation cuvette and applied one pulse at the above settings. The cells were allowed to recover for 1 hour at 37°C in 1ml of S O C and were plated onto L B plates containing 150ug/ml ampicillin. The plates were incubated overnight at 37°C and the . 5 9 following day colonies were picked, allowed to grow in liquid medium, and the plasmid D N A isolated. c) Construction of the K R Subclone: The 5.2 kbp Hind III subcloning vector was digested with Age I and BlpI and the resulting 4kbp fragment was gel purified. This was dephosphorylated with C l A P (Calf Intestinal Alkal ine Phosphatase for N E B ) and ligated to the 1.2kbp P C R fragment containing the A > G mutation. Ligated D N A was electroporated into WM1100 E. coli cells as described previously. Plasmids obtained from colonies grown on L B +Ampici l l in plates were screened by size and then sequenced to ensure the A > G mutation was present. d) Construction of the p C V B 3 ( K R ) Clone A 1.8kbp fragment containing the A > G mutation was gel purified from the K R subclone digested with B g l II and Bst B L Similarly an 8.8kbp fragment was gel purified and dephosphorylated by C I A P from the full length infectious clone pCVB3(T7) digested with B g l l l and Bst B L These two fragments were ligated with T4 D N A ligase, and subsequently electroporated into WM1100 cells which were plated and grown overnight at 37°C on L B + Ampic i l l in plates. Plasmid D N A was isolated from various colonies and screened by size. The expected size of a full length clone was 10.6kbp. Clones of the appropriate size were screened by sequence analysis to ensure the presence of the A > G mutation. 4.20 Isolation of Virus from cDNA Clones By In Vitro Transcription 5ug of pCVB3(T7) or p C V B 3 (KR) D N A was linearized with Sal I for 3 hours at 37°C. The linear D N A was ethanol precipitated and resuspended in 10p.l of D E P C - d d H 2 0 . A l p l sample 6 0 was separated by electrophoresis on a 0.7% agarose gel next to standards in order to determine the concentration, l p g of the linear D N A was used as the template in an in vitro transcription reaction. The final reaction volume was 20ul and contained l u l R N A s e inhibitor (10U), 2ul of 25mM N T P mixture, 2ul lOx reaction buffer (containing DTT) , 0.5ul (5U) T7 R N A polymerase (NEB) . This was allowed to react for 2 hours at 37°C. Fol lowing transcription the reaction was diluted with 80ul of D M E M . F 1 2 medium which was combined with lOul of Lipofectamine Reagent ( G I B C O - B R L ) diluted in 90 ul of D M E M . F 1 2 medium. The mixture was incubated for 40 minutes at room temperature at which time it was diluted to 1ml with D M E M . F 1 2 containing 0.5% H IFBS and applied to Vero cells (70-80% confluent) pre washed with P B S . Vero cells were treated with the transfection solution for 6-8hours at 37°C in a humidified 5% C 0 2 atmosphere at which time the mixture was removed and replaced with 4ml D M E M . F 1 2 containing 5% H IFB S and 1% gentamicin. The cells were monitored for C P E over the next 4 days. 61 Chapter Five: Experimental Results and Discussion 5.1 Isolation of Antibody Escape Mutants Previous work in the laboratory lead to the isolation of a C V B 3 antibody escape mutant, E M I , which exhibits an amyocarditic phenotype in male A / J mice. E M I replicates 10-50 fold less in the hearts of infected mice while replicating at wild-type levels in pancreatic tissue (Sadeghi, 1997). The objective of this experiment was to repeat the isolation procedure used for E M I in order to isolate a panel of antibody escape mutant (EM) strains which could be compared with E M I and CVB3(RK) (parental strain) in terms of c D N A sequence, in vitro growth characteristics, and pathogenic potential in A / J mice. As described in Chapter 4, the parental wild-type virus stock, CVB3(RK) , was pretreated with a neutralizing monoclonal antibody and the residual non-neutralised fraction was allowed to infect Vero cells. Following the development of C P E , the tissue culture medium containing the E M viruses was assayed for the development of plaques by conventional methods. Two days post infection, the petri dishes were examined for plaque formation and the virus-containing medium beneath the agar overlay was removed from 11 plaques into a tube of fresh D M E M . F 1 2 . These were then used to infect 11 flasks of Vero cells in order to make plaque-purified antibody escape mutant stocks. O f the 11 plaques picked, 9 produced significant C P E in Vero cells. These nine antibody escape mutant stocks were labeled EM2-EM10 . 62 5.2 Viral Sequence Analysis In order to correlate a given viral phenotype, such as the ability to escape neutralization by an antibody or the inability to induce myocarditis, with a specific genotype sequence, comparisons were carried out. The parental strain, CVB3(RK) , virus derived from the C V B 3 infectious clone, pCVB3(T7), E M I , and EM10 were sequenced. In addition the PI region of the remaining escape mutants was also sequenced. As described in the methods section, viral R N A was isolated from E M I - E M I 0 and CVB3(RK) and used in an R T - P C R reaction to generate c D N A copies of the viral genome. This c D N A was used as the template in 5 different P C R reactions in order to amplify the 5'NTR, PI, P2, P3, and 3 'NTR genomic regions [Figure 5.1]. The P C R products were purified using the Qiagen PCR Clean-Up Kit and were sent to the U B C N A P S unit for sequencing. Figure 5.1 PCR Primer Location and Products Figure 5.1 PCR primers and products. (A) PCR primer pairs are indicated by matching color arrow sets. (B) PCR products spanning the following genomic regions: 5'NTR, PI , P2, P3 and 3'NTR. Samples were run on a 0.7% agarose gel next to X D N A digested with Hindl l l as a molecular weight marker (MW). MW 5'NT PI P2 P3 3'NT kbj3 g m II jjjawd mum •I 63 E M I Sequencing Data Initially the sequence of E M I was compared with that of the parental CVB3(RK) strain and is shown in Appendix 1 and Appendix 2. While most of the mutations in E M I are silent (do not change the amino acid sequence), there are three loci at which the amino acid sequence differs from the parental CVB3(RK) strain. There were no nucleotide differences in the 5'or 3' N T R regions. The first two mutations occur in the nonstructural region of the genome. A n A to G transition at nucleotide position 6826 results in an isoleucine to valine mutation at amino acid 309 of the 3D polymerase gene product. Similarly, a T to C transition at nucleotide position 7025 results in a valine to alanine mutation which corresponds to amino acid 375 of the 3D polymerase. Due to the nature in which E M I was isolated, it is unlikely that the mutations in the 3D polymerase are responsible for the antibody escape phenotype. One would expect this i phenotype to be a product of mutations in the structural genes. However, as demonstrated in previous studies (Sadeghi, 1997), E M I has a reduced ability to replicate at 39°C indicated by its small plaque phenotype. Bouchard et al. have demonstrated that some of the determinants of temperature sensitivity in the poliovirus type I Sabin vaccine strain are located in the 3D polymerase gene (Bouchard et al., 1995). Therefore it is possible that these mutations may play a role in the temperature sensitivity phenotype of E M I . The third mutation in E M I , that results in an amino acid change, is located in the VP2 gene of the PI region which encodes the structural gene products. A n A to G transition at nucleotide 1421 produces a lysine to arginine mutation at amino acid 158 of the VP2 protein. Amino acid 158 lies in the E F loop;,the largest, most variable surface loop of VP2 [Figure 5.2]. The E F loop 64 of VP2, otherwise referred to as the 'puff region', comprises amino acid residues 129-180 and is shorter than the puff region described for of poliovirus I (Mahoney strain) (PVI/M) while being slightly longer than that of human rhinovirus 14 (HRV14) (Muckelbauer et al., 1995). The puff loop contains a major neutralizing/immunogenic site in both the polio- and rhinoviruses (Page et al., 1988; Sherry et al., 1986; Sherry et al., 1985). It has been suggested that the puff region plays a role in maintaining the surface topology surrounding the canyon region thereby playing a role in receptor and/or antibody recognition. Therefore, it is likely that this lysine to arginine mutation in E M I is responsible for its antibody escape phenotype. Interestingly, Knowlton et al. have described an antibody escape mutant exhibiting an attenuated myocarditic phenotype which was linked to an asparagine to aspartate mutation at amino acid 165 of VP2; also within the puff region (Knowlton et al., 1996). This would suggest that the puff region may also play a role in determining the tissue tropism and thus virulence characteristics of a given strain. In this light, it is possible that the lysine to arginine mutation is also responsible for the attenuated myocarditic potential of E M I . PI Sequencing Data For Mutants E M 2 - EM10 In order to determine if this lysine to arginine mutation found in VP2 of E M I was also responsible for the antibody-escape phenotype of the other variant strains, the PI region of the mutants EM2-EM10 was sequenced. O f these nine variant strains, six contained the lysine to arginine mutation found in E M I suggesting it plays a strong role in the ability of these virus strains to avoid neutralization by the monoclonal antibody while remaining viable. Two isolates, E M 7 and EM10, contain a novel mutation at nucleotide position 1916. A n A to G transition at 65 this locus results in a glutamate to glycine substitution at amino acid 60 of the VP3 protein [Figure 5.3]. Amino acid residues 58-69 of VP3 form a major protrusion on the capsid surface; referred to as the 'knob' region (Muckelbauer et al., 1995). Like the puff of VP2, the knob of VP3 contains a major neutralization/immunogenic site for both PV I /M and HRV14 (Page et al., 1988; Sherry et al., 1986; Sherry et al., 1985). Thus, this mutation may be responsible for the antibody escape phenotype of E M 7 and EM10. The fact that the lysine to arginine mutation of E M I and the glutamate to glycine mutation of E M 10 both reside in close proximity on external regions of the capsid suggests that the neutralizing monoclonal antibody recognizes a tertiary conformational epitope formed by multiple capsid proteins [Figure 5.4]. It should be noted that the sequence of one isolate, E M 3 , could not be determined through the puff region using the same primers as for E M I . No further analysis of this isolate has occurred. 66 Figure 5.2 Location of the Lysine to Arginine Mutation in the VP2 Protein of EMI Figure 5.2 Computer generated image of the coxsackievirus VP2 protein. The location of the lysine to arginine mutation at amino acid 158 within the VP2 capsid protein of EMI is indicated. Computer images were constructed with the help of Dr. Michael Murphy in the Department of Biochemistry at the University of British Columbia using methods described in (Kraulis, 1991; Merrit, 1997) Figure 5.3 Location of the Glutamate to Glycine Mutation in the VP3 Protein of EM10 Figure 5.3 Computer generated image of the coxsackievirus VP3 protein. The location of the glutamate to glycine mutation at amino acid 60 within the VP3 capsid protein of EM 10 is indicated. The computer image was generated with the help of Dr. Michael Murphy in the Department of Biochemistry at the University of British Columbia 68 Figure 5.4 Location of EMI and EM10 Capsid Mutations Figure 5.4 Computer generated image of a coxsackievirus protomer containing one of each of the viral capsid proteins VP1 (purple), VP2 (green), VP3 (red) and VP4 (yellow). The location of the E M I VP2 lysine to arginine mutation is indicated by the number 158 while the E M 10 VP3 glutamate to glycine mutation is indicated by the number 60 representing the amino acid position within each respective protein. As this image depicts the two mutation sites are in close proximity with one another. The distance spanning the gap between these two residues, as positioned by this computer image, ranges between 6-12 Angstroms depending upon the rotation of the functional groups of the amino acid residues. This image was generated with the help of Dr. Michael Murphy in the Department of Biochemistry at the University of British Columbia. 69 Since both E M 7 and E M 10 contain the same mutation in the PI region, only E M 10 was selected for a complete sequence analysis for comparison with E M I and the parental C V B 3 ( R K ) strain (Appendix 1 and Appendix 2). As with E M I , E M 10 has multiple silent mutations throughout its genome. Unlike E M I it does not contain amino acid mutations within its 3D polymerase gene. Instead, E M 10 contains a C to T nucleotide transition at position 119 of its 5' N T R . Nucleotide 119 lies within stem-loop C of the 5 'NTR according to the secondary structures predicted by the method of Zuker (Yang et al., 1997; Zuker, 1989). Mutations within the 5 'NTR have been shown to affect virulence of several picornaviruses. For example, previous work on poliovirus has defined sites within the 5 'NTR responsible for its neurovirulent phenotype (Evanset al., 1998; LaMonica et A l . , 1987; LaMonica et al., 1986; Omata et al., 1986). More recently, a single nucleotide mutation at position 234 of the C V B 3 5 'NTR has been found to be associated with cardiovirulence (Tu et al., 1995). A U at position 234 is associated with cardiovirulence while a C at this position is associated with an attenuated phenotype. Interestingly the noncardioirulent strain, when compared to the cardiovirulent C V B 3, demonstrated a tenfold lower viral R N A synthesis efficiency while viral protein translation remained at wild-type levels (Tu et al., 1995). The 5' N T R is known to have a role in both viral R N A synthesis and translation due to the stem loop structures which have promoter activity, in addition to their role as an internal ribosome entry site. Extrapolating from this, it is possible that the C to T transition at nucleotide 119 of the 5 'NTR may contribute to the pathogenic phenotype of E M 10. 70 pCVB3(T7) Sequence Data The entire viral sequence of the CVB3 infectious clone, pCVB3(T7), was sequenced for comparison with the published CVB3 sequence(Klump et al., 1990) and that of the wild-type strain CVB3(RK) [Appendix 1 and Appendix 2]. When compared with the sequence of CVB3(RK), pCVB3(T7) contains two mutations which may be of interest. These include a C at position 609 within the IRES of the 5' NTR and an alanine at amino acid residue 375 of the 3D polymerase. These mutations may play a role in the temperature-sensitive phenotype of pCVB3(T7) derived virus discussed in section 5.5. 71 5.3 Monoclonal Antibody Neutralization Assay In order to determine the level of neutralization by the mAb used in the isolation of the escape-mutants, virus stocks of CVB3(RK) , E M I and EM10 were incubated on ice for one hour with or without the addition of the mAb. Following the neutralization step, half of the stock sample incubated with the mAb was treated with Zysorbin for 45 minutes to remove Ab-virus complexes/The titre of each virus sample was determined by plaque assay [Table 5.1]. Table 5.1 Neutralization by the mAb aCVB3-948 Virus Strain Virus Titre pfu/ml Virus + mAb pfu/ml Virus+MAb+Zy sorbin pfu/ml CVB3(RK) 2 x 107 2.5 x 104 <10 3 E M I 5 x l 0 7 5 x l 0 7 3 x l 0 5 EM10 7 x 107 6 x l 0 7 1.5 x l O 6 The wild-type C V B 3 ( R K ) strain is neutralized by the mAbs indicated by the 1000-fold reduction of the stock titre following treatment with the antibody. Unlike the wild-type strain, E M I and E M 10 were not neutralized by the mAb, consistent with the manner by which they were isolated. Viral neutralization is defined as a decrease in infectivity of a given suspension of purified virions. There are several mechanisms whereby viruses may be neutralized by antibodies (Dimmock, 1993). Antibody binding may sterically block the virion from recognizing and/or 72 binding to its specific cell surface receptor. Alternatively, antibody attachment could cause a capsid conformational change which would inhibit the virion's ability to recognize its cell surface receptor. Bivalent antibody binding may cross-link capsid pentameric subunits thereby preventing the conformational changes required for delivery of the viral R N A into the host cell. Finally bivalent inter-virion antibody attachment could result in the aggregation of virions leading to a reduction in the amount of infectious virus particles within a given sample. The fact that the addition of Zysorbin to the virus suspensions, following treatment with the antibody, resulted in a further 10-100 fold reduction in the titre of all strains suggests that some of the virus-antibody complexes are still infectious. In terms of E M I and EM10, this can be interpreted to mean that although the mAb may be able to bind to these strains, it is unable to prevent the conformational changes required for infection 5.4 EMI Replication in Cardiomyocytes In order to determine if the reduced potential of E M I to induce myocarditis in A / J mice was the product of an inability to replicate in heart tissue, cardiomyocytes were cultured from neonatal Balb/c and A / J mice as described in the Methods chapter. The myocytes were infected with either E M I or CVB3(RK) and monitored for viral growth over the course of 24 hours. Samples were taken at 3, 6, 9 and 24 hours post-infection, and the viral titre determined by plaque assay [Figure 5.5 and 5.6]. Figure 5.5 Replication of CVB3(RK) and EMI in Balb/c Cardiomyocytes Fig.5.5 Replication of CVB3(RK) and E M I virus strains in Balb/c cardiomyocytes. Virus was harvested from Balb/c cardiomyocytes at 3,6,9 and 24 hours post infection and titred by plaque assay. Titres are expressed as pfii/ml on a logarithmic scale. Figure 5.6 Replication of CVB3(RK) and EMI in A/J Cardiomyocytes 3 6 9 24 Hours Post Infection — CVB3(RK) — EM1 Fig. 5.6 Replication of CVB3(RK) and E M I virus strains in A/J cardiomyocytes. Virus was harvested from A / J cardiomyocytes at 3, 6, 9 and 24 hours post infection and titred by plaque assay. Titres are expressed as pfu/ml on a logarithmic scale. 74 As illustrated in Figures 5.5 and 5.6 there is no difference between CVB3(RK) and E M I replication in cardiomyocytes cultured from either Balb/c or A / J neonatal mice. Therefore, the reduced level of E M I cardiovirulence cannot be attributed to an inability of this mutant to bind to or replicate in cultured cardiomyocytes. 5.5 Temperature Sensitivity Analysis In order to determine the degree of temperature sensitivity of CVB3(T7) , E M I and EM10 relative to the parental CVB3(RK) strain, a sample of each virus stock was serially diluted and titred by plaque assay at either 37°C or 39°C as described in Chapter Four. The titres were graphed for comparison [Figure 5.7] and the plaque size of each strain at both temperatures was examined [Figure 5.8]. CVB3(T7) exhibits the greatest degree of temperature sensitivity with a 1000-fold reduction in it's titre at 39°C. On the other hand, EM10 appears to grow equally well at both temperatures, while CVB3(RK) and E M I display between 5-10 fold reductions in their titres at the higher temperature. CVB3(RK) and CVB3(T7) exhibit little, if any, reduction in plaque size at 39°C while E M I and E M 10 both display a small plaque phenotype at the higher temperature. This result is suprising. Since CVB3(T7) growth is highly restricted at 39°C, one would expect its plaque size to decrease; however, its plaque size remains unchanged. Meanwhile E M 10 produces similar yields at both temperatures, but displays a small plaque phenotype at 39°C. Thus viral yield, in this case, does not correlate with plaque phenotype. Previously there has been shown to be a loose correlation between virulence and plaque size for picornaviruses (Nakano et al., 1978; Ramsingh et al., 1995) with large-plaque variants being generally more virulent than small-plaque variants. This correlates with the in vivo attenuated 75 phenotypes of E M I and EM10; both of which exhibit small plaques at 39°C (see Section 5.9 for in vivo analysis). This small plaque phenotype may be a product of decreased viral replication at the higher temperature due to the mutations in the 3D polymerase and 5 'NTR of E M I and EM10, respectively. As discussed in the Introduction, both of these regions play important roles in the viral replication cycle, and both have been shown to contain temperature sensitivity determinants in other picornaviruses (Bouchard et al., 1995; Ramsingh et al., 1995). The fact that CVB3(T7) does not exhibit a small-plaque phenotype at either temperature, but displays a 1000-fold reduction in its titre at 39°C suggests this strain may be restricted at the level of viral receptor binding and/or entry into the host cell as a result of the A to G mutation at nucleotide 2024 of VP3 relative to the CB3(RK) sequence which results in an asparagine to serine amino acid change. Binding studies would be required to confirm that the growth restriction of CVB3(T7) occurs at the level of virus entry into the host cell. It is possible that the mutation in VP3 leads to capsid instability at higher temperatures which is manifested as a slight conformational change thereby decreasing the virus's affinity for the cellular receptor. As a result, less virus is able to gain entry into the host cells and the viral titre is reduced. It should be noted that the determinants of attenuation, such as a reduction in myocarditic potential, and temperature sensitivity may be genetically separated (Bouchard et al., 1995). Therefore those mutations in E M I and E M 10 responsible for their small plaque phenotype may not be responsible for their attenuated phenotype in vivo. 76 Figure 5.7 Temperature Sensitivity Assay • 3 7 C • 3 9 C RK T7 EM1 EM10 Virus Strain Figure 5.7 Temperature sensitivity assay comparing CVB3(RK), CVB3(T7), E M I , and E M 10 titres at both 37°C and 39°C. Titres are expressed as pfu/ml on a logarithmic scale and are an average of two identical experiments. 77 Figure 5.8 Viral Plaque Phenotypes at 37°C and 39°C 37°C 39°C CVB3(RK) CVB3(T7) E M I EM10 Figure 5.8 Viral plaque phenotypes of CVB3(RK), CVB3(T7), E M I , EM10 at 37°C and 39°C in Vero cells. Monolayers were stained with Coomassie Blue stain in order to reveal the plaques. 78 5.6 Viral Decay Curve In order to analyze the stability of CVB3(RK), E M I and E M 10, each virus strain was incubated at either 37°C or 39°C for 10 days. Samples were taken prior to incubation and again on days 1, 3, 5, 7, and 10. The virus titre in each sample was determined by plaque assay and plotted against time to determine the rate of inactivation for each strain at both temperatures [Figure 5.9]. When repeated, this experiment produced data exhibiting similar trends as shown in Fig 5.9. Figure 5.9 Viral Decay Curves A. 37°C — CVB3(RK) -"-EMI — EM10 0 + 0 1 3 5 7.5 Timepoint (Days) 79 Figure 5.10 Inactivation of CVB3 (RK) , EMI, and EM10 at 37°C (A) and 39°C (B) over 10 days. Titres are expressed as pfu/ml on a logarithmic scale. All strains decay at a greater rate at the higher temperature. CVB3 (RK) and EMI decay at similar rates when compared to each other while EM 10 is more stable decaying at a slower rate than both CVB3 (RK) and EMI at both temperatures. When compared to each other, CVB3(RK) and E M I decay at approximately the same rate at both temperatures while EM10 appears to be more stable decaying at a slower rate. A l l strains are less stable at 39°C when compared to their decay rates at 37°C. The fact that EM10 is more stable than the parental CVB3(RK) strain may be a product of the glutamate to glycine mutation in its VP2 protein. This mutation may confer additional capsid stability thereby decreasing the rate of viral inactivation. 80 5.7 Timecourse of Infection of Susceptible A/J Mice with CVB3(RK) The objective of this experiment was to determine when the maximal viral titres occurred in various organs during C V B 3 infection of susceptible A / J mice. As described in Chapter 4, male A / J mice were injected intraperitoneally with 105 pfu of CVB3(RK) . Three animals were sacrificed daily for 6 days and serum, heart, spleen and pancreas samples were removed for histopathological assessment and viral titration. Sections stained with Masson's Trichrome stain were examined for signs of tissue damage while viral titres in the organs were determined by plaque assay. Viral titres in selected tissues within each group were expressed as an average and plotted as a bar graph [Figure 5.10]. Both the serum and pancreas displayed maximal viral titres one day post infection while the spleen and heart reached maximal titres two and three days-post infection, respectively. Virus remained at high levels (>105 pfu/g) in the hearts of infected animals up to 6 days post-infection while being cleared to levels beneath detection in the other tissues examined by 3-4 days post-infection. None of the animals survived past six days post-infection indicating the high degree of virulence of the CVB3(RK) strain. These data formed the basis for the remaining animal experiments in which animals were sacrificed on days 3 and 6 post-infection in order to analyze the hearts for maximal viral titres (day 3), myocarditis and the beginning signs of fibrosis (day 6). 81 Figure 5.10 Timecourse of CVB3(RK) Infection HEART SPLEEN Days Post Infection Figure 5.10 Analysis of CVB3(RK) infection in the heart, pancreas, spleen and serum of male A/J mice. Viral titres are expressed as an average value calculated from three animals per timepoint. Error bars express the range of data. 82 Figure 5.11 Histological Assessment of A/J Mice Infected With CVB3(RK) Figure 5.11 Histological assessment of male A/J mice infected with 10$ pfu CVB3(RK). Tissue sections are stained with Masson's Trichrome stain to reveal regions of fibrosis. Panels [A] and [D] display the control pancreas and heart, respectively, from a mouse injected with PBS. Panels [B] and [C] display the pancreas on days 1 and 3 post infection' respectively. Panels [E] and [F] display the heart on days 3 and 6 respectively. It should be noted that 6 days post infection does not provide sufficient time for substantial fibrosis to develop in the hearts of infected animals. As a result there is limited blue staining (indicative of the presence of fibrosis) on these tissue sections. 83 5.8 In Vivo Analysis of CVB3(T7) in Comparison With CVB3(RK) The objective of this experiment was to compare the in vivo effects of infection with virus derived from the infectious clone pCVB3(T7) {CVB3(T7)} with those produced by the wild-type CVB3(RK) strain. Like CVB3(T7) , CVB3(RK) was derived from the infectious clone however it was passaged several times in vitro (in Hela cells) and in vivo (in the hearts of infected mice) in order to enhance its cardiovirulent phenotype. This experiment was therefore carried out to assess the cardiovirulence of the original T7 strain, information that was considered crucial for the analysis of C V B 3 mutants derived by site-specific mutagenesis of the infectious clone (see Section 5.10). As explained in the methods, A / J mice were infected intraperitoneally with 105 pfu of either CVB3(T7) or CVB3(RK) . Animals were sacrificed on days 3 and 6 post infection and serum, heart, pancreas and spleen tissue samples were analyzed by plaque assay to determine the viral titres within each [Figure 5.12]. As shown in the graphs below, there is no significant difference in the ability between CVB3(T7) and CVB3(RK) to replicate in any of the tissues thereby establishing the wild-type phenotype of virus produced from the infectious clone. 84 Figure 5.12 Infection of A/J Mice With CVB3(T7) • CVB3(RK) • CVB3(T7) Spleen Serum Days Post-Infection 6 Days Post-Infection Pancreas "3 Q. O Heart 7- I 6 • w 5 / • 4 / • 3 A • 2 / • 1 0 / A • 6 Days Post-Infection 8 7-6 A 3 5 Q. I o 4 Bo 3 -I J 2 1 0J •1 Days Post-Infection Figure 5.12 Infection of male A/J mice with CVB3(T7) and CVB3(RK). Viral titres in the various tissues from each animal were assayed by plaque assay and expressed as an average for each group. Error bars represent the range of data used to calculate the average. The sensitivity of the plaque assay cannot detect viral titres less than 100 pfu/ml as represented by the dotted line on the graph of serum titres. 85 5.9 Comparison of the Virulence In Vivo of EMI, EM10 and CVB3(RK) The next research objective of this project was to examine the in vivo phenotype of EM 10 as compared to EMI and CVB3(RK). Five week old male A/J mice were injected intraperitoneally with 105 pfu of CVB3(RK), EMI or EM10 and sacrificed on either day 3 or 6 post-infection. The serum, heart, pancreas and spleen were removed from each animal for plaque assay and histological analysis. Serum There is no significant difference between the serum viral titres of the three different strains as shown in Figure 5.13. This is an important control because it indicates an equivalent amount of virus was inoculated into each animal. By day six post-infection serum viral titres have fallen beneath the level of detection. Figure 5.13 Serum Viral Titres • CVB3 (RK) • EM1 • EM10 Day 3 Day 6 Days Post Infection Figure 5.13 Serum viral titres of A/J mice infected with CVB3(RK), E M I or E M 10. Values are expressed as an average calculated from the animals within each group. Error bars represent the range of data. 86 Spleen E M 10 does not show a diminished tropism for the spleen as compared to CVB3(RK) and E M I [Figure 5.14]. A l l three viruses were detected in the spleen at high levels (10 5-10 6 pfu/g) and no significant difference was determined in the amounts of any of the 3 strains analyzed. By day six post-infection the virus was no longer detected in the spleen. Figure 5.14 Spleen Viral Titres 6 5 O) "3 S 4 o at °, 3 6 5 5 5 4 5 3 5 2 • CVB3(RK)| • EM1 • EM10 Days Post-Infection Figure 5.14 Spleen viral titres of A/J mice infected with either CVB3(RK), E M I or EM10. Titres for mice infected with either CVB3(RK) or EM10 are expressed as an average calculated from three similar experiments. Error bars represent the range of data. 87 Pancreas There is no difference in the ability of E M 10 to replicate in the pancreas as compared to C V B 3 ( R K ) and E M I [Figure 5.15]. Histological analysis shows extensive destruction of the pancreas while in-situ hybridization confirms the presence of virus [Figure 5.16]. Figure 5.15 Pancreas Viral Titres • CVB3(RK) • EM1 • EM10 Days Post-Infection Figure 5.15 Pancreas viral titres in A/J mice. Viral titres in animals inoculated with either C V B 3 ( R K ) or E M 10 are expressed as an average calculated from three similar experiments. Error bars represent the range of data. 8 8 Figure 5.16 Histological Analysis and In-Situ Hybridization of Pancreas Infected With Either CVB3(RK) or EM10 Figure 5.16 Sections of pancreas from male A/J mice 3 days post infection. Animals were injected intraperitoneally with 105 pfu of either CVB3 (RK) |A] [C] or EM10 [B] [D]. The upper panels display sections stained with Masson's Trichrome. The lower panels display sections analyzed by in-situ hybridization in which the dark staining regions indicate the presence of viral genome in the tissue. There is a minimal difference in the level of pancreas damage caused by either CVB3(RK) or E M 10 as indicated by this histological and immunohistochemical analysis. This is consistent with the pancreas viral titre data. 89 Heart The heart tissue samples contain 100-fold lower titres of E M 10 as compared to wild-type C V B 3 ( R K ) levels on day three and 100-fold lower levels on day 6 post infection. E M I exhibits an intermediary phenotype between C V B 3 ( R K ) and E M 10 producing approximately a tenfold lower titre than wild-type levels on both days 3 and 6 post infection [Figure 5.17]. These data suggest that E M 10 replicates to a lower level in the hearts of infected mice and is cleared from the cardiac tissue quicker than the other two strains. Histological analysis supports this conclusion as more extensive myocyte damage is observed in the animals infected with C V B 3 ( R K ) [Figure 5.18]. Figure 5.17 Heart Viral Titres o • CVB3(RK) • EM1 • EM10 3 6 Days Post-Infection Figure 5.17 Heart viral titres of animals infected with either C V B 3 ( R K ) , E M I or EM10. Titres from animals inoculated with either C V B 3 ( R K ) or E M 10 are expressed as an average calculated from three similar experiments. Error bars represent the range of data. 90 Figure 5.18 Histological Analysis and In Situ Hybridization of Heart Infected With Either CVB3(RK) or EM10 Figure 5.18 Sections of heart from male A/J mice 6 days postinfection. Animals were injected intraperitoneally with 10^ pfu of either CVB3(RK) [A] |C] or EM10 [B] [D]. The upper panels display sections stained with Masson's Trichrome stain. Those regions stained blue identify regions of fibrosis within the tissue. By day 6 post infection there is minimal fibrosis present in the hearts of animals infected with either CVB3(RK) or EM10. The bottom panels display sections of heart analyzed by in-situ hybridization. Animals infected with CVB3(RK) [C] contain significantly more viral genome in their hearts, as indicated by the dark blue-black staining, than those animals infected with EM 10 [D). The lack of viral genome in the hearts of animals infected with EM 10 is consistent with the tissue titre data. 91 5.10 Site-Directed Mutagenesis of pCVB3(T7) As shown in Section 5.8, the infectious clone, pCVB3(T7), produces a wild-type infection in the animal model associated with equivalent myocarditis to that found for CVB3(RK) . This finding opened up the possibility of engineering mutations in this clone, and studying their effects in the progeny viruses produced. Our objective was therefore to perform site-directed mutagenesis on the infectious clone in order to incorporate the E M I lysine to arginine mutation, found within the VP2 coding region, and to study its effects on the resulting progeny virus. In this way it was hoped to correlate a single mutation with a given phenotype such as the reduced ability to replicate in the heart leading to the limited development of myocarditis. As described in the methods, an A to G mutation responsible for the lysine to arginine mutation was incorporated into a P C R primer used to generate a 1.2 kbp P C R product. This was subsequently ligated into a 5.2 kbp subcloning vector, derived from pCVB3(T7) , to produce the K R subclone which was sequenced to ensure the presence of the A to G mutation. The strategy to incorporate the A to G mutation into a subclone instead of directly into the full length infectious clone was a product of the minimal number of unique restriction sites in pCVB3(T7); a reflection of the size of the coxsackievirus genome. Following the generation of the K R subclone, a 1.8 kbp fragment was cut out of the full length infectious clone pCVB3(T7), and replaced with the corresponding region from the K R subclone containing the A to G mutation [Figure 5.19]. The ligation mixture was electroporated into E.coli cells which were grown overnight to produce colonies which 92 maintain plasmid DNA containing the A to G mutation. Plasmid DNA was isolated from multiple colonies and subjected to size analysis. Twelve clones which were the correct size (10.6kbp) were sent for sequence analysis to ensure the presence of the A to G mutation. A 10.6 kbp clone containing the appropriate DNA sequence was used as the template in an in vitro transcription reaction to produce a full length RNA transcript of the CVB3 genome containing the A to G mutation which was subsequently transfected into Vero cells. Despite multiple attempts using varying amounts of RNA transcript and lipofectamine reagent, the KR clones constructed were unable to generate infectious virus. The lack of infectivity of the KR clone may be explained in several ways. The A to G mutation, by itself, engineered onto the wildtype background of pCVB3(T7) may be lethal. This is very unlikely since EMI contains this mutation and is still viable. Alternatively, during the engineering process a lethal mutation may have occurred at a site distant from position 1421 which was not detected during sequence analysis of this region. This is the more likely explanation for the noninfectious phenotype of pCVB3(KR), and may only be confirmed by a complete sequence analysis of the entire clone including the plasmid region containing the transcription promoters and the antibiotic resistance genes. 93 Figure 5.19 A. Strategy for pCVB3(KR) Construction Kpnl BstBI Agel BsiWI Kpnl Digest with Kpnl and BsiWI Digest with Hindll l and ligate 5.2kbp fragment Kpnl BlpI BlpI BsiWI PCR A>G Digest with Agel and BlpI Agel Ligate PCR product and digested HIII subclone Kpnl Hindlll Kpnl , Hindlll' Kpnl BstBI Agel BsiWI Bglll Digest HIII subclone with Agel and BlpI. Ligate with PCR product. Kpnl jstBI T7 BlpI rAMPR KR Subclone 5.2kpb •BsiWI 94 Digest with Bglll and BstBI. Purify 1.8 kbp fragment and ligate with the 8.8 kbp fragment cut out from pCVB3(T7) Digest with Bglll and BstBI. Purify 8.8kbp fragment and ligate with the 1.8 kbp fragment cut out from the HIII subclone Figure 5.19 Cloning strategy for introducing the A>G mutation found in the VP2 gene of E M I into the coxsackievirus infectious clone pCVB3(T7). A: Strategy for the construction of the K R subclone. B: Strategy for the construction of the full length K R clone pCVB3(KR). 95 Chapter Six: Conclusions The long term objective of this research was to identify attenuating mutations within the C V B 3 genome which one day may be utilized in the development of a vaccine. Previous work in the laboratory has led to the isolation of an antibody escape mutant, E M I , which exhibits an amyocarditic phenotype producing approximately tenfold lower viral titres in the hearts of infected animals. Sequence analysis of the entire E M I genome has identified three point mutations resulting in amino acid changes as compared to the sequence of the wild-type strain CVB3(RK) . The first results in an arginine residue at amino acid 158 within the puff region of VP2. This residue lies within a neutralizing/immunodominant site, and is likely the site responsible for the antibody escape phenotype of E M I . It is also likely that this mutation is responsible for the reduction in the myocarditic potential of E M I . While E M I produces lower cardiac viral titres and induces limited myocarditis in vivo, it replicates to wild-type levels in cultured murine cardiomyocytes which suggests the reduced level of E M I cardiovirulence cannot be attributed to an inability to bind to or replicate in the cultured cardiac cells. Alternate explanations for the difference in tissue tropism between CVB3(RK) and E M I may relate to differences in affinity of each strain for the viral receptor on the cardiomyocyte in the context of the organ in which there is connective tissue around each cell. It is not uncommon for cells derived from a tissue to demonstrate a higher degree of permissiveness for virus than the organ in vivo. For example Yoon et al. have demonstrated that C V B 3 has the capacity to infect beta cells in human pancreatic cell cultures while the endocrine pancreas is generally not damaged during an in vivo infection (Yoon et al., 1978). Unfortunately it is not possible to produce organ cultures of heart tissue to test this idea. A n 96 alternative explanation may lie in the heterogeneity of the vascular endothelial cells (VEC) found throughout the body. Vascular endothelial cells act as a barrier between the vascular space and the organs within the body. VEC are heterogeneous displaying unique properties depending upon the organ in which they are found (Kumar et al., 1987; Turner et al., 1987). For example, expression of CVB3 receptor molecules has been shown to vary between endothelial cells derived from the heart, liver and lung (Huber et al., 1990). Similarly CVB3 virus isolated from either the heart or liver of infected mice replicated to greater levels in VEC isolated from the same tissue (Huber et al., 1990; Lodge et al., 1991). This suggests that VEC play a role in determining viral tissue tropism since the virus must pass through the vascular endothelial layer in order to gain access to the various organs throughout the body. In this regard, it is possible that the lysine to arginine in VP2 of EMI causes a capsid conformational change which restricts the ability of the virus to gain entry into the heart via the cardiac vascular endothelial cells. This limited access manifests as an inability to induce myocarditis to the same degree as the parental CVB3(RK) strain. The other two mutations in EMI result in a valine and an alanine at amino acids 309 and 375 of the 3D polymerase enzyme, respectively. EMI is temperature-sensitive at 39°C indicated by its small plaque phenotype at an elevated temperature. Valine 309 may be responsible for this phenotype as temperature-sensitivity determinants occur within this region of the poliovirus genome (Bouchard et al., 1995). Following the characterization of EMI a panel of antibody escape mutants was isolated (EM2-EM10) and their PI regions sequenced. Six of the nine variants contain the same 97 arginine 158 mutation as found in EMI. This adds further support to the theory that this residue lies within a neutralizing epitope on the capsid surface. In contrast, EM7 and E M 10 contain a novel mutation resulting in a glycine at amino acid 60 within the knob region of VP3. This locus also resides within a neutralizing epitope on the capsid surface, and is likely responsible for the antibody escape phenotype of these two variant strains. The close juxtaposition of arginine 158 and glycine 60 on the predicted three dimensional surface of the capsid provides an insight into the monoclonal antibody binding site. Previous work in the laboratory was aimed at characterizing the epitope recognized by the monoclonal antibody used in the isolation of EMI -EM 10 by immunoprecipitation assays. Data obtained from these experiments were inconclusive since all three capsid proteins (VP 1-3) coprecipitated. Furthermore, the monoclonal antibody did not detect any viral protein on Western blots [Kavoosi and Chantler unpublished observation]. These facts taken together with the position of the VP2 and VP3 mutations of EMI and E M 10 suggest that the monoclonal antibody recognizes a three dimensional conformational epitope formed by the interaction of multiple capsid subunits. Complete sequence analysis of E M 10 identified a second mutation at nucleotide position 119 of the 5' NTR. As discussed in the Introduction, the 5'NTR plays an important role in the replicative cycle of picornaviruses. It contains the internal ribosomal entry site (IRES) essential for translation of the viral genome, and hence production of the proteins required for viral replication. In addition, a ribonucleoprotein complex is thought to form on the cloverleaf structures of the 5'NTR thereby initiating viral plus strand RNA synthesis (discussed in Section 1.10 of the Introduction). In this light, it is conceivable that mutations 98 within this region could restrict the ability of a virus to replicate which could be manifested as an attenuated pathogenic phenotype. Like E M I , E M 10 produces small plaques at 39°C indicative of a temperature- sensitive phenotype. Temperature-sensitivity determinants occur within the 5 'NTR of the C V B 4 genome (Ramsingh et al., 1995) therefore it is not unlikely that the mutation in the 5 'NTR of E M 10 may also contribute to its small plaque formation at 39°C. Infection of mice with either CVB3(RK) , E M I or EM10 confirmed the reduced myocarditic potential of E M I . More interestingly these experiments revealed that E M 10 has an even greater attenuated myocarditic phenotype producing 100-1000 fold lower viral titres in the hearts of infected mice. While EM10 is evidently amyocarditic, it produces an extensive infection within the pancreas and spleen. This suggests its restricted tissue tropism is limited to cardiac tissue. Like E M I this may be a product of the differences in the vascular endothelial cells within the various organs throughout the body. Previous studies have shown that attenuating determinants leading to an amyocarditic phenotype lie within the 5 'NTR and the PI regions of the C V B 3 genome. Tu et al. have identified a C to U transition at nucleotide position 234 of the 5 'NTR responsible for the cardiovirulent phenotype of a C V B 3 strain (Tu et al., 1995). In addition, Knowlton et al. have identified an asparagine to aspartate mutation at amino acid 165 within the puff region of VP2 as being responsible for the amyocarditic phenotype of an isolated C V B 3 antibody-escape mutant (Knowlton et al., 1996). Interestingly this is only 7 residues upstream from the lysine to arginine mutation found in the VP2 protein of E M I . Therefore it is likely that the 99 amyocarditic phenotype of E M I and E M 10 are a product of their mutations within the PI and 5 'NTR regions of the genome. This project has identified several potentially attenuating mutations within the 5 'NTR, PI and P3 regions of the C V B 3 genome. In order to determine the individual effects these mutations play in the development of an attenuated C V B 3 pathogenic phenotype, each mutation will need to be engineered onto a wild-type genetic background and the resulting virus analyzed for its in vitro and in vivo characteristics. In an attempt to characterize the arginine 158 mutation of E M I site directed mutagenesis was performed on the C V B 3 infectious clone pCVB3(T7) thereby incorporating an A to G transition responsible for the lysine to arginine mutation. This was unsuccessful in that infectious virus could not be recovered from the full length K R clone, possibly a product of a lethal mutation event distant from nucleotide position 1421. In the future, protocols intended to engineer clones containing these mutations should be designed in an effort to reduce the number of steps which could result in the incorporation of an additional mutation. Regardless of the fact that the in vitro mutagenesis did not produce an infectious clone, the limited number of mutations identified in E M I and EM10 make the 5 'NTR and the PI loci strong candidates for attenuating determinants. Further analysis of these mutants, such as their ability to replicate in cultured vascular endothelial cells from different organs will no doubt give an insight into their mechanisms of pathogenesis. As discussed in the Introduction, the study of pathogenic variants, such as E M I and EM10, provides the framework for understanding the molecular determinants of viral pathogenesis. 100 Currently there are no effective antivirals for the prevention or treatment of enteroviral diseases, and the only enterovirus vaccines available are those for the three types of polioviruses. The development of C V B vaccines will require the identification of multiple attenuating mutations within each type ( C V B 1-6). As has been shown for the live poliovirus vaccines, the necessity for multiple attenuation sites is a result of mutations which may revert the virus back to its virulent phenotype (Melnick, 1962). 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Science, 244, 48-52. 113 Appendix I: Sequence Comparison (The numbered row is the published C V B 3 sequence (Klump, 1990) 1 TTA AAA CAG CCT GTG GGT TGA TCC CAC CCA CAG GCC CAT TGG GCG R K T7 EMI EM10 46 CTA GCA CTC TGG TAT CAC GGT ACC TTT GTG CGC CTG TTT TAT ACC R K : T 7 _ EMI • : EM10 91 CCC TCC CCC AAC TGT AAC TTA GAA GTA ACA CAC ACC GAT CAA CAG R K T 7 _ EMI : '• EM10 : . -- ATA : 136 TCA GCG TGG CAC ACC AGC CAC GTT TTG ATC AAG CAC TTC TGT TAC R K — T 7 — . : _ — EMI • : EM10 : • 1 8 1 CCC GGA CTG AGT ATC AAT AGA CTG CTC ACG CGG TTG AAG GAG AAA R K T 7 : _ EMI EM10 — 2 2 6 GCG TTC GTT ATC CGG CCA ACT ACT TCG AAA AAC CTA GTA ACA CCG R K T 7 _ EMI EM10 2 7 1 TGG AAG• TTG CAG AGT GTT TCG CTC AGC ACT ACC CCA GTG TAG ATC R K • T 7 _ EMI EM10 3 1 6 AGG TCG ATG AGT CAC CGC ATT CCC CAC GGG CGA CCG TGG CGG TGG R K '• T 7 . _ EMI EM10 3 6 1 CTG CGT TGG CGG CCT GCC CAT GGG GAA ACC CAT GGG ACG CTC TAA R K .. . - - -T 7 . .__._ EMi --- : EM10 : ______ 4 0 6 TAC AGA CAT GGT GCG AAG, AGT CTA TTG AGC TAG TTG GTA GTC CTC R K - - — — : : T 7 _ : _ _ ;__ EMI '-• ______ EM10 -114 4 5 1 CGG CCC CTG AAT GCG GCT AAT CCT AAC TGC GGA GCA CAC ACC CTC .RK : : T 7 :__ _ : ________ EMI : '• • E M 1 0 4 9 6 AAG CCA GAG GGC AGT GTG TCG TAA CGG GCA ACT CTG CAG CGG AAC RK T 7 EMI EMIO 5 4 1 CGA CTA CTT TGG GTG TCC GTG TTT CAT TTT ATT CCT ATA CTG GCT RK T 7 EMI • EMIO 5 8 6 GCT TAT GGT GAC AAT TGA GAG ATC GTT • ACC ATA TAG CTA TTG GAT RK ATT - : T 7 EMI ATT • EMIO : ; ATT 6 3 1 TGG CCA TCC GGT GAC TAA TAG AGC TAT TAT ATA TCC CTT TGT TGG RK T 7 EMI EMIO 676 GTT TAT ACC ACT TAG CTT GAA AGA GGT TAA AAC ATT ACA ATT CAT RK T 7 EMI EMIO 7 2 1 TGT TAA GTT GAA TAC AGC AAA ATG GGA . GCT CAA GTA TCA ACG CAA RK T 7 _ EMI EMIO 7 6 6 AAG ACT ' GGG GCA CAT GAG ACC AGG CTG AAT GCT AGC GGC AAT TCC RK T 7 EMI : '• EMIO 8 1 1 ATC ATT CAC TAC ACA AAT ATT AAT TAT TAC AAG GAT GCC GCA TCC RK • T 7 EMI EMIO .--8 5 6 AAC TCA GCC AAT CGG CAG GAT TTC ACT CAA GAC CCG GGC AAG TTC RK • T 7 EMI EMIO 115 9 0 1 ACA GAA CCA GTG AAA GAT ATC ATG ATT AAA TCA CTA CCA GCT CTC RK GTA T 7 EMI GTA - —-EM10 GTA 9 4 6 AAC TCC CCC ACA GTA GAG GAG TGC GGA TAC AGT GAC AGG GCG AGA RK • T 7 — _ : _ — EMI -- ' EM10 • 9 9 1 TCA ATC ACA TTA GGT AAC TCC ACC ATA ACG ACT CAG GAA TGC GCC RK • — T 7 : EMI EM10 1 0 3 6 AAC GTG GTG GTG GGC TAT GGA GTA TGG CCA GAT TAT CTA AAG GAT RK : : T 7 . . _ EMI - -EM10 1 0 8 1 AGT GAG GCA ACA GCA GAG GAC CAA CCG ACC CAA CCA GAC GTT GCC RK T7 . : EMI EM10 1 1 2 6 ACA TGT AGG TTC TAT ACC CTT GAC TCT GTG CAA TGG CAG AAA ACC RK :--T 7 _ — EMI EM10 1 1 7 1 TCA CCA GGA TGG TGG TGG AAG CTG CCC GAT GCT TTG TCG AAC TTA RK T 7 EMI EM10 1 2 1 6 GGA CTG TTT GGG CAG AAC ATG CAG TAC CAC TAC TTA GGC CGA ACT RK T 7 EMI EM10 1 2 6 1 GGG TAT ACC GTA CAT GTG CAG TGC AAT GCA TCT AAG TTC CAC CAA RK T 7 _ EMI : E M 1 0 1 3 0 6 GGA TGC TTG CTA GTA GTG TGT GTA CCG GAA GCT GAG ATG GGT TGC RK GTG T 7 EMI GTG EM10 GTG 116 1 3 5 1 GCA ACG CTA GAC AAC ACC CCA TCC AGT GCA GAA TTG CTG GGG GGC RK • T 7 _ _ _ _ _ _ _ •____ . EMI EMIO '• 1 3 9 6 GAT ACG GCA AAG GAG TTT GCG GAC AAA CCG GTC GCA TCC GGG TCC RK AGC AAA T7 - - - - AGC EMI AGC. AAA AGA - - . EMIO AGC AAA _ 4 4 1 AAC AAG TTG GTA CAG AGG GTG GTG TAT AAT GCA GGC ATG GGG GTG RK : T 7 EMI . EMIO 1 4 8 6 GGT. GTT GGA AAC CTC ACC ATT TTC CCC CAC CAA TGG ATC AAC CTA RK • T 7 . EMI '• : EMIO 1 5 3 1 CGC ACC AAT AAT AGT GCT ACA ATT GTG ATG CCA TAC ACC AAC AGT RK = T 7 EMI EMIO 1 5 7 6 GTA . CCT ATG GAT AAC ATG TTT AGG CAT AAC AAC GTC ACC CTA ATG RK • T 7 EMI ; EMIO • 1 6 2 1 GTT ATC CCA T T T GTA CCG CTA GAT TAC TGC CCT GGG TCC ACC ACG RK '• T 7 : EMI ; • EMIO 1 6 6 6 TAC GTC CCA ATT ACG GTC ACG ATA GCC CCA ATG TGT GCC GAG TAC RK T 7 : EMI EMIO 1 7 1 1 AAT GGG TTA CGT TTA GCA GGG CAC CAG GGC TTA CCA ACC ATG AAT RK AAC T 7 EMI AAC EMIO AAC 1 7 5 6 ACT CCG GGG AGC TGT CAA T T T CTG ACA. TCA GAC GAC TTC CAA TCA RK • T 7 ; . EMI • ' EMIO ' 117 1 8 0 1 CCA TCC GCC ATG CCG CAA TAT GAC GTC ACA CCA GAG ATG AGG ATA RK T 7 EMI EM10 : 1 8 4 6 CCT GGT GAG GTG AAA AAC TTG ATG GAA ATA GCT GAG GTT GAC TCA RK T 7 EMI EM10 1 8 9 1 GTT GTC CCA GTC CAA AAT GTT GGA GAG AAG GTC AAC TCT ATG GAA RK T 7 EMI EM10 666 1 9 3 6 GCA TAC CAG ATA CCT GTG AGA TCC AAC GAA GGA TCT GGA ACG CAA RK EMI : T 7 _ EM10 1 9 8 1 GTA TTC GGC T T T CCA CTG CAA CCA GGG TAC TCG AGT GTT T T T AGT RK 1 AAT T 7 EMI - . AAT EM10 AAT 2 0 2 6 CGG ACG CTC CTA GGA GAG ATC TTG AAC TAT TAT ACA CAT TGG TCA RK T 7 EMI ; : EM10 2 0 7 1 GGC AGC ATA AAG CTT ACG TTT ATG TTC TGT GGT TCG GCC ATG GCT RK • -T 7 EMI EM10 2 1 1 6 ACT GGA AAA TTC CTT TTG GCA TAC TCA CCA CCA GGT GCT GGA GCT RK T 7 EMI • : EM10 -2 1 6 1 CCT ACA AAA AGG GTT GAT GCT ATG CTT GGT ACT CAT GTA ATT TGG RK : GCC T 7 _ : . EMI •— GCC ' EM10 GCC 2 2 0 6 GAC GTG GGG CTA CAA TCA AGT TGC GTG CTG TGT ATA CCC TGG ATA RK T 7 EMI : EM10 118 2 2 5 1 A G C C A A A C A C A C T A C C G G T T T G T T G C T T C A G A T G A G T A T A C C G C A R K — : T 7 • _ _ _ _ E M I • G C G E M I O - — ; - - . — 2 2 9 6 G G G G G T T T T A T T A C G T G C T G G T A T C A A A C A A A C ' A T A G T G G T C C C A R K •• T 7 : _ . : ; : _ _ E M I • : — '• - - • E M I O ; ' • • 2 3 4 1 G C G G A T G C C C A A A G C T C C " T G T T A C A T C A T G T G T T T C G T G T C A G C A R K . : : - . T 7 ! _ _ _ E M I • '• — . — ; G C G E M I O - - : '- — : 2 3 8 6 T G C . A A T G A C T T C T C T G T C A G G C T A T T G A A G G A C A C T C C T T T C A T T R K ' ' _ _ T 7 :—___ _ _ ___ E M I : - - . E M I O : — — , — - . • - : : - -2 4 3 1 T C G C A G C A A A A C T T T T T C C A G G G C C C A G T G G A A ' G A C G C G A T A A C A R K - - - - - — . - : : _ _ T 7 ___ . : ._: __ E M I . . E M I O — — : '• — • 2 4 7 6 G C C G C T A T A G G G A G A G T T G C G G A T A C C G T G G G T A C A G G G C C A A C C R K : . : - - • T7: • • — — — ; • ; - -E M I ' - • : • — E M I O — • - 2 5 2 1 A A C T C A G A A G C T A T A C C A ' G C A C T C A C T G C T G C T G A G A C G G G T C A C R K T 7 _ E M I • : E M I O ; • ' • -2 5 6 6 A C G T C A C A A G T A G T G C C G G G T G A C A C T A T G C A G A C A C G C C A C G T T R K T7 ' ' ; • • ' E M I . • -r : E M I O • — : — 2 6 1 1 ' A A G A A C T A C C A T T C A A G G T C C G A G T C A A C C A T A G A G A A C T T C C T A R K : : ••: - — T 7 . _ :_ _ E M I ' . • • : : E M I O - • : _ _ 2 6 5 6 T G T A G G T C A G C A T G C G T G T A C T T T A C G G A G T A T A A A A A C T C A G G T R K • T 7 _ ' E M I , ; ' — E M I O " - - - • 119 2701 GCC AAG CGG TAT GCT GAA TGG GTA TTA ACA CCA CGA CAA GCA GCA RK T 7 __ . EMI GAG EM10 '• 2746 CAA CTT AGG AGA AAG CTA GAA TTC TTT ACC TAC GTC CGG TTC GAC RK T 7 EMI EM10 2791 CTG GAG CTG ACG TTT GTC ATA ACA AGT ACT CAA CAG CCC TCA ACC RK T 7 : EMI ' EM10 2836 ACA CAG AAC CAA GAT GCA CAG ATC CTA ACA CAC CAA ATT ATG TAT RK T 7 EMI EM10 2881 GTA CCA CCA GGT GGA CCT GTA CCA GAT AAA GTT GAT TCA TAC GTG RK T 7 EMI EM10 2926 TGG CAA ACA TCT ACG AAT CCC AGT GTG TTT TGG ACC GAG GGA AAC RK T 7 EMI EM10 2971 GCC CCG CCG CGC ATG TCC ATA CCG TTT TTG AGC ATT GGC AAC GCC RK T 7 EMI EM10 3016 TAT TCA AAT TTC TAT GAC GGA TGG TCT GAA TTT TCC AGG AAC GGA RK T 7 EMI EM10 3061 GTT TAC GGC ATC AAC ACG CTA AAC AAC ATG GGC ACG CTA TAT GCA RK T 7 EMI : EM10 3106 AGA CAT GTC AAC GCT GGA AGC ACG GGT CCA ATA AAA AGC ACC ATT RK T 7 _ EMI EM10 120 3 1 5 1 AGA ATC TAC TTC AAA CCG AAG CAT GTC AAA GCG TGG ATA CCT AGA R K T 7 _ E M I EM10 3 1 9 6 CCA CCT AGA CTC TGC CAA TAC GAG AAG GCA AAG AAC GTG AAC TTC R K • T 7 E M I EM10 3 2 4 1 CAA CCC AGC GGA GTT ACC ACT ACT AGG CAA AGC ATC ACT ACA ATG R K • T 7 _ E M I EM10 3 2 8 6 ACA AAT ACG GGC GCA TTT GGA CAA CAA TCA GGG GCA GTG TAT GTG R K T 7 _ : E M I : EM10 3 3 3 1 GGG AAC TAC AGG GTG GTA AAT AGA CAT CTA GCT ACC AGT GCT GAC R K T 7 __ E M I EM10 3 3 7 6 TGG CAA AAC TGT GTG TGG GAA AGT TAC AAC AGA GAC CTC TTA GTG R K T 7 E M I ' EM10 - ^ : 3 4 2 1 AGC ACG ACC ACA GCA CAT GGA TGT GAT ATT ATA GCC AGA TGT CAG R K - - ' — • T 7 __ . E M I -EM10 — 3 4 6 6 TGC ACA ACG GGA GTG TAC TTT TGT GCG TCC AAA AAC AAG CAC TAC R K . T 7 . _ : E M I EM10 '• 3 5 1 1 CCA ATT TCG TTT GAA GGA CCA GGT CTA GTA GAG GTC CAA GAG AGT R K T 7 : _ E M I - -EM10 : 3 5 5 6 GAA TAC TAC CCC AGG AGA TAC CAA TCC CAT GTG CTT TTA GCA GCT R K T 7 E M I : -EM10 121 601 GGA TTT TCC GAA CCA GGT GAC - TGT GGC GGT ATC CTA AGG TGT GAG RK T 7 _ EMI : EMIO i 3646 CAT GGT GTC ATT GGC ATT GTG ACC ATG GGG GGT GAA GGC GTG GTC RK . . T 7 EMI : EMIO 3691 GGC TTT GCA GAC ATC CGT GAT CTC CTG TGG CTG GAA GAT GAT GCA RK '• T 7 EMI : EMIO 3736 ATG GAA CAG GGA GTG AAG GAC TAT GTG GAA CAG CTT GGA AAT GCA RK '• T 7 . EMI . EMIO : , 3781 TTC GGC TCC GGC T T T ACT AAC CAA ATA TGT GAG CAA GTC AAC CTC RK" ' T 7 : : _ _ EMI EMIO 3826 CTG AAA GAA TCA CTA GTG GGT ' CAA GAC TCC ATC TTA GAG AAA TCT RK • T 7 . EMI : : - -EMIO — 3871 CTA AAA GCC TTA GTT AAG ATA ATA TCA GCC TTA GTA ATT GTG GTG RK TTG • T 7 : -r EMI TTG EMIO TTG ; — 3916 AGG AAC CAC GAT GAC CTG ATC ACT GTG ACT GCC ACA CTA GCC CTT RK - -T 7 . _ _ _ : EMi — ; • • EMIO • • 3961 ATC GGT TGT ACC TCG TCC CCG TGG CGG TGG CTC AAA CAG AAG GTG RK : • T 7 _ :'. EMI • • ; • EMIO : '• : •— ; 4006 TCA CAA TAT TAC GGA ATC CCT ATG GCT GAA CGC CAA AAC AAT AGC RK -T 7 _ _ _ _ _ _ _ _ _ _ _ EMI EMIO . 122 4 0 5 1 TGG CTT AAG AAA T T T ACT GAA ATG ACA AAT GCT TGC AAG GGT ATG RK ACG T 7 EMI ACG : EM10 ACG 4 0 9 6 GAA TGG ATA GCT GTC AAA ATT CAG AAA TTC ATT GAA TGG CTC AAA RK : T 7 EMl — : EM10 : 4 1 4 1 GTA AAA ATT TTG CCA GAG GTC AGA GAA AAA CAC GAG TTC CTG AAC RK AGG T 7 EMI . AGG EM10 • AGG • 4 1 8 6 AGA CTT AAA CAA CTC CCC TTA TTA GAA AGT CAG ATC GCC ACA ATC RK • T 7 EMI EM10 4 2 3 1 GAG CAG AGC GCG CCA TCC CAA AGT GAC CAG GAA CAA TTA T T T TCC RK ' T 7 EMI EM10 4 2 7 6 AAT GTC CAA TAC T T T GCC CAC TAT TGC AGA AAG TAC GCT CCC CTC RK T 7 EMI EM10 4 3 2 1 TAC GCA GCT GAA GCA AAG AGG GTG TTC TCC CTT GAG AAG AAG ATG RK T 7 EMI EM10 4 3 6 6 AGC AAT TAC ATA CAG TTC AAG TCC AAA TGC CGT ATT GAA CCT GTA R K . T 7 _ EMI EM10 '• 4 4 1 1 TGT TTG CTC CTG CAC GGG AGC CCT GGT GCC GGC AAG TCG GTG GCA RK _ _ _ _ _ T 7 EMI EM10 4 4 5 6 ACA AAC TTA ATT GGA AGG TCG CTT GCT GAG AAA CTC AAC AGC TCA RK T 7 EMI EM10 123 4501 GTG TAC TCA CTA CCG CCA GAC CCA GAT CAC TTC GAC GGA TAC AAA RK CTG - -T7- .-. EMI '• CTG : -EM10 CTG 4546 CAG CAG GCC GTG GTG ATT ATG GAC GAT CTA TGC CAG AAT CCT GAT RK T 7 EMI - ; EM10 : 4591 GGG AAA GAC GTC TCC TTG TTC TGC CAA ATG GTT TCC AGT GTA GAT RK -T 7 _ EMI EM10 4636 TTT GTA CCA CCC ATG GCT GCC CTA GAA GAG AAA GGC ATT CTG TTC RK T 7 . EMI EM10 4681 ACC TCA CCG TTT GTC TTG GCA TCG ACC AAT GCA GGA TCT ATT AAT RK T 7 EMI EM10 ' 4726 GCT CCA ACC GTG TCA GAT AGC AGA GCC TTG GCA AGG AGA TTT CAC RK : T7 EMI EM10 4771 TTT GAC ATG AAC ATC GAG GTT ATT TCC ATG TAC AGT CAG AAT GGC RK T7 ' . EMI EM10 4816 AAG ATA AAC ATG CCC ATG TCA GTC AAG ACT TGT GAC GAT GAG TGT RK T 7 ' EMI • EM10 • 4861 TGC CCG GTC AAT TTT AAA AAG TGC TGC CCT CTT GTG TGT GGG AAG RK •.. , T7 . _____ :_ EMI ' EM10 • 4906 GCT ATA CAA TTC ATT GAT AGA AGA ACA CAG GTC AGA TAC TCT CTA RK • . T7 . _: ; EMI EM10 : 124 4951 GAC A T G C T A G T C A C C G A G A T G T T T A G G G A G T A C A A T C A T A G A C A T RK T 7 E M I EMIO 4996 A G C G T G GGG A C C A C G C T T GAG G C A C T G T T C C A G G G A C C A C C A G T A RK T 7 _ E M I EMIO 5041 T A C A G A G A G A T C A A A A T T A G C G T T G C A C C A G A G A C A C C A C C A C C G RK T 7 E M I EMIO 5086 C C C G C C A T T G C G GAC C T G C T C A A A T C G G T A GAC A G T G A G G C T G T G RK T 7 E M I EMIO 5131 A G G GAG T A C T G C A A A G A A A A A G G A T G G T T G G T T C C T GAG A T C A A C RK T 7 _ E M I EMIO . 5176 T C C A C C : C T C C A A A T T GAG A A A C A T G T C A G T C G G G C T T T C A T T T G C RK T 7 E M I . EMIO 5221 T T A C A G G C A T T G A C C A C A T T T G T G T C A G T G G C T G G A A T C A T A T A T RK T 7 E M I EMIO 5266 A T A A T A T A T A A G C T C T T T G C G G G T T T T C A A G G T G C T T A T A C A G G A RK T 7 : E M I • - - • EMIO T -5311 G T G C C C A A C C A G A A G C C C A G A G T G C C T A C C C T G A G G C A A G C A A A A RK • T 7 E M I EMIO : 5356 G T G C A A G G C C C T G C C T T T G A G T T C G C C G T C G C A A T G A T G A A A A G G RK T 7 E M I EMIO G G T 125 5401 AAC TCA AGC ACG GTG AAA ACT GAA TAT GGC GAG T T T ACC ATG CTG RK - - : T 7 ____ — _ _ EMI .— EMIO ; - -5446 GGC ATC TAT GAC AGG TGG GCC GTT TTG CCA CGC CAC GCC AAA CCT RK T 7 _ : EMI ' EMIO 5491 GGG CCA ACC ATC TTG ATG AAT GAT CAA GAG GTT GGT GTG CTA GAT RK T 7 EMI EMIO CTG 5536 GCC AAG GAG CTA GTA GAC AAG GAC GGC ACC AAC T T A GAA CTG ACA RK — . T 7 EMI • EMIO ; 5581 CTA CTC AAA TTG AAC CGG AAT GAG AAG TTC AGA GAC ATC AGA GGC RK AGA T 7 . EMI AGA EMIO AGA • 5626 TTC TTA GCC AAG GAG GAA GTG GAG GTT AAT GAG GCA GTG CTA GCA RK T 7 EMI EMIO 5671 ATT AAC ACC AGC AAG TTT CCC AAC ATG TAC ATT CCA GTA GGA CAG RK ; T 7 EMI AAT EMIO 5716 GTC ACA GAA TAC GGC TTC CTA AAC CTA GGT GGC ACA CCC ACC AAG RK T7 . . EMI EMIO 5761 AGA ATG CTT ATG TAC AAC TTC CCC ACA AGA GCA GGC CAG TGT GGT RK T 7 EMI EMIO 5806 GGA GTG CTC ATG TCC ACC GGC AAG GTA CTG GGT ATC CAT GTT GGT RK T 7 EMI EMIO : 126 5851 GGA AAT GGC CAT CAG GGC TTC TCA GCA GCA CTC CTC AAA CAC TAC RK T 7 EMI EM10 '• - : 5896 TTC AAT GAT GAG CAA GGT GAA ATA GAA T T T ATT GAG AGC TCA AAG RK T 7 EMI -EM10 5941 GAC GCC GGG T T T CCA GTC ATC AAC ACA CCA AGT AAA ACA AAG TTG RK T 7 ' EMI EMIO 5986 GAG CCT AGT GTT TTC CAC CAG GTC TTT GAG GGG AAC AAA GAA CCA RK T 7 EMI EMIO • : . 6031 GCA GTA . CTC AGG AGT GGG GAT CCA CGT CTC AAG GCC AAT T T T GAA RK T 7 EMI EMIO 607 6 GAG GCT ATA T T T TCC AAG TAT ATA GGA AAT GTC AAC ACA CAC GTG RK T 7 . EMI EMIO • 6121 GAT GAG TAC ATG CTG GAA GCA GTG GAC CAC TAC GCA GGC CAA CTA RK T 7 EMI EMIO : 6166 GCC ACC CTA GAT ATC AGC ACT GAA CCA ATG AAA CTG GAG GAC GCA RK T 7 _ EMI -• EMIO • • 6211 GTG TAC GGT ACC GAG GGT CTT GAG GCG CTT GAT CTA ACA ACG AGT RK - : : T 7 — EMI • EMIO - - • 6256 GCC GGT TAC CCA TAT GTT GCA CTG GGT ATC AAG AAG AGG GAC ATC RK - - : ' T 7 EMI EMIO 127 6301 CTC TCT AAG AAG ACT AAG GAC CTA ACA AAG TTA AAG GAA TGT ATG RK T 7 EMI EMIO 6346 GAC AAG TAT GGC CTG AAC CTA CCA ATG GTG ACT TAT GTA AAA GAT RK T 7 EMI : EMIO 6391 GAG CTC AGG TCC ATA GAG AAG GTA GCG AAA GGA AAG TCT AGG CTG RK EMI EMIO 6436 ATT GAG GCG TCC AGT TTG AAT GAT TCA G T G ' GCG ATG AGA CAG ACA RK '• T7 EMI : EMIO 6481 T T T GGT AAT CTG TAC AAA ACT TTC CAC CTA AAC CCA GGG GTT GTG RK • - -T 7 EMI : EMIO • 6526 ACT GGT AGT GCT GTT GGG TGT GAC CCA . GAC CTC T T T TGG AGC AAG RK • T 7 _ . EMI EMIO • 6571 ATA CCA GTG ATG TTA GAT GGA CAT CTC ATA GCA TTT GAT TAC TCT RK T 7 _ EMI EMIO 6616 GGG TAC GAT GCT AGC TTA AGC CCT GTC TGG T T T GCT TGC CTA AAA RK • . T 7 _ __ EMI . - : - — . EMIO '• • • • 6661 ATG TTA CTT GAG AAG CTT GGA TAC ACG CAC AAA GAG ACA AAC TAC RK T 7 EMI : EMIO 6706 ATT GAC TAC TTG TGC AAC TCC CAT CAC CTG TAC AGG GAT AAA CAT RK T 7 EMI EMIO -•• 128 6751 TAC TTT GTG AGG GGT GGC ATG CCC TCG GGA TGT TCT GGT ACC AGT RK T7 EMI ' '•-EMIO .— • 6796 ATT TTC AAC TCA ATG ATT AAC AAT ATC ATA ATT AGG ACA CTA ATG RK : T7 ... : — EMI ' . ' GTT EMIO • 6841 CTA AAA GTG TAC AAA GGG ATT GAC TTG GAC CAA TTC AGG ATG ATC RK . T 7 - K _ _ _ ^ _ EMI ' EMIO ; ' 6886 GCA TAT GGT GAT GAT GTG ATC GCA. TCG TAC CCA TGG CCT ATA GAT •RK : '•-T 7 _ : : EMI , : - -EMIO — - ' : 6931. GCA TCT TTA CTC GCT GAA GCT GGT AAG GGT TAC GGG CTG ATC ATG RK • T 7 _ _ _ . i EMI -r EMIO . : : 6976 ACA CCA GCA GAT AAG GGA GAG TGC TTT AAC GAA GTT ACC TGG ACC RK ' : - -T 7 EMI • T-EMIO ' 7021 AAC GCC ACT TTC CTA AAG AGG TAT TTT AGA GCA GAT GAA CAG TAC RK GTC T 7 : _ _ — EMI .— EMIO GTC 7066 CCC TTC CTG GTG CAT CCT GTT ATG CCC ATG AAA GAC ATA CAC GAA RK T 7 .__ EMI • EMIO .-7111 TCA ATT AGA TGG ACC AAG GAT CCA AAG AAC ACC CAA GAT CAC GTG RK T 7 . _ EMI EMIO — • 7156 CGC TCA CTG TGT CTA TTA GCT TGG CAT AAC GGG GAG CAC GAA TAT RK T 7 EMI EMIO 129 7 2 0 1 gag gag t t c a t c c g t a a a a t t a g a agc g t c c c a g t c gga c g t t g t r k . T 7 EMI EMIO 7 2 4 6 t t g a c c c t c c c c g c g t t t t c a a c t c t a c g c a g g a a g t g g t t g gac r k T 7 EMI EMIO 7 2 9 1 t c c t t t t a g a t t a g a gac a a t t t g a a a t a a t t t a g a t t g g c t t a a r k T 7 EMI EMIO 7 3 3 6 c c c t a c t g t g c t a a c c g a a c c a g a t a a c g g t a c a g t agg g g t a a a r k T 7 EMI EMIO : 7 3 8 1 t t c t c c g c a t t c g g t g c g g r k T 7 __ EMI EMIO Appendix II: Sequence Comparison Summary 130 T7 RK EMI EMIO 5'NTR: 119ACA>ATA 609 ATC>ATT 609 ATC>ATT 609 ATC>ATT VP4: 912 GTG>GTA 912 GTG>GTA 912 GTG>GTA VP2: 1329 GTA>GTG 1329 GTA>GTG 1329 GTA>GTG 1400 ACG>AGC (Thr> Ser) 1400 ACG>AGC (Thr>Ser) 1400 ACG>AGC (Thr>Ser) 1400 ACG>AGC (Thr>Ser) 1407 AAG>AAA 1407 AAG>AAA 1407 AAG>AAA 1713 AAT>AAC 1421 AAA>AGA (Lys>Arg) 1713 AAT>AAC 1713 AAT>AAC VP3: 2024 AGT>AAT (Ser>Asn) 2024 AGT>AAT (Ser>Asn) 2181 GCT>GCC 2181 GCT>GCC 1916 GAG>GGG (Glu>Gly) 2024 AGT>AAT (Ser>Asn) 2181 GCT>GCC 2295 GCA>GCG 2385 GCA>GCG VP1: 2718 GAA>GAG P2: 3882 TTA>TTG 3882 TTA>TTG 3882 TTA>TTG 4077 ACA>ACG 4077 ACA>ACG 4077 ACA>ACG 131 T7 RK EMI EMIO P2: 4164 AGA>AGG 4164 AGA>AGG 4164 AGA>AGG 4512CTA>CTG 4512CTA>CTG 4512CTA>CTG P3: 5364GGC>GGT 5588 AAA>AGA (Lys>Arg) 5588 AAA>AGA (Lys>Arg) 5694 AAC>AAT 5503 TTOCTG 5588 AAA>AGA (Lys>Arg) 6826 ATT>GTT (IloVal) 7025 GCC>GTC (Ala>Val) 7025 GCC>GTC (Ala>Val) 

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