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Specific protein interactions with the 5’- and 3’-untranslated regions of coxsackievirus B3 RNA Cheung, Paul Kim-ming 2004

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Specific Protein Interactions with the 5'- and 3'-Untranslated Regions of Coxsackievirus B3 RNA By PAUL Kim-ming CHEUNG B.Sc, The University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES Department of Pathology and Laboratory Medicine; Faculty of Medicine We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 2004 © Paul Kim-ming CHEUNG, 2004 A B S T R A C T In recent years, studies of the molecular pathogenesis of coxsackievirus B3 (CVB3) have revealed that the viral 5' and 3' untranslated regions (UTRs) contain determinants of viral tissue tropism, translation efficiency, and cardiovirulence. However, the mechanism that links the UTRs to these phenotypic observations remains unknown. Thus, the study of protein-UTR interactions is important to understand the underlying mechanism of functions mediated by the CVB3 UTRs. My dissertation addresses this subject by the identification and characterization of cellular proteins that bind to the CVB3 UTRs. To achieve these goals, I sub-cloned the CVB3 5' and 3'-UTR by a PCR-based strategy. The 5'-UTR sequences of nt 1-209, 210-529 and 530-630 were cloned in consideration of their respective structural and functional significance. At the same time, the 3'-UTR wild type sequence of nt 7299-7399 [+ poly(A) tail] and five mutated 3'-UTR clones were generated. Mutations in these 3'-UTR clones were previously reported to cause tertiary (but not secondary) structural changes in the 3'-UTR, leading to specific inhibition of viral replication. By using radiolabeled RNA transcripts of these UTR sequences in gel mobility shift and competitive UV cross-linking assays, fifteen specific CVB3 5'-UTR-binding proteins in the HeLa cell were identified. The molecular weight of six important 5'-UTR-binding proteins resembled those of the eukaryotic translation initiation factors (eIF4A, 4B, 4G), the death associated protein-5, La autoantigen and the polypyrimidine tract binding proteins. Using the same method, protein interactions with the 3'-UTR and its various mutants were also investigated. Two small proteins (22 and 24 kDa) that bound specifically to the mutated 3'-UTRs were observed. As these corresponding mutations were previously shown to inhibit viral replication, a functional role of these two protein-3'-UTR interactions was inferred. Also, the poly(A) tail of the 3'-UTR was found to be important in mediating HeLa cell protein ii interactions. As the poly(A) tail can interact with the 3'-UTR sequence to maintain a stable secondary RNA structure, my data suggests an important role of the stem-loop RNA structures in mediating protein-3'-UTR interactions. In the A/J mouse model, by using in situ hybridization and UV cross-linking assays, the correlation between CVB3 tissue tropism and protein-5'-UTR interaction was investigated. Among the various UTR-binding proteins in different tissues, a 28 kDa kidney protein was found to bind to the antisense 5'-UTR sequence of nt 210-529 and correlated well with the low viral infectivity in the kidneys, thereby suggesting a possible inhibitory function of this 28 kDa protein. Moreover, a specific 5'- and 3'-UTR-binding protein that shares the same molecular weight (52 kDa) with the La autoantigen, was chosen for further investigation. Using recombinant GST-La fusion protein from purified from E. coli, interactions between the La protein and the 5' or 3'-UTR radiolabeled probes was verified. Specificity of interaction was determined from competitive UV cross-linking experiments. It was observed that the La protein had the highest affinity towards nt 210-529, followed by the nt 530-630 (internal ribosomal entry site, IRES) and then nt 1-209. The lack of primary sequence consensus among these 5'-UTRs suggests that the La protein recognizes secondary RNA structures during interaction. This is also supported by the observation that the La protein interacted with both the wild type and mutated 3'-UTR RNAs, both of which share identical secondary structures. Analysis of the data suggests that each La protein may bind either one of the 5' and 3'-UTR at a time. iii TABLE OF CONTENTS A B S T R A C T ii L I S T O F T A B L E S viii L I S T O F F I G U R E S ix A B B R E V I A T I O N S xii L I S T O F P U B L I C A T I O N S , A B S T R A C T S A N D P R E S E N T A T I O N S xv A C K N O W L E D G E M E N T S xx CHAPTER I BACKGROUND 1 1.1 Discovery of Coxsackieviruses 1 1.2 Epidemiology of viral myocarditis and CVB3 3 1.3 CVB3-induced myocarditis 7 1.4 Chronic infection by CVB3 10 1.4-1 Myocarditis and dilated cardiomyopathy 10 1.5 Life cycle of CVB3 11 1.6 Cellular receptors 15 1.6-1 The Coxsackievirus-Adenovirus Receptor 15 1.6- 2 The Decay-Accelerating Factor 18 1.7 Genetics of CVB3 19 1.7- 1 VPg protein 23 1.7-2 C V B 3 proteases 2 A p r o and 3 C p r o 23 1.7-2a Protease 2A (2A p r o ) 25 1.7-2b Protease 3C (3C p r 0) 28 1.7-2c Cytopathic effects induced by the proteases 30 1.7-3 Molecular determinants of C V B 3 cardiovirulence 32 1.7-3a Relationship between virulence and IRES 32 iv 1.7- 3b Virion capsid proteins and C V B 3 virulence 34 1.8 Expression and replication of viral genome 36 1.8- 1 Classification of picornaviral IRES 38 1.8-2 Cap-independent translation initiation of eukaryotic m R N A 40 1.8-3 Apoptosis and IRES mediated translation initiation 44 1.8-4 Transcription of Viral R N A 45 1.8-4a Tertiary R N A structure of the 3 ' -UTR and C V B 3 replication 45 1.8- 4b Requirement of host proteins in picornaviral replication 46 1.9 Functional roles of host protein-CVB3 UTR interactions 48 1.9- 1 Participation of eukaryotic translation initiation factors 49 1.9-2 Functional roles of the L a autoantigen and P T B 51 1.9-3 Role of protein-RNA interaction in stimulating R N A transcription 55 1.9-4 Characterization of other U T R binding proteins 56 1.9-5 Research Focus 57 CHAPTER II HYPOTHESIS AND SPECIFIC AIMS 59 CHAPTER III RESEARCH QUESTIONS AND STRATEGY 60 CHAPTER TV 62 IDENTIFICATION OF SPECIFIC CVB3 UTR-BINDING PROTEINS 62 4.1 EXPERIMENTAL STRATEGIES 62 4.2 RESULTS 75 Specificity of HeLa protein-CVB3 U T R interactions (aiml) 75 Tissue tropism of C V B 3 in A / J mice by ISH (Aim 2) 81 Profiles of the protein-5'UTR interactions (Aim 2) 90 HeLa cell protein-5'-UTR interactions (Aim 3) 105 v HeLa cell protein-3'-UTR interactions (Aim 4) I l l 4.3 DISCUSSION 115 Interactions of A / J mouse proteins with the viral 5 ' -UTR 115 Common-UTR binding proteins in cell lysates of A / J mouse tissues 116 The 28kDa protein interaction with antisense nt 210-529 118 HeLa cell protein vs 5 ' -UTR 119 The elF4F family and C V B 3 5' -UTR 120 Significance of the Poly-(A) tail in mediating protein-interaction 121 Protein interactions unique to the replication defective mutants 122 CHAPTER V 124 Characterization of the La autoantigen - CVB3 UTR interactions 124 5.1 EXPERIMENTAL STRATEGIES 124 5.2 RESULTS 130 Purification of the La autoantigen 130 Interaction of the 5 ' -UTR with the La protein 132 Specific La protein - 3 ' -UTR interaction - hyperbolic saturation curve 138 Specific La protein - 3 ' -UTR interaction - hyperbolic saturation curve 140 Stoichiometry of interaction (1:1) and the 90% saturation quantity 140 Mutations in the 3 ' -UTR do not affect interaction with the La protein 141 Each La protein molecule can only bind to either one of the 5' / 3 ' -UTR at a time 145 Apparent dissociation constants of the L a - U T R interactions 148 5.3 DISCUSSION 152 Role of secondary structures in the interaction of L a with the 5 ' -UTR probes 152 Functional significance of the La protein-5'-UTR interaction 153 Interaction of L a protein with the 3 ' -UTR did not depend on tertiary structures 154 Affinity of the L a protein - 3 ' -UTR interaction 154 Circularization of C V B 3 R N A 156 vi Possible dimerization of L a protein-UTR complexes 157 Method developments and experimental limitations 159 CHAPTER VI CONCLUSION AND FUTURE DIRECTIONS 162 6.1 Conclusions and scientific contributions 162 6.2 Strategies for future directions 164 To separate the endocrine and exocrine cell types by L C M 165 To sequence identify U T R binding proteins by 2D P A G E and Mass Spectrometry (MS) 168 To verify UTR-protein interactions by standard molecular techniques 168 To enhance the biochemical analysis of protein-RNA interaction by large format SDS-PAGE.. . .170 6.3 Final remarks 171 CHAPTER VII MATERIALS AND METHODS 172 7.1 Plasmid construction 172 7.2 In vitro transcription 172 7.3 Preparation of HeLa cell extracts 176 7.4 Preparation of lysates from A/J mouse organs 176 7.5 RNA mobility shift assays 177 7.6 UV cross-linking experiments 177 7.7 Virus, animal and tissue processing 178 7.8 In situ hybridization 179 7.9 Protein purification 180 7.10 Estimation of the apparent Kd and stoichiometry of La-UTR interaction. 181 7.11 Calculations of the Scatchard Plot transformation 186 REFERENCES 187 vii LIST OF T A B L E S Table 1. Families of viruses infecting human and animals 5 Table 2. Classifications of the three type of IRESes 39 Table 3. Summary of host genes translated by the IRES mechanism 43 Table 4. Summary of the correlation between ISH observations and the p28 interactions. 101 Table 5. Summary of A/J mouse tissue protein interactions with viral 5'-UTR sequences.... 103-4 Table 6. Summary of Protein-RNA interactions between CVB3 5'-UTR sequences and the HeLa cell Lysate 110 Table 7. List of primers and conditions used in PCR cloning 172-4 Table 8. Experimental conditions for quantifying the La protein-3'-UTR interactions 184 Table 9. Transformation of saturation plot data to scatchard plot the La-3 '-UTR IRES interaction 185 viii LIST OF FIGURES Figure la Phylogenetic relationship of major enteroviruses 4 Figure lb CVB3 epidemiology. 6 Figure 2 Triphasic disease development of CVB3 induced myocarditis 9 Figure 3. The CVB3 virion structure and viral entry 13 Figure 4. The life cycle of CVB3 14 Figure 5. The CAR and DAF receptors 17 Figure 6. Genome organization (P1-P3) and proteolytic cleavage of the polyprotein. 20 Figure 7. Major cellular cleavage events mediated by the 2Apro and 3Cpro 24 Figure 8. Homologies between eIF4G I and 4G II 27 Figure 9. The CVB3 5'-UTR secondary structures and homology of the IRES with host rRNA 37 Figure 10. The 3'-UTR tertiary structure and the kissing pair interactions 47 Figure 11a. Summary of steps in cap-dependent translation initiation 50 Figure 1 lb. Current model of picornaviral IRES mediated translation initiation 52 Figure 12a. The overall strategy employed in my experiments 61 Figure 12b. The cloning scheme for the 5'-UTR 64 Figure 12c. The cloning scheme for the 3'-UTR 65 Figure 12d. The strategy of the mobility shift assay 67 Figure 12e The strategy of the AJ mice experiments 69 Figure 12f. The strategy of UV cross-linking experiments 72 Figure 13a. Mobility shift assay of 5'-UTR nt 1-209 77 ix Figure 13b. Mobility shift assay of 5'-UTR nt 210-529 78 Figure 13c. Mobility shift assay of 5'-UTR nt 530-630 79 Figure 13d. Mobility shift assay of 3'-UTR sense and antisense sequences 80 Figure 14a. ISH data of A/J hearts 83 Figure 14b. ISH data of A/J pancreas 84 Figure 14c. ISH data of A/J livers 85 Figure 14d. ISH data of A/J brain 87 Figure 14e. ISH data of A/J spleen 88 Figure 14f. ISH data of A/J kidneys 89 Figure 15. Layout of the UV cross-linking profiles with A/J lysates 90 Figure 16a-f. UV cross-linking profiles of individual A/J lysates 94-99 Figure 17. The p28 interactions with the antisense nt 210-529 100 Figure 18a. HeLa cell protein interactions with 5'-UTR nt 1-209 106 Figure 18b. HeLa cell protein interactions with 5'-UTR nt 210-529 107 Figure 18c. HeLa cell protein interactions with 5'-UTR nt 530-630 108 Figure 19. HeLa cell protein interactions with the 3' -UTRs 112 Figure 20. The strategy of characterizing La -5' and 3'-UTR interactions 125 Figure 21. Schematic representation of the GST-La protein plasmid 126 Figure 22. Strategy in quantifying La protein - probe complexes 127 Figure 23. Purification of the La protein 131 Figure 24. UV cross-linking between La protein and 5'-UTRs 135-7 Figure 25. Saturation plot of the La-wild type 3'-UTR interaction (cpm vs concentration)... 139 x Figure 26. Two-stages UV cross-linking between La protein and 3'-UTR probes 144 Figure 27. Two-stages UV cross-linking between La protein and 5'-UTR probes 147 Figure 28. Scatchard plot of the La-3 '-UTR interaction 150 Figure 29. The strategy of using LCM 166 Figure 30. Preliminary LCM data 167 Figure 31. The use of 2D PAGE to identify UTR binding proteins 169 xi ABBREVIATIONS AIF Apoptosis Inducing Factor BIP Immunoglobulin Heavy-Chain Binding Protein CAR Coxsackievirus Adenovirus Receptor CPE Cytopathic Effect CREB Cyclic-Amp-Response-Element-Binding-Protein CVA9 Coxsackievirus A9 CVB3 Coxsackievirus B3 CVB4 Coxsackievirus B4 DAF Death Accelerating Factor DAP 5 Death Associated Protein 5 DCM Dilated Cardiomyopathy elF Eukaryotic Translation Initiation Factor eIF4G Eukaryotic Translation Initiation Factor 4G EMCV Encephalomyocarditis Virus ER Endoplasmic Reticulum FGF-2 Fibroblast Growth Factor 2 FMDV Foot And Mouth Disease Virus GAD Glutamate Decarboxylase GPI Glycophosphatidylinositol GSK-3 Glycogen Synthase Kinase-3 HAV Hepatitis A Virus HCV Hepatitis C Virus HIV Human Immunodeficiency Virus HRV 14 Human Rhinovirus 14 HTLV-1 Human T-Cell Lymphotropicvirus Type 1 IGTP Interferon-Gamma-Inducible Guanosine Triphosphatase IL2 Interleukin 2 INF- y Interferon y IPTG Isopropyl-p-D-thiogalactopyranoside xii IRES Internal Ribosomal Entry Site ISH In Situ Hybridization Kd Dissociation constant MAPK Mitogen Activated Protein Kinase MHC Major Histocompatibility Complex MKK 6 Map Kinase Kinase 6 MOI Multiplicity Of Infection mRNA Messenger RNA NTP Nucleoside Triphosphate OCT-1 Octomer Binding Transcription Factor -1 ODC Ornithine Decarboxylase ORF Open Reading Frame P.I. Post Infection PABP Poly-A Tail Binding Protein PARP Poly-Adp Ribose Polymerase PCBP Poly-Rc Binding Protein •PKB Protein Kinase B PTB Poly-Pyrimidine Tract Binding Protein PV Poliovirus RdRP RNA dependent RNA polymerase RNP Ribonucleo-Protein Complex RRM RNA Recognition Domain rRNA Ribosomal RNA RT-PCR Real Time Polymerase Chain Reaction SCID Severe Combined Immunodeficiency Disease SCR Short Consensus Repeat SD Shine-Dalgarno SN RNA Small Nuclear RNA TNF-a Tumor Necrosis Factor a tRNA Transfer RNA TUNEL Terminal Deoxynucleotidyl Transferase Mediated Nick End Labeling xiii Untranslated Region Vascular Endothelial Growth Factor World Health Organization x i v L I S T O F P U B L I C A T I O N S . A B S T R A C T S A N D P R E S E N T A T I O N S 1. Cheung, P.. Zhang, M . , Yuan, J., Chau, D., Yanagawa, B . , McManus, B and Yang, D C . Specific interactions of HeLa cell proteins with coxsackievirus B3 R N A : L a autoantigen binds differentially to multiple sites within the 5' untranslated region. (2002). Virus Research 90, 23-26 2. Cheung, P.. Yanagawa, B . , Zhang, H . , Wang, A . , Luo, H . , Esfandiarei, M . , Saurez, A . , Wilson, J., Carthy, C , McManus, B . and D. Yang. Molecular Mechanisms of Cardiovirulence and Host Cel l Responses in Coxsackievirus B3 induced Myocarditis. (2002). Recent Research Developments in Virology 3, 427-454. 3. Cheung. P.. Zhang, M . , Yuan, J., Chau, D. , Yanagawa, B . , McManus, B . and Yang D C . Specific binding of L a protein within the Coxsackievirus B3 3' U T R and the significance of the poly-(A) tail in mediating HeLa cell protein interactions. Under revision. 4. Cheung, P.. Yuan, J., A . Suarez, McManus, B . and Yang, D C . Correlation between pathology of C V B 3 infection in A / J mouse and interactions of cellular proteins with the viral 5' untranslated sequence. Under revision. 5. Yang, D C , Cheung, P.. Sun, Y . , Yuan, J., Zhang, H . , Carthy, C M . , Anderson, D.R. Bohunek, L . , Wilson, J.E., McManus, B . M . A Shine-Dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 R N A . (2003). Virology 305, 31-43 6. Yang, D. , Cheung. P.. Yanagawa. B. , Cheung, Caroline, Chow, D. , and McManus, B . Picornaviral proteases in viral replication and pathogenesis. (2003). Current Topics in Virology 3,17-31. 7. Zhang, H . M . , Cheung, P., Yanagawa, B . , McManus, B . M . and Yang, D. . BNips: A group of pro-apoptotic proteins in the Bcl-2 family. (2003). Apoptosis. 8:229-236. 8. Zhang, H . M . , Yuan, J., Cheung, P., Luo, H . , Yanagawa, B . , Chau, D. , Tozy, N . S., Wong, B . , Zhang, J., Wilson, J.E., McManus, B . M . and Yang, D.. Over-expression of interferon-gamma -inducible GTPase inhibits coxsackievirus B3-induced apoptosis through the activation of the PI3-K / A k t pathway and inhibition of viral replication. (2003). J. Biol. Chem. 278:33011-19. 9. Yanagawa, B . , Spiller,O.B., Choy,J., Luo,H., Cheung,P., Zhang,H.M.; Goodfellow,I.G.; Evans,D.J.; Suarez,A.; Yang, D . Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice. (2003). Lab Invest. 83: 75-85. 10. Zhang, H . M . , Yanagawa, B . , Cheung. P.. Luo, H . , Yuan, J., Chau, D. , Wang, A . , Bohunek, L . , Wilson, J. E. , McManus, B . M . and Yang, D . Nip21 gene expression reduces coxsackievirus B3 replication by promoting apoptotic cell death via a mitochondria-dependent pathway. (2002). Circ. Res. 90,1251-1258. 11. Yanagawa, B . , Esfandiarei, M . , Carthy, C , Cheung, P.. Luo, H . , Granville, D. , Yang, D. , Choy, J., Lu i , A . , Dabiri, D. , Wilson, J.E., Wang, A . , Zhang, M , Sinn, S., McManus, B . , Wei, K . , and xv Laher, I. Life and death signaling pathways in C V B 3 induced myocarditis. (2002). In "Myocarditis: Bench to the Bedside" Ed. L T Cooper (Rochester, M N ) . Humanan Press. August, 2002. ISBN: 1-58829-112-X. 12. Wang, A . , Cheung, P.K.. Zhang, H . , Carthy, C. M . , Bohunek, L . , Wilson, J. E . , McManus, B . M . and Yang, D. Specific inhibition of coxsackievirus B3 translation and replication by phosphorothioate antisense oligodeoxynucleotides. (2001). Antimicrob. Agents Chemother. 45:1043-1052. 13. L i u , Z . , Carthy, C. M . , Cheung. P.. Bohunek, L . , Wilson, J. E. , McManus, B . M . and Yang, D . Structural and functional analysis of the 5' untranslated region of coxsackievirus B3 R N A : In vivo translational and infectivity studies of full-length mutants. (1999). Virology 265:206-217. 14. Taylor, L . A . , Carthy, C. M . , Yang, D. , Saad, K . , Wong, D. , Schreiner, G. , Stanton, L . W. and McManus, B . M . Host gene regulation during coxsackievirus B3 infection in mice: assessment by microarrays. (2000). Circ. Res. 87: 328-334. Highlights of Abstracts and Presentations 1. Cheung, P., Zhang, M . , Yuan, J., Chau, D., Yang, D. L a autoantigen interacts with multiple sites in the 5 ' -UTR of Coxsackievirus B3 . Annual Conference 2002, American Society of Virology, University of Kentucky, Lexington, US. 2. Cheung, P., Yanagawa B , Chau, D., Zhang M , Lai , J., Yuan, J., McManus B M and Yang D . 2002. Differential Interactions of L a autoantigen with the 5' untranslated region of coxsackievirus B3 R N A . Annual Pathology Day, 2002, Department of Pathology and Laboratory Medicine, U B C , Vancouver, Canada 3. Cheung. P., Yanagawa B , Wang, A . , Zhang, M . , La i , J., Carthy C, Bohunek L , Luo H , Wilson JE, McManus B M , Yang D. (2001) Host-proteins-coxsackievirus B3 R N A interactions. Frontiers in Cardiovascular Research Conference, 2001, Seattle, Washington, US. 4. Cheung, P., Yanagawa, B . , Wang, A . , Zhang, M . , La i , J., Carthy, C M . , Bohunek, L . , Luo, H . , Wilson, J.E., McManus, B . M . , Yang, D. Host proteins-coxsackievirus B3 R N A interactions: implications for systemic pathogenesis. Annual Pathology Day 2001, Department of Pathology and Laboratory Medicine, Faculty of Medicine, U B C Vancouver, Canada. 5. Cheung, P., Yanagawa, B . , Wang, A . , Zhang, M . , La i , J., Carthy, C M . , Bohunek, L . , Luo, H . , Wilson, J.E., McManus, B . M . , Yang, D . Specific interactions of HeLa cell proteins with the internal ribosome entry site and the 3' untranslated region of coxsackievirus B3 R N A . Annual Pathology Day 2001, Department of Pathology and Laboratory Medicine, U B C , Vancouver, Canada. 6. Cheung. P.. Carthy, C , Yanagawa, B . , Wang, A . , Zhang, M . , Bohunek, L . , Wilson, J.E., McManus, B . M . , and Yang, D. Specific interactions of HeLa cell proteins with the 5' and 3' untranslated regions of coxsackievirus B3 R N A . Annual Pathology Day 2000, Department of Pathology and Laboratory Medicine, U B C , Vancouver, Canada. xvi 7. Cheung, P., Yanagawa, B . , Wang, A . , Zhang, M . , La i , J., Carthy, C M . , Bohunek, L . , Luo, H . , Wilson, J.E., McManus, B . M . , Yang, D . Host protein interactions with the 5' and 3' untranslated regions of coxsackievirus B3 . Annual Pathology Day 1999, Department of Pathology and Laboratory Medicine, U B C , Vancouver, Canada. 8. Cheung, P., Carthy, C M . , Watson, K . , Bohunek, L . , Wang, A . , Wilson, J.E., McManus, B . M . Host protein interactions with the 5' and 3' untranslated regions of coxsackievirus B3 . Annual Conference 1999, American Society of Virology, Boston, U S A . Oral Presentation. 9. Cheung. P.. Carthy, C M . , Watson, K . , Bohunek, L . , Wang, A . , Wilson, J.E., McManus, B . M . Differential interaction of organ specific proteins with the 5" untranslated region of coxsackievirus B3 R N A . Annual Meeting 1998, American Society of Microbiology, Vancouver, Canada. 10. McManus, B . , Luo, H . , Yanagawa, B . , Esfandiarei, M . , Carthy, C , Yang, D. , Wilson, J.E., Cheung, P.. Wang A . Structural and biochemical basis of cardiac myocyte survival and death: A dynamic tension. Published for the 2nd International Congress on Heart Disease, Washington, D C (2001). 11. Yang, D. , Zhang, M . , Cheung. P.. Chau, D., Yuan, J., Yanagawa, B . , Luo, H . , Zhang, J., Wilson, J.E., McManus, B . M . Coxsackievirus B3 2A and 3C proteases induce cell death through apoptosis. Annual Conference 2002, American Society of Virology, University of Kentucky, Lexington, US. 12. Zhang, M , Yanagawa, B . , Luo, H . , Cheung, P., Wang, A . , Yuan, J., Yen, C , Granville, D. , Choy, J., Luo, Z . , Bohunek, L . , Sinn, S., Wilson, J.E., McManus, B . M . , Yang, D . Nip 21 is a pro-apoptotic gene and its product inhibits virus replication by promoting cell death. Keystone symposia, Colorado, U S A , 2001. 13. Zhang, M . , Yanagawa, B . , Cheung. P.. Yuan, J., Esfandiarei, M . , Chau, D. , Luo, H . , Zhang, J., Dabiri, D. , Wilson, J.E., McManus, B . M . and Yang, D . Functional Roles of IP10 and IGTP Expression in Coxsackievirus B3 (CVB3) Replication. 2002. Providence Health Research Symposium, Vancouver, Canada. 14. Yang, D. , Zhang, M . , Cheung. P.. Chau, D. , Yuan, J., Yanagawa, B . , Luo, H . , Dabiri, D . , Toma, M . , Wilson, J.E., McManus, B . M . Coxsackievirus B3 2A and 3C proteases induce cell death through apoptosis. Frontiers in Cardiovascular Research Conference, Oct 2001, Seattle, W A . 15. Zhang, M . , Yanagawa, B . , Yuan, J., Esfandiarei, M . , Dabiri, D. , Luo, H . , Cheung, P. , Wang, A . , Wilson, J.E, McManus, B . M . , and Yang D. Functional role of IP10 and IGTP in cell survival, fetal gene re-expression and coxsackievirus B3-induced disease. Frontiers in Cardiovascular Research Conference, Oct 2001, Seattle, W A . 16. Spiller, O.B. , Yanagawa, B . , Choy, J., Luo, H . , Cheung. P.. Zhang, M . , Suarez, A . , Yang, D. , McManus, B . M . Soluble D A F abrogates CVB3-induced myocarditis in mice. Presented at the X I X International Complement Workshop, September 2002, Palermo, Italy xvii 17. Yanagawa , B . , C h o y , J . , L u o , H . , Cheung, P.. Suarez, A . , Esfandiarei , M . , Zhang , M . , W a n g , A . , Spi l le r , O . B . , and M c M a n u s , B . M . A Potential Soluble V i r u s Receptor Therapeutic A g e n t for E a r l y C V B 3 Infection. A n n u a l Research D a y , Department o f Pathology and Laboratory M e d i c i n e , U B C , Vancouver , M a y 2001. A l s o selected as a finalist (1 o f 12) and for Presentation at C I H R / B i o c o n t a c t , Quebec C i t y , Quebec, October 2001 Presented at Frontiers i n Cardiovascular Research Conference, Oc t 2001, Seattle, W A . 18. Zhang , M . , Yanagawa , B . , L u o , H . , Cheung. P.. W a n g , A . , Y u a n , J . , Y e n , C , Granv i l l e , D . , C h o y , J . , L u o , Z . , Bohunek , L . , S inn , S., W i l s o n , J .E . , M c M a n u s , B . M . and Y a n g , D . C . N i p 2 1 Is a Pro-apoptotic Gene and Its Product Inhibits V i r u s Repl ica t ion by Promot ing C e l l Death. Frontiers i n Cardiovascular Research Conference, Oc t 2001, Seattle, W A 19. Yanagawa , B . , L u o , H . , Reza i , N . , B u c k l e y , J . , Tr iche , T. , Zhang , J . , Zhang , M . , Cheung, P.. Y u a n . J . , Y a n g , D . , and M c M a n u s , B . M . Gene prof i l ing i n C V B 3 - i n f e c t e d mouse hearts. L a J o l l a - R o m e - Y a m a g u c h i Research Conference, "Therapeutic Hor izons i n Heart Fa i lu re" , San D i e g o , C A , December 2002. 20. Yanagawa , B . , Spi l ler , O . B . , Proctor, D . G . , Goodfe l l ow , I.G., C h o y , J., L u o , H . , Cheung. P.. Zhang , M . , Suarez, A . , Y a n g , D . , and M c M a n u s , B . M . A r e Soluble C A R and D A F M a g i c Bul le t s to Treat Coxsack iev i rus B 3 Infection? C I H R Nat iona l Poster Compet i t ion , W i n n i p e g , M A , Canada. June 2002. (one o f four abstracts chosen to represent U B C i n national compet i t ion -A w a r d for Exce l lence : Si lver) 21 . Yanagawa , B . , L u o , H . , Car thy, C , Cheung, C , C h o y , J . , W a l i n s k i , H . , Zhang , M . , Cheung, P., Zhang , J . , C h a u , D . , Y a n g , D . C , and M c M a n u s , B . M . The R o l e o f Apoptos i s i n M y o c a r d i t i s . Presented at Providence Heal th Research Sympos ium. M a r c h , 2002 and the Department o f Pathology and Laboratory M e d i c i n e , Facul ty o f M e d i c i n e , Univers i ty o f B r i t i s h C o l u m b i a , A n n u a l Research D a y , Vancouver , Canada. 22. Yanagawa , B . , Spi l ler , O . B . , Proctor, D . G . , C h o y , J . , L u o , H . , W a l i n s k i , H . , Cheung, P.. Zhang , M . , Suarez, A . , Zhang , J . , Y a n g , D . C , and M c M a n u s , B . M . C A R - F c T o Treat E a r l y C V B 3 -Induced Myoca rd i t i s . Presented at Providence Heal th Research. Sympos ium. M a r c h , 2002, and the Department o f Pathology and Laboratory M e d i c i n e , Facul ty o f M e d i c i n e , Un ive r s i ty o f B r i t i s h C o l u m b i a , A n n u a l Research D a y , Vancouver , Canada 23. Y a n g , D . , Cheung, P., Car thy, C , Bohunek, L . , W i l s o n , J .E . , M c M a n u s , B . M . H e L a ce l l proteins interact wi th the 5 ' untranslated region o f coxsackievirus B 3 genomic R N A . International conference on medica l v i ro logy , Be i j ing , Ch ina . 1999. xviii Highlights of Awards 1. Doctoral Research Award, year 2001-2003, Heart and Stroke Foundation, BC and Yukon. 2. Research Traineeship Award, year 1999-2001, Heart and Stroke Foundation, BC and Yukon. 3. Student Travel Grant, 1999, American Society for Virology, - for oral presentation of research findings at the Annual Meeting of American Society for Virology, University of Massachusetts, Boston, USA. 4. Student Travel Award, 1999, Faculty of Medicine, UBC, for poster presentation at the American Society for Virology, University of Massachusetts, Boston, USA. 5. Student Travel Award, 2001, Faculty of Medicine, UBC, for poster presentation at the Frontier in Cardiovascular Research Meeting, Seattle, WA., US. xix A C K N O W L E D G E M E N T S Over the past few years, there have been tremendous changes in the landscape of virus research. New technologies and novel publications are rapidly released every month, accelerating advances in the field of viral pathogenesis. It would be impossible to keep up with these new knowledge and technologies without the help of my mentors, collaborative scientists, laboratory technicians, fellow graduate students, computing and other laboratory support staff at the MRL/iCAPTUR 4E centre. Thus I would like to express my sincere gratitude to all of these people. In addition, I would like to express my sincere appreciation to the funding agencies, the Heart and Stroke Foundation of B.C. and Yukon and the St. Paul's Hospital Foundation. Their financial assistance has ensured the continuation of my program. Of particular importance to my academic development is Dr. Decheng Yang. I am most grateful for his mentorship and his willingness to accept me as his first graduate student. His leadership, patience, and attention have been most crucial to my academic development. His encouragement and his support have helped me endured difficult times. I thank him for the countless hours of dedications in my projects, in refining my manuscripts and in the improvement of my research methods and strategies. As well, I am deeply thankful to my mentor Dr. Bruce McManus. As director of the cardiovascular laboratory, Dr. McManus has always encouraged and challenged me to be creative in thinking, be precise in experimental work, be insightful in analysis and be strong in undertaking difficult assignments. Dr. McManus has also provided me with opportunities to explore new techniques and collaborations with other excellent research personnel. Dr. McManus is highly dedicated to the progress of science and the nurture of young apprentices. xx I am also most thankful to my supervisory committee members, Dr. Gregory Bondy (chairman), Dr. Shirley Gillam and Dr. Caroline Astel, who have generously provided me with inputs and guidances. Throughout my program, their advice and criticisms have been much appreciated and most essential to the incorporation of new ideas into my work, to the refinement of my research skills and to the generation of the thesis. At the same time, I would like to thank other scientists and collaborators: Dr. Mary Zhang, Dr. Ed Pryzdial, Dr. Aikun Wang, and Dr. Zewei Liu, who were visiting scientists to the laboratory and provided me with help and guidance in various aspects of my research; Dr. Delbert Dorscheid who has provided me with expertise in laser capture microdissection; Dr. Derek Smith, Dr. Bob Olafson and other staff at the Genome B.C. center, who have enlightened my eyes to the science of proteomics analysis. A special person whom I would like to mention in my acknowledgement is Ms. Patricia Sham who has been an understanding and altruistic friend during my years of graduate studies. I am indebt to all her support and companionship, during all difficult times and especially my recovery period from the surgical repair of my broken Achilles' tendon. Last but not the least, I would like to thank my parents for their infinite love and their patience with the time it took to complete my graduate studies. I am deeply grateful for their dedication to my upbringing, their sacrifice and their willingness to put my education ahead of their interests. Since childhood, they have planted in my heart the concept of becoming a better person everyday, and to be courageous in defeating challenges. All these have been the source of my energy to excel in research and to overcome all hardships and challenges. xxi CHAPTER I BACKGROUND The concept of viruses was first described by Jacob Henle of Gottingen in 1840, as "extremely small infectious agents". Eventually, these agents were characterized as "non-filterable" and "toxin-like", eventually leading to the recognition that 'viruses' as pathogenic materials that are significantly smaller in size than bacteria 3 1. In 1900, the United States Army presented the first report on a human pathogenic virus: the yellow fever virus; and established the link to patients with acute febrile illness An important landmark in the study of viruses as causative agent of diseases was the development of unified diagnostic criteria by A. S. Evans in 1982 1 0 4 At the present day, viruses are understood as parasitic organisms that can only replicate within cellular environments. Viruses utilize enzymes and other metabolic machineries of a host to complete their life cycles. Consequently, interactions of cytoplasmic proteins and viral genetic material are important in the pathogenesis of many viruses. Knowledge of these interactions helps understand the process of disease development. In this dissertation, I shall present findings of my investigations on the interactions between host proteins and the untranslated region (UTR) of the coxsackievirus B3 (CVB3). 1.1 Discovery of Coxsackieviruses Coxsackievirus was discovered in 1948 by Dalldorf and Sickles 9 1 when a filterable agent was isolated from children suffering paralysis that resembled poliomyelitis in the village of Coxsackie, New York. At the present time, coxsackieviruses are divided into two main groups based on the types of pathology that they cause in murine models. Group A coxsackieviruses Paul Cheung Page 1 (serotypes 1-22, 24) infect mostly striated muscles causing flaccid paralysis 9 1 , while the group B (serotypes 1-6) viruses cause local myositis as well as meningitis, pancreatitis, myocarditis and hepatitis " 8 . Genetically, coxsackieviruses are very similar to the poliovirus (PV) and other enteroviruses (Figure la), all of which display highly similar physical and chemical properties: single stranded positive sense genomic RNA, commonly with a length of 7,500 nucleotides (nts) and that is flanked by UTRs on both the 5' and 3' ends. Enterovirus is a genus in the family of Picornaviridae. Picornaviridae is a family of small, icosahedral, non-enveloped viruses with a single-stranded RNA genome of positive polarity. To date, more than 230 serotypes of picornaviruses have been recognized and they are classified into six genera 1 7 6 ' 3 0 7 ; enteroviruses (e.g. PV and coxsackievirus), rhinoviruses (the most important etiological agent of the common cold), parechoviruses (e.g. human echovirus 22), hepatoviruses (e.g. hepatitis A virus, HAV), aphthovirus (e.g. foot-mouth-disease virus, FMDV), cardiovirus (e.g. encephalomyocarditis virus, EMCV) (Table 1). Together, these viruses cause numerous human syndromes, ranging from fatal paralysis, encephalitis, meningitis, myocarditis, diabetes, hepatitis and the foot-and-mouth disease. The name "enterovirus" originates from the ability of these viruses to infect human and animals via the alimentary tract. Enteroviruses share high degree of sequence homology, and it has been postulated that the whole genus of enteroviruses resemble more a pool of genetic variants than genetically discrete species 2 4 6 , 3 3 9 . The rise of genetic variations in this group of viruses is likely a consequence of the inaccurate RNA transcription performed by the enteroviral RNA polymerase which has an error rate of roughly 10"3 to 10"4 substitutions per base 2 4 6 . Such an error rate translates into an average of one mutation incorporated per genome-replication: a rate that is almost detrimental to the continued existence of the species 2 4 6 . Yet, this nearly fatal Paul Cheung Page 2 error rate contribute to the evolutionary advantage of the enteroviruses: to diversify into strains that can thrive in a wide range of host environments 2 4 6 . 1.2 Epidemiology of viral myocarditis and CVB3 In numerous studies, CVB3 is characterized as a major viral agent causing acute myocarditis and its late phase dilated cardiomyopathy (DCM) H4,i66,i67,2i8,226,340 Various studies, using real time-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) of enteroviral RNA have demonstrated the presence of an enteroviral genome in as much as 70% of myocarditis patients 2 5 ' . In a global surveillance conduced by the World Health Organization (WHO) from 1975 to 1985, the group B coxsackieviruses have been diagnosed as the most common causal agent, with 34.6 cardiovascular diagnoses per 1,000 documented virus infections 1 2 6 (Figure lb). This is followed by the influenza B virus (17.4 per 1,000 infections), influenza A virus (11.4 per 1,000 infections), coxsackie A virus (9.1 per 1,000 infections) and cytomegalovirus (8.0 per 1,000 infections). Other viruses such as coxsackievirus B4 (CVB4), adenoviruses , hepatitis C virus (HCV) , and human immunodeficiency virus (HIV) are also implicated in causing myocarditis or myocardial decompensation, although their contributions may not be as dominant as that by the CVB3 158>321>333. Paul Cheung Page 3 Figure la. Phylogenetic relationship of major enteroviruses. The dendrogram is constructed by comparing nucleotide alignments of coat protein sequences for the polioviruses, coxsackieviruses, enterovirus 70, and bovine enterovirus (BEV). Percent nucleotide identity is given by the vertical bar connecting virus pairs (Fitch et.al. 1977, Rico-Hesse 1987). High homology is defined as more than 50% sequence identity. Thus, poliovirus and coxsackievirus share high homologies in the virion capsid sequences (60~70%), while the enterovirus 70 (EV70) and the bovine enterovirus (BEV) are more distally related (50-60%). (Adapted from B.N. Fields et. al. (ed)., Fields Virology Lippincott-Raven Publishers, Philadelphia, Pa., 1996) Virus Percent nucleotide identity in the aligned PI sequence (Virion capsid protein sequence) 100 90 80 70 60 50 40 Polio-Is Polio-2s Polio-3s CVA21 CVB1 CVB3 CVB4 CVA9 EV70 BEV Paul Cheung Page 4 Table 1. The family of Picornaviruses Adapted from B.N. Fields et. al. (ed)., Fields Virology (Lippincott-Raven Publishers, Philadelphia, Pa., 1996 and from The International Committee on Taxonomy of Viruses http://www.ncbi.nlm.nih.gov/ICTVdb/ Genus No. Serotypes Members Rhinovirus 102 3 Human rhinoviruses 1A-100, IB, "Hanks'' Bovine rhinoviruses 1,2,3 Enterovirus 3 23 6 28 Aphthovirus Cardiovirus Hepatovirus Parechovirus 4 1 18 2 8 7 1 2 Human polioviruses 1,2,3 Human coxsackieviruses A1-22, 24 Coxsackieviruses A23 = echovirus 9 Human coxsackieviruses Bl-6 Human echoviruses 1-7, 9, 11-21, 24-27, 29-34 Echovirus 1 = echo 8 Echovirus 10 — reovirus type 1 Echovirus 28 = rhinovirus 1A Human enterovirus 68-71 Vilyuisk virus Simian enterovirus 1-18 Bovine enterovirus 1,2 Porcine enterovirus 1,2 Foot-and-mouth disease virus 1-7 (serotypes A,C,0, SAT-l,2,3,Asia-l) Encephalomyocarditis virus, theiler's virus murine encephalomyelitis Human hepatitis virus A Human parechoviruses 1, 2 (Formerly human echoviruses 22 and 23) Paul Cheung Page 5 *> "Si / - v a a> o G g 2 * .is -c «a > o *H O ^ o ft. VC. T3 .fe N SO < C3 . 4> 3 2 V. Ix •2 ^  ° ? ~ 60 u >, « w J= d o 5^ il) . C e3 8 «t »! _ i © M "S "S « OJ 60 „ ft 2 oj <3 I 2 <u .a o - & CO „ QJ 60 2 00 CO O o 1/-) <-<-) o CN 09 C o .5 O o o 0> CH og aj oa o c 6X S3 CJ 09 • e « pa <c cu 2 u CS (O X e U 1 U 3 e c CJ 3 c o u Q. c 3 i X o O o tso .5 1) CO E S3 O Q . > o tu | | I 1 I I 1 o Cu > o e i c s o U o £ -a < oN X S a. Q. E a. b o I CJ e-CJ o a: E < c CJ X 1.3 CVB3-induced myocarditis Direct CVB3 involvement in the injury of cardiomyocytes has been proven by histological analysis of murine hearts and the observation of myocardial death and destruction in virus infected myocytes at early post-infection (p.i.) time points 64>68>184'226. Pathologic effects are common before the infiltration of immune cells. The host inflammatory response then causes further injuries to the organ 206>219>282. CVB3-induced myocarditis is a poorly understood disease because it progresses through different stages with distinctly different mechanisms and manifestations. Recent analysis 2 0 6 of available data has concluded that the disease can be summarized by a triphasic model: Phase 1) Direct viral injury by viral replication in the heart muscles and subsequently the triggering of immune responses; Phase 2) Activation of immune responses to achieve viral clearance and recovery but possible development of immune and autoimmune injuries; Phase 3) Remodeling of the myocardium by mechanisms that eventually lead to dilated cardiomyopathy (Figure 2). During phase I of the infection, CVB3 enters host via the alimentary tract. The virus is harbored in immune cells of lymphoid organs, temporarily escapes immune clearance and is then transported to target organs such as the heart and pancreas 1 4 4 . Viral replication and direct cellular injury occurs rapidly in infected cells, causing cell lysis and viral dissemination. The viral mediated injury may not be as damaging to the myocardium as are the secondary immune responses that serve to eliminate viruses. Specifically, T-cells are activated by the classic cell-mediated pathway: via presentation of viral peptide fragments by the major histocompatibility complex (MHC) at the cell surface. This results in the destruction of the infected cell by cytokine and or perforin-mediated cell cytolysis. In the heart, the rapid cytolytic effect causes significant reduction in the number of effective myocytes in the contractile units. At the same Paul Cheung Page 7 time, molecular mimicry by viral peptides to host antigens on the myocyte surface will result in persistent T-cell activations, TH-2 responses and CD8 killer cells that are directed against healthy cardiomyocytes. Such is commonly described as CVB3-induced autoimmune injury and further amplifies the structural injury of the myocardium l 4 4 ' 2 0 6 . The significance of autoimmune injury in the course of CVB3-induced myocarditis has been demonstrated by the data that survival-rate of CD4/CD8- or p561ck-knockout animals is significantly better than that of the wild type mice during a CVB3 challenge ' ' . On the other hand, while autoimmune injury is a pathogenic consequence of CVB3 infection, the benefit of an intact host immune system in clearing CVB3 infection can not be ignored: CVB3 infection can result in higher mortality when the host is immunocompromised 1 8 1 . Events such as T-cell activation 1 7 8 , nitric oxide synthesis 211,293,354 a n ( j i m m u n 0 g i 0 f c . m m production 1 7 7 - 1 7 9 ' 1 8 0 a r e a i s o important in reducing disease severity and viral dissemination. In summary, a delicate balance between immune reactivity and viral clearance is essential to limit the severity of a CVB3 infection. Paul Cheung Page 8 Figure 2. Triphasic disease development of CVB3 induced myocarditis. C V B 3 is the prime agent in causing myocarditis. Acute infection, as well as cell death as a result of host inflammatory responses and autoimmune responses contribute to the injury inflicted upon the myocardium, is followed by which can progress to autoimmune injury (phase II) despite viral clearance. Autoimmune injury is often diagnosed by endomyocardial biopsy and serological markers. Death of myocytes and possible activity of proteases induce the remodeling of the myocardium, eventually leading to the condition of dilated cardiomyopathy (phase III). Repeated exposure to the virus can enhance the likelihood of developing dilated cardiomyopathy. (Model adapted from Liu, P. P. and J. W. Mason. 2001. Circulation 104:1076-1082.) Phase I Viral infection and direct tissue injury Recovery Phase II Viral Clearance Immune - and autoimmune mediated injury (infection resolved eventually) Repeated Infection -> Phase III Dilated cardiomyopathy Paul Cheung Page 9 1.4 Chronic infection by CVB3 Typically, CVB3 infection of murine models resolves rapidly after immune clearance and the initial phases of infection. However, there are isolated instances when viral RNA can persist in myocytes for as long as 42 days post infection, in immune-competent mice 1 8 5 . Using sensitive diagnostic methods such as PCR, CVB3 RNA has been detected at 90 days p.i. without significant levels of inflammation ' . Such are interesting observations as CVB3 is a single-stranded RNA and cytopathic virus that requires continuous RNA-dependent RNA replication in order to persist. Persistence of viral replication without cytopathic effect and apparent pathogenic markers in immune-competent animals suggests that there are yet other undiscovered reservoirs or mechanisms of viral persistence. 1.4-1 Myocarditis and dilated cardiomyopathy Extended periods of myocarditis will lead to the condition of diked cardiomyopathy. During myocarditis, the CVB3 protease 2A p r o can directly cleave the cytoskeletal proteins such as the dystrophin and sarcoglycan complexes in myocytes 2 3 ' 2 6 , causing myocardial remodeling and ventricular dilation, which are often observed soon after viral infection 24"26>199. Compounded to other events such as the activation of matrix metalloproteinases (gelatinase, collagenases, and elastases) 2 5 4 and repeated viral infections 2 4 3 , 3 3 6 s persistent heart muscle injury is resulted. Extended periods of tissue damage will lead to the condition of dilated cardiomyopathy 243>254>336 Cardiomyopathy is a term that describes the general illness of the heart muscles which can lead to clinically significant dysfunction. Heart muscle diseases due to infarcts from coronary atherosclerotic diseases are excluded from such a definition. The dilated, hypertrophic and restrictive forms of cardiomyopathy can be responsible for heart failure 9 7 ' 1 0 9 . Paul Cheung Page 10 Phenotypic pathology of dilated cardiomyopahty includes observations of significant calcification, fibrosis, myocardium remodeling and ventricular dilation 166>226. Murine models are often chosen to study the molecular mechanism of CVB3 induced myocarditis and dilated cardiomyopathy in vivo because the pathology observed in the mouse model is strikingly similar to those of the human conditions. Typically in CVB3-infected A/J mice, prominent changes to the myocardium occur within 2 days of infection, including coarse granularity and multivesicular vacuolation 183>184>226. From days 3 to 5, large areas of myocytes would appear damaged, with signs of calcifications °°> 1 0^°. The use of terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and ISH for viral RNA reveals that the presence of viral RNA can be associated with the pathology of cellular injury 6 4 ' 6 8 . Data from TUNEL-staining has been 78 125 186 regarded as a general indicator of cells that are apoptotic, necrotic or proliferating ' ' . At this stage of disease, significant changes in gene expression in the tissue can be detected by the method of differential mRNA display 3 5 °. As well, remodeling of the heart tissue begins in order to compensate for the reduced contractile capability. On day 7 and beyond, inflammatory infiltrates are common 6 8 ' 8 1 . Often, necrotic myocytes are indicative of the presence of viral mediated cellular destruction as well as the presence of auto-immune injury. For mice that survive past this point, infection should be resolved on day 9 and onwards. L5 Life cycle ofCVB3 The virion of CVB3 is extremely compact and the structure is simple in that a single molecule of a 7,399 nt long positive sense RNA, coupled to a 22 amino-acids protein (VPg), is enclosed in a 30 nm hexagonal protein coat. Similar to other picornaviruses, the CVB3 virion structure is composed of sixty protomer subunits. The process of CVB3 virion binding to host receptor has not been verified but is believed to be highly similar to that of other picornaviruses Paul Cheung Page 11 (Figure 3). Upon cellular entry, the genomic RNA is immediately expressed by the mechanism of internal ribosomal entry, producing a poly-protein of ~250 kDa. Autolytic cleavage of this poly-protein results in the generation of the individual virion capsid proteins and the viral proteases. The viral proteases are mostly responsible for "reprogramming" the cellular environment to achieve three major goals: 1. Exclusive translation of the viral genome RNA; 2. Inhibition of the majority of host protein translation; 3. Replication of viral genome RNA and the subsequent packaging of viral RNA into infectious virions. Replication of the viral genome and packaging of the new virions occur in the late stage of infection but before the lysis of the host cell. The life cycle of CVB3 is highly similar to that of other enteroviruses, such as the poliovirus, and a schematic representation of the cycle is illustrated in Figure 4. Paul Cheung Page 12 Figure 3. The picornaviral virion structure and viral entry (Adapted from B . N . Fields et.al. (ed)., Fields Virology, Lippincott-Raven Publishers, Philadelphia, Pa., 1996) Arrangement of VP1, 2, 3 on the surface of a picornaviral virion. VP4 is buried beneath the surface. Each asymmetric face of the virion is illustrated by a triangle. Each molecule of PI is autocleaved to generate one set of V P 1,2,3,4 asymmetric triangular units (yellow). A total of 60 asymmetric subunits integrate to form a complete virion. The structure and organization of the C V B 3 virion is expected to be highly similar. receptor(CAR) Entry of viral genome STEP 1: Virion binding to coxsackie-adenovirus receptor (CAR) internalization receptor (red triangle). A coated pit on the cellular membrane is formed. Acidic conditions inside the coated pits result in VP4 release and the fusion of lipid membrane with hydrophobic patches inside the virion. STEP 2: Fused virion and membrane permits the injection of the R N A genome (blue line) into the cytoplasm. Pau l Cheung Page 13 o > 41 JO T J i i 00 CD £ « g Cd 3 oo l l 2 a CL) Ou c cu cS I § o c 'ft 5 (50 o cu > .S » * 3 g .a > c CD CD c o U o. CL) CJ CD — < Q T J c C3 < u CL> CJ > JJ a c o T3 CJ 'ZZ cd 13 S I £ < cd CD 8 .2 >I cd — o o a O . OH cd i— 9.s o ^ a CD *o CU ^ i cj C3 a! oo CJ E CD CJ -a < CD CO +2 "S o & o >^  X) T J CJ cd CD TJ cd Ui In oo CD a , o g T J a cd < u r>> O « *-a> Id -C oo H « CJ . t Tf cd OH cd >-T J 3 00 CJ -C 1 i 00 CD C O c T J 4) S -s s > B -a oo a JS 3 t i CJ 00 o 2 CD JS 1.6 Cellular receptors Generally speaking, the study of cellular receptors of a virus is important to the understanding of pathogenesis and tissue tropism. For picomaviruses, at least nine receptors and "Kf\ ins 1 R attachment proteins have been identified ' ' . For CVB3, two specific receptors have been discovered and studied in detail: the 46 kDa coxsackie-adenovirus receptor (CAR) and the -70 kDa decay accelerating factor (DAF). 1.6-1 The Coxsackievirus-Adenovirus Receptor Both the human and mouse CAR were demonstrated to mediate attachment and infection of coxsackie B group viruses and adenovirus 2 and 5 ' ' . The importance of CAR in mediating infection has been demonstrated by studies that show that Chinese hamster ovary cells become susceptible to all six serotypes of coxsackie-B group viruses only after they are transfected by the CAR mRNA 2 2 0 . The murine CAR protein is 91% similar in sequence to the human C A R 3 6 . CAR is a transmembrane adhesion molecule of the immunoglobulin superfamily 3 6 (Figure 5). The trans-membrane domain does not play a role in the internalization of the virus 3 3 2 , and the endogenous function of CAR is uncertain, as are the downstream signaling events following the binding of extracellular substrates (and virus). The extracellular domain of the receptor is sufficient for virus binding and internalization. Current knowledge of the relationship between CAR expression and CVB3 infection is not sufficient to fully understand the cardiovirulence of the virus. For instance, the use of an anti-CAR monoclonal antibody does not attenuate the binding of CAR to some isolates of CVB3 2 2 0 . This suggests the presence of co-receptors or chaperone molecules that may participate in virus binding and presentation to the CAR receptor. The CAR receptor has been cloned and Paul Cheung Page 15 sequenced by various methods 35>61>62'318>342'343 Northern-blot analysis of the RNA expression indicates that the CAR mRNA is strongly expressed in human (and mouse) heart, brain, and pancreas, but is below the level of detection in the placenta or skeletal muscle 3 5 ' 3 1 8 . The patterns of mRNA expression correlate in part with the patterns of CVB3-induced pathology in mouse, with pancreas and heart being the most susceptible organs 2 3 0 . Yet, high levels of CAR expression in the kidney do not correlate with the general resistance of the organ towards CVB3. It is possible that transcription of the CAR mRNA may not truly reflect the amount of functional protein. At present, there is no definitive data regarding receptor protein expression and CVB3 tissue tropism. In light of other findings on the cardiovirulence determinants of CVB3 in the viral genomic RNA sequence (Section 1.7-3), it is reasonable to assert that the expression of CAR may only partially determine the tissue tropism of CVB3. Paul Cheung Page 16 09 U O a u a> s-< Q TS a S3 < u 43! H O l S -9 >>.£ "° £ cj C C cu £ u a C J § 43 £ H fe •£ JH 3 3 £ « -§ C J CU o o T 3 cj f < •o C3 CU cu Cw 42 ° £ <u cu eg § * I co .p cu rel •S cS O > +-> CU •a | S | o -2 •S s § £ CO £ T O Q _g U o •a g c C3 B CO g X O cu CO CU Oi cS C/3 R C" flj JS a P 5 fa »-'G & CO rel £ T3 -a C J T3 CO 43 C o = 3 a-cu cj S J S J C J -3 a g su a 43 [Id O Q C « 15 5 ° T» t S J = l T ccj CQ > U C Q. cj CJ CJ t-cu 43 •a ~r o < 2 l - l CU o 2 Qi 9 ^ a C N •§ 06 «>& O t/3 E H I • . c « cu CO C '5 E o Q a o — < -a c C3 < U CJ co S ret 43 •— o £| ° c cu O <u% £ !S J2 o P *? cj S J CN £ -a JS § 3 o > g 2 .2 S 1 I cu ™ £ 2 co t r -O >> C U 2 . x ,o S J o J8 S J 23 So  •a <s S J ? CN O C l CN Erf o co co & '5 B 1 o So 'J3 O ca 43 a a. B O a CJ CJ oi S o a CJ 43 O O 4 P oo ao CU u 1 a cr < C J o cn U o > • £ -i M .is S > u r 43 « S J J «« H « § u i C/3 cs cu 2 rel «B 1. 6-2 The Decay-Accelerating Factor DAF is a membrane-bound glycoprotein, which is also known as CD55. It serves as an attachment protein for a number of different human enteroviruses including enterovirus type 70 and coxsackievirus A21 83'168>224>296>299. Interaction of CVB3 with DAF appears to occur with the short consensus repeat (SCR) sequence 2 and 3 on the DAF protein 3 4 (Figure 5). The normal physiological function of this protein is to inhibit complement-mediated cellular damage at the C3 convertase level, thus preventing the generation of the membrane attack complex and other cytopathic molecules such as anaphylotoxins and opsonins 2 1 2 . The expression of DAF on the cell surface is an important mechanism to protect cells from complement-mediated lysis 2 4 8 . DAF is expressed in most human cells types, including the blood cells and the vascular endothelium 2 4 8 . Human and mouse DAF are both functionally and genetically similar 93,175,304,305 ^  a i m o u g n CVB3 only interacts with the human DAF and not with the mouse counterpart. The glycosylphosphotidylinositol (GPI) moiety on the DAF is critical for its 9 9 4 9 9 7 attachment to the cellular membrane ' but not for interaction with CVB3. The GPI moiety also allows the DAF molecule to interact with other membrane bound glycoproteins. For instance, dimerization of DAF or interaction with other unidentified proteins via the phosphatidylinositol domain can lead to interleukin 2 (IL2) secretion and T cell activation ' . The GPI domain of DAF is a post translational modification that occurs in the SCR 3 region. The mature form of DAF is about 70 kDa, although the MW of DAF can vary with the extent of glycosylation. Interestingly, while the GPI and SCR 3 are involved in initiating T-cell activation 8 6 , the same domain is also involved in interaction with the CVB3 virion (Figure 5). Thus, it is believed that CVB3 binding with DAF can have immunological consequences. Paul Cheung Page 18 The role of DAF in the pathogenesis of CVB3 is now believed to be that of a co-receptor: membrane-bound DAF captures a CVB3 virion from the extracellular environment and delivers the virion to the transmembrane CAR, which then mediates the internalization of the virus 2 9 9 . Shafren et al. showed that DAF expression in rhabdomyosarcoma cells does not permit viral infection unless CAR is co-expressed. Monoclonal antibodies against DAF are required to protect permissive cell lines against high input multiplicities of infection (MOI) 3 7 ' 2 9 9 . A similar mechanism of viral entry has also been reported in the lytic infection by coxsackievirus A21, which utilizes ICAM-1 and DAF as receptor molecules 2 9 7 , 2 9 8 . The use of soluble DAF in preventing CVB3 infection was demonstrated by our research group 3 4 1 . 1.7 Genetics of CVB3 Like other enteroviruses, the entire coding region of CVB3 is a single open reading frame (ORF) encoding one polyprotein. The coding region of CVB3 starts from nt 741 to 7299. This single polyprotein is organized into three regions: PI, P2 and P3 (Figure 6). Structural proteins (VP1-VP4) are encoded in PI, and non-structural proteins (2A-2C and 3A-3D) in domains P2 and P3, respectively. PI is encoded at the N-terminus of the polyprotein and the 2A protease (2Apro) is located immediately downstream of PI. Upon translation, the 2A p r o cz's-cleavage activity occurs between the C-terminus of capsid protein VP1 and the N-terminus of the 2A p r o itself, which then releases the PI precursor peptide (capsid proteins) from the P2-P3 precursor peptide. Most of the remaining cleavages of the picomaviral polyprotein are mediated by the viral enzyme, 3C protease (3Cp r 0 ) (Figure 6). The "primary" cleavage event by protease 2A p r o occurs very rapidly while the polyprotein chain is still nascent. In some polioviruses, protease Paul Cheung Page 19 S I CU O 60 ^ ed -cd <u cu CO 75 cd O. 1| o ^ O H-H g o < -s cu . s -^ 0) CU _g cu J3 cn k> cd c5 u D. 2-e o CJ '5b 3 *•« cu cu -T J S cd U <N <^  OH « >> ao _o o — O > cu e 'M -3 =L w CT g"TJ 3 CU C/3 O £ &.o cu .3 -8 « O JS 2 °1 « *3 O Q. O ,g cu m * -° J < « .y oo E s § •tt cu 8 M " 2 CU C3 oSi) c 'Sp «. 5 a CO cd K c o Q . 3 2 2 £ i l a E S 8 « •C T J cd S .S e « u o £ o c o | IB o > Hi Cd OH CU H ^ O o tn o, •— o .2 J* ^ . bD +•» > » " o » • S P H C ^ <= H -o o. 'I & O T J o O l M « pis*, Q f*> PQ < ca fN ft* > ft* fN ON > PH IT; cn 0_ cn C o u CH o 3 s :n I a o Z CM CU en C 8 — °-cu C/3 O -> £3 cu ep cd > cd cj U c B o H PH o a. cn 0) M ca > cd cj T J CU CJ 3 2 CJ 3 T J o cd C OX s 3 2A p r o recognizes another cleavage site on the 3 CD-precursor protein, producing non-functional products 3C and 3D' proteins instead of the viral protease 3C p r o and polymerase 3LV01. These alternative cleavages of the 3CD protein by protease 2A p r o appears to be strain-specific and the cleavage products are not essential for viral replication 1 9 0 , 2 8 9 . Thus, the "secondary" cleavage and formation of the 3C and 3D' proteins were speculated to modulate the ratio of functional capsid proteins and viral enzymes in an infected cell 1 9 0 . The PI region of the genome encodes four virion proteins, known as VP1, 2, 3, and 4. Each of the 60 subunits of the virion is constructed from these four viral proteins. Data from X-ray crystallography indicates that the structure of the viral capsid proteins VP 1, VP2 and VP3 is highly conserved among CVB3 and other picornaviruses 2 3 9 . Structural differences in the capsids between CVB3 and other enteroviruses are located mostly on the virion surface. The canyon and major surface depressions of the virion are predicted to be the primary and secondary receptor-binding sites, respectively. Assembly of the virion capsid from VP 1-4 occurs by the following process 1 0 8 ' 2 8 8 ' 3 3 9 : the PI capsid precursor is myristoylated to form 5S promoters, which are then cleaved by the protease 3CD p r o to result in VPO, VP3, and VP1 proteins 1 6 1 . The VPO protein is the combined form of VP4 and VP2 proteins. Repeating units of 5S promoters are then assembled into 14S pentamers. The 14S pentamers are further assembled into 75S hexagonal empty capsids. Packaging of the genomic RNA into the capsid forms the 160S provirions. The VPO protein in the pro-virion is then cleaved by the protease 3Cpro to produce VP4 (inside virion) and VP2 (on the surface), effectively closing the capsid and forming a mature progeny virus. The P2 region of the genome encodes three polypeptides: 2A p r 0, 2B and 2C proteins. 2A p r o is a major protease that is responsible for many pathogenic events, and the functionality of Paul Cheung Page 21 this protease will be discussed in detail in section 1.7-2a. The 2B protein modifies the permeability of cellular membranes, by creating amphipathic pore structures which disrupt cellular Ca 2 + concentrations. This modification can lead to the release of viral progeny from infected cells ' ' . The 2C protein is postulated to participate in viral replication ' . It contains three motifs that are characteristic of an RNA transcription function: an amino-terminal amphipathic helix 2 6 ' , a putative zinc finger 1 3 7 , and a nucleoside triphosphate (NTP)-binding site 9 8 . Other possible functions of the 2C protein may include RNA encapsidation 2 0 1 and molecular mimicry. Sequence homology between the 2C protein (amino acids. 32-47) of several members of CVB and the glutamate decarboxylase (GAD, amino acids. 247-279), which is a major diabetes antigen, has been observed 1 5 ' 1 6 9 . These cellular immunological findings suggest an inductive role for Coxsackie B viruses in autoimmunity 1 5 , 1 6 9 . The P3 region of the enterovirus genome encodes the 3A protein, 3B (Vpg) protein, protease 3C p r o, and 3D polymerase. Stable intermediates of 3AB protein and protease 3CD p r o are also present and functional during infection. The functional 3AB protein and protease 3CD p r o localize in membranous replication complexes 40>314>315. Based on data from mutational studies 3 8 , the 3A and 3AB proteins are postulated to mediate viral replication. Also the 3AB protein and 3D polymerase cooperate to initiate the activity of RNA polymerase 192>193-260. The protease 3CD p r o polypeptide is a proteinase that is responsible for RNA transcription as well as cleaving the polyproteins 133'134>161. 3C p r o is also a major CVB3 proteinase and its functionality is discussed in section 1.7-2b. The 3B (Vpg) protein is a short, 22 amino acid residue protein that is attached to the 5' end of the genomic RNA. The Vpg is proposed to be an essential feature in forming the replicative complex of the virus. It is present on genomic RNA of the virus, while viral RNA that is undergoing translation does not necessarily possess the Vpg. Paul Cheung Page 22 1.7-1 VPs protein A unique feature of the picornaviral genomic RNA is the covalent linkage of a twenty-two amino acid Vpg protein at its 5' end. This is in contrast to the host mRNA which contains the 7-methylated guanosine triphosphate cap-structure. The functional role of the Vpg remains unclear, but it may serve as an anchor for the formation of a replication complex 3 0 , 3 1 5 , 3 1 7 . The viral Vpg protein, however, cannot substitute the function of the 7-methylated guanosine triphosphate cap structure. Without the 7-methylated cap structure, the CVB3 RNA cannot be recognized and translated by the normal method of host translation initiation, the ribosomal scanning mechanism. Instead, translation of the CVB3 RNA occurs by direct ribosomal entry into a specific viral sequence known as the internal ribosomal entry site (IRES). The general mechanism of internal ribosomal entry has also been observed in many other RNA viruses and in a small number of eukaryotic mRNAs. 1.7-2 CVB3 proteases 2Apr0 and 3C?ro The proteases 2A p r o and 3C p r o are crucial in the life cycle of CVB3. In addition to processing the viral polyprotein, they also cleave major host proteins and result in the severe loss of cellular homeostasis. So far, identified targets of cleavage include translation proteins 3, anti-apoptotic proteins l l 9 , caspases 2 9 , and structural proteins such as dystrophin 2 6 (Figure 7). Beyond causing immediate cell death, such cleavages are also related to the remodeling of the infected target organs such as the myocardium, wherein dilated cardiomyopathy can be a consequence of acute CVB3 infection 90>245. The structures and functional significances of these two proteases, 2A p r o and 3C p r o, are discussed below in detail. Paul Cheung Page 23 CJ -(-» o ft "o3 B f> .2 m g 00 o ft' „ CO fe o J 1 ca -a | o 00 *H O 5 3 ° — "5, 5 g « S CJ .5 co o f : § r#i t*"\ OT CO CO E 2 S a. .3 o •a ft H 03 03 CJ T3 o e eo o o. E cu 4= E o o ca ft C N cj 60 CO > 03 CU f> CJ 43 s <-8 «g cu '35 oo 60 c — c & ° T3 S J 2 « 6 .S fi tj o < oo o b « ! cu 4= o ft cm CU f e s a T3 a» c >• 03 CO v -a esjD .5 88 ft I F I • CN fij cu 93 cm s § ? 2 « ft J - CO .2, 2 S3 > 5j SJ ^ i ~ CO 2 O s u 60 cu J2 •g cu 03 > cu § J ? cs W 03 5 § 5. 03 SJ •E 5 2 pq cj II ftO 0 cfl fe « •§> I CU I 2 1 03 ft O ft f« 03 i i 03 g "3 j 3 a eg •a cj < 43 3 SJ 60 03 03 CJ aj c m SJ r— | I ° I ± •£ > .a B CL — o iS I CO £ CO C ft u O e _o c/5 '-*-» 'en 03 o > ft Act apo on « a. a o c o ft tu I— ~5 c a *c o c« c 03 60 C "SJ -a o E S J 3 CJ CJ +^ 43 CJ 43 1 ft O >> E o | 03 CJ &2 aj Cfl -*-* C« 03 O ^3 ft -a DO c '•5 03 SJ M B 3 cu 4= u 0. 1.7-2a Protease 2A (2A p r oi Coxsackievirus protease 2A (2Apro) is a small cysteine protease, about 17 kDa, which shares sequence homology with bacterial trypsin-like serine proteases 2 8 9 , 3 5 3 . in the case of the entero- and rhino viruses, the catalytic triad of protease 2A p r o was identified as His-20, Asp-38 and Cys-109 ' . The thiol group of the cysteine residue acts as a nucleophile and its catalytic • • 111 activity can be strongly inhibited by thiol alkylating reagents ' , as shown by inhibitor studies using iodoacetamide and N-ethylmaleimide, compounds that are known to be active against thiol proteases 2 8 9 . The role of these residues in catalysis was confirmed by site-directed mutagenesis. These data also suggests that protease 2A p r o is structurally more related to a small trypsin-like serine protease than to a typical papain-like cysteine protease ' . A proposed model of the protease 2A p r 0 of human rhinovirus type 2 suggested that the overall structure of viral protease 2A p r o is similar to that of Streptomyces griseus protease A: both proteases were found to share the same components in the domains of the catalytic center 2 9 5 . Recent data has characterized the protease 2A p r o as a zinc-containing enzyme, in which the metal ion, Zn 2 + stabilizes the protein rather than being directly involved in catalyzing proteolysis ' ' . As mentioned earlier, the cw-cleavage activity of protease 2A p r o takes place between the C-terminus of capsid protein VP1 and the N-terminus of 2A p r o itself. Tra/w-cleavage by protease 2A p r o also occurs in an increasing list of cellular factors. Over the past decade, a major focus of studies on protease 2A p r o has been its cleavage of eukaryotic translation initiation factor 4G (eIF4G). The focus on eIF4G cleavage is due to the probable association of this activity with picornaviral-induced inhibition of translation of host proteins. Investigations have demonstrated that purified recombinant protease 2A p r 0 of CVB4 and human rhinovirus type 2, as well as PV, can cleave eIF4G between amino acid residues Arg485-Gly486, in vitro 54>195. Given the known Paul Cheung Page 25 functional role of eIF4G as a bridge between the ribosome and mRNA, the cleavage of eIF4G is believed to inhibit normal host protein synthesis. During viral infection, cleavage of eIF4G occurs very rapidly, in an average of two hours post-infection 1 9 5 . Timing of this cleavage event coincides with that of the reduction of cap-dependent translation of cellular mRNA and the rise of cap-independent translation of viral mRNA. On the other hand, studies of PV infection in cell culture environments have shown that host protein translated by the cap-dependent mechanism is suppressed by only 50% or less despite the complete cleavage of eIF4G 2 0 8 , 2 6 9 . Hence, the general inhibition of host protein synthesis during a picornaviral infection may require proteolysis of factors in addition to eIF4G. Recently, a novel human homologue of eIF4G, called eIF4GII (while the original eIF4G was renamed as eIF4GI), has been reported as the second event required for the complete inhibition of host protein synthesis 123>124. eIF4GII appears to be functionally equivalent to eIF4GI, but is only 46% identical at the amino acid level 1 2 3 (Figure 8). Both poliovirus and human rhinovirus protease 2A p r o were found to cleave eIF4GII, with slower kinetics as compared to the cleavage of eIF4GI. The cleavage can result in the greater inhibition of host protein synthesis 1 1 9- 3 1 0. These data may explain several earlier reports documenting the lack of correlation between eIF4GI cleavage and the partial inhibition of cellular mRNA translation after 9 0 S 9ftO enterovirus infections ' . More significantly, the cleavages of both eIF4GI and eIF4GII may trigger host cell apoptosis, either directly by inhibiting the cap-dependent expression of proteins required for maintaining normal cell viability or by enhancing the cap-independent expression of important regulatory proteins such as c-Myc 1 5 9 and death associated protein (DAP5) 1 3 8 , which may play roles in the activation of cell death responses. Paul Cheung Page 26 Figure 8. Homology between eIF4G I and 4G II. Both proteins are functionally similar, with an overall 46% a.a. sequence homology. Sequence homology in the eIF3 binding domain (blue) is 79%. The eIF4GII is 1585 a.a. long and the eIF4G I is 1560 a.a. long. Both can be cleaved by picornaviral 2APro and the cleavage of both eIF4G variants correlates with the general inhibition of host protein synthesis during an infection. Other protein-interaction domains are also indicated. (Adapted from Gingras, A . C , B . Raught, and N . Sonenberg. 1999. Annu. Rev. Biochem. 68:913-963). |- 2Apro Cleavage site t PABP binding domain I t eIF4E binding domain t eIF3 / 4A Binding domain I 2Apro Cleavage site t eIF4A binding domain I eIF4G I eIF4G II Paul Cheung Page 27 1.7-2b Protease 3C (3Cpro) The protease 3C p r o is a 20 kDa cysteine protease that is phylogenetically conserved among picornaviruses. Protein cleavage mediated by the poliovirus protease 3C p r o occurs exclusively at glutamine and glycine bonds, although in other picornaviruses the glycine residue may be substituted by other amino acids such as serine and alanine 14>257>258-278. The viral protease has been cloned and characterized as a cysteine protease that can be inhibited by N-ethylmaleimide and iodoacetamide 2 6 5 . The histidine and cysteine residues are believed to form the active site, as confirmed by X-ray crystallography of protease 3C p r o from the HRV14 and HAV 6 ' 2 1 5 ' 2 2 2 . The protease 3C p r o is generated from the autolytic cleavage of 3 CD (Figure 6). Alternative cleavage of 3CD by the polio protease 2A p r o can give rise to 3C and 3D' 1 8 2 , which is not required for the generation of infectious virus particles 1 7 0 . It is suggested that the alternate cleavage of 3CD serves as a possible regulatory mechanism for the replication and maturation of pro-virions 1 9 0 . The protease 3C p r o is capable of cleaving a number of host proteins (Figure 7). The 3D domain is an RNA-dependent RNA polymerase but in the uncleaved form (3CD), the presence of the 3D domain serves to alter the substrate specificity of protease 3C p r 0 in the processing of capsid precursors 1 6 1. Recombinant protease 3C p r o 4 2 has been used to study its effect on host protein cleavages • ' ' . Of note, a number of translationally important host proteins are affected by the protease 3C p r o. Although a significant body of evidence suggests that cleavage of the eIF4G family is mainly executed by the protease 2A p r o , there is also evidence that protease 3C p r o induces cleavages of eIF4A and 4G in the FMDV system 3 3 . Our research group has also observed cleavages of eIF4G by the CVB3 protease 3C p r o 3 5 1 . Another important target protein Paul Cheung Page 28 of the protease 3C p r o is the La autoantigen, a 408-amino acid residue, cellular protein that has \fi 1 1 0 1 ^ tft 7R7 been demonstrated to stimulate internal ribosomal entry ' ' ' . The normal physiological functions of the La protein includes tRNA folding, synthesis of polymerase II transcripts and formation of snRNP complexes as well as ribosomal binding ' ' ' . Cleavage of La by the poliovirus protease 3C p r o occurs in the C-terminal region, between Gln358 and Gly359 3 0 1 . This protease modification of the nuclear-bound La protein can cause it to relocate into the cytoplasm, and such relocation is observed in a number of other independent studies 1 7 2 1 (also discussed in section 1.9-2). The Picornaviral translation take place mostly in the cytoplasm, and thus the cleavage of the La autoantigen is believed to improve the translational efficiency of the virus. The poly-pyrimidine tract binding protein (PTB) is another major nuclear protein cleaved by the protease 3Cpro. The PTB is pertinent to picornaviral translation initiation because it has been shown to bind viral 5'-UTR sequences and cooperate with the La autoantigen in stimulating translation initiation of the viral genome 147>,64>165 Interestingly, cleavage of the PTB by poliovirus protease 3C p r o occurs in 3 different positions, at amino acid residues Ala-X-X-Gln/Ala, generating multiple peptides which appear to inhibit IRES-dependent translation in a cell-based assay system 2 2 . The cleavage results in inhibition of its capability to bind to another translation stimulatory molecule : poly-rC-binding protein (PCBP) 1 7 ' . The RNA-recognition motiff (RRM) of PTB resides in RRM-3 and RRM-4 2 6 8 . The protease 3C p r o can cleave all forms of the PTB protein (including isoforms: PTB1, PTB2, and PTB4), and the cleaved products are redistributed to the cytoplasm from the nucleus 2 2 . The fragments of PTBs are hypothesized to interfere with the binding of intact PTBs to the PV IRES. Analysis of the cleavage event reveals 22 that the cleaved PTB protein can stimulate replication of the viral RNA genome Consequently, the cleavage event has been suggested as a molecular switch from viral protein Paul Cheung Page 29 expression to viral genome replication . However, the molecular mechanisms signaling the appropriate time of transition remain unknown. In a picornaviral infection, successful expression of viral proteins increases the amount of functional protease 3C p r o (along with other viral structural and non-structural proteins) in the cytoplasmic environment, and the rising cellular concentration of protease 3C p r o may increase the overall likelihood that the protease will cleave proteins that are already bound to the viral 5'-UTR RNA (such as the La, PTB and other proteins). Thus, the overall transition from the stage of viral protein translation to the stage of genome replication may be achieved in a gradual manner 1 1 . This assertion requires further studies and verification. Nonetheless, it underscores the significance of the viral protease 3C p r 0 in the pathogenesis of the picornaviruses. 1.7-2c Cytopathic effects induced by the proteases Activity of the CVB3 proteases can directly lead to cytopathic effects (CPE). The term CPE is used in multiple models of virus infection when significant cell death is observed in lieu of morphological changes. In the case of CVB3, rapid CPEs are observed in HeLa cell cultures, typically resulting in the severe loss of cellular homeostasis, rupturing of mitochondrial or plasma membranes and the alteration in cellular morphology. These observations occur usually from 7-9 hrs post infection in HeLa cell cultures 6 6 ' 6 8 . These CVB3-specific CPEs are consistent with the classical definition of necrotic cell death 214>322. in the picornaviral family, the first and best studied viral protease causing CPE is the protease 2A p r o . Over-expression of recombinant protease 2A p r 0 alone in a cell line is sufficient to induce cell death 1 1 9 . Structural damage by the protease 2A p r o in a cellular environment can be related to the cleavage of intermediate filament family members, dystrophin and cytokeratin 8, leading to cell lysis 2 3 ' 2 5- 2 6 . These structural proteins may also have scaffolding or docking functions Paul Cheung Page 30 that are especially important in the formation of intricate signaling pathways . Furthermore, depletion of the dystrophin-glycoprotein complex is the primary cause of Duchenne muscular dystrophy n ' 5 5 , and has been identified in patients with viral myocarditis leading to DCM 2 6 . Hence, there is evidence of the causal effect of the CVB3 protease activity in remodeling of the myocardium, which eventually leads to the condition of dilated cardiomyopathy. Equally significant in causing cell death is the protease 3C p r o. The PV protease 3C p r o induces drastic morphological alterations and apoptotic cell death by a caspase-dependent mechanism 2 9 . The protease 3C p r o has been demonstrated to cleave the cyclic-AMP-response-element-binding-protein (CREB/ATF) 3 4 4 , transcription activator octomer-binding-transcription-factor (OCT-1) 3 4 6 , the TATA-binding protein and TFIIIC 8 2 ' 3 4 5 , and the histone protein (H3) 106,316 ( p j g U r e 7 ) -p/he deavage of these major transcription factors and host proteins reduces the efficiency of host mRNA expression and hence, leads to eventual cell death by inhibition of host protein expression. In addition to the protease 2A p r o , and 3C pro, the viral protein 2B can modify the permeability of the plasma membrane, mitochondria and endoplasmic reticulum (ER) ' ' . This will disrupt the delicate balance between selective membrane permeability and cellular homeostasis. Damage to the ER will release calcium into the cytosol, and contributes to further cellular injury 4 . Ukimura et al. 3 2 4 have provided electron microscopic evidence of mitochondrial swelling and rupture in infected mouse hearts. The direct consequence of losing mitochondrial membrane integrity is the release of calcium and death pathway "trigger molecules" such as cytochrome C, AIF, and caspase-2 and -9 39'65>66) leading to apoptotic responses. This is in contrast to other protease activity that leads to rapid cell lysis. Of note, the cap-independent translation of some host proteins continues (discussed in section 1.8-3). The Paul Cheung Page 31 apoptotic response of the CVB3-infected host is both interesting and important in the course of disease development. Detailed discussion of the relationship between apoptotic responses and CVB3 infection can be found in recent articles that our group has published 67>355'356. 1.7-3 Molecular determinants of CVB3 cardiovirulence Among the six serotypes of the coxsackievirus B group viruses, there are high homologies in the genomic RNA sequences. In spite of these similarities, each of the six viruses exhibits very different tissue tropism. For instance, CVB4 is a well studied agent in diabetes, while CVB3 is notorious for its cardiovirulence. Within the CVB3 serotype, several strains exist that demonstrate specific differences in cardiovirulence. For example, the CVB3/0 is a noncardiovirulent strain and unable to induce inflammatory heart diseases, while CVB3/m is cardiovirulent and can cause severe myocarditis. Persistent infection with CVB3/m may also result in the development of dilated cardiomyopathy 5 6 , 3 1 9 . Upon close inspection, the coding sequences of all CVB3 strains are very similar. This raises the question as to what determines the specific cardiovirulence and pathogenicity of coxsackieviruses. 1.7-3a Relationship between virulence and IRES Studies from the tissue tropism of PV has provided some insights into the molecular mechanisms of CVB3 cardiovirulence. One of the remarkable properties of the PV is its capability to infect motor neuronal cells (as opposed to sensory cells) in specific areas of the neural system, namely the spinal cord of the cervical and lumber regions while the cerebral cortex, the olfactory and the thoracic regions are spared 149>249'341. The attenuated PV vaccine strains have provided possible explanations for this tissue tropism. In this attenuated strain of PV, mutations were mapped to a specific region in the 5'-UTR of the viral RNA 1 5°. This region was subsequently identified as the domain V of the IRES 3 3 9'. Within this domain, nts 480, 481 Paul Cheung Page 32 and 472 were mutated in PV types 1, 2 and 3, respectively 1 2 8 . Point mutations in domain V significantly reduced translation efficiencies of the corresponding viral RNA as compared to wild-type templates. This is the first evidence that the translation efficiency of a viral genome is directly related to its virulence. Recently, in order to determine the structures responsible for neurovirulence, Gromeier et al ' used a chimeric virus strategy to dissect the PV and human rhinovirus-2 IRES elements. They found that both domains IV and V of the PV 5'-UTR are required to produce a neurovirulent phenotype. It appears that specific UTR sequences are coordinately responsible for the neuropathogenicity (neurovirulence) of PV. Following the work in the PV system, molecular determinants of the tissue tropism of CVB serotypes were also discovered within the 5'-UTR. CVB1, a less pathogenic strain than CVB3, has virulence determinants in nts 117 to 161 of its 5'-UTR 2 8 4 . Also, Tu et al 7 5 ' 3 2 3 found that an U to C mutation at nt 234 within the CVB3 5'-UTR results in attenuation of the cardiovirulent phenotype in mice. Replacement of the cardiovirulent CVB3/m or CVB3/20 5'-UTR with that of avirulent strain CVB3/0 attenuates the ability of the resultant viruses in causing 197 198 myocarditis ' . These investigators subsequently analyzed multiple clinical CVB3 isolates as well as other enteroviruses and demonstrated that nt 234 is always U regardless of the cardiovirulence phenotype of the virus 7 4 ' 7 5 . Thus, they concluded that the previously reported U-C mutation at nt 234 is most likely the result of a rare artificial mutation, as opposed to a naturally occurring genetic variation. However, their findings do imply the likelihood of discovering other sites in the CVB3 5'-UTR that can account for the variations in virulence as seen in the natural isolates of CVB3. In the search for these determinants, Gauntt and Pallansch 1 1 5 performed partial sequence analysis of the 5'-UTR (nts 300-599) of 15 CVB3 wild-type strains and found that nt 565 was A Paul Cheung Page 33 in cardie-virulent strains and U or C in avirulent strains. These data suggest that translation efficiency is related to CVB3's tissue tropism because this position is located at the center of the CVB3 IRES 3 4 9 . Recently, Dunn et al. 1 0 2 used two phenotypically and genotypically different clinical isolates to examine the genetics of CVB3 cardiovirulence. By constructing of a chimeric virus and testing it in a mouse model, these investigators finally confirmed that the 5'-UTR harbors natural genetic elements which regulate the cardiovirulent phenotype of CVB3: a chimeric virus, constructed by replacing the entire 5'-UTR of a cardiovirulent CVB3 strain with that of the PV strain, cannot induce heart disease in young mice. However, inoculation of this non-virulent chimeric virus does stimulate protective immunity against inflammatory heart and pancreatic diseases that are triggered by subsequent virulent CVB3 challenge 73'. Further work has also identified the high order secondary structures in the 5'-UTR as possible determinants of cardiovirulence 1 0 1 . These findings are very interesting and strongly highlight the fact that CVB3 cardiovirulence is related to the functions of the 5'-UTR. The functions mediated by the 5'-UTR are in turn dependent on interactions of host proteins with the UTR sequence. Currently, there are no data on such interactions. It is thus an objective of my dissertation to discover and characterize these host protein- CVB3 UTR interactions. 1.7-3b Virion capsid proteins and CVB3 virulence In general, the efficiency of interaction between virion and receptor is a determinant of virulence. For enteroviruses, the conformation and structure of the capsid proteins contain determinants contributing to the pathogenic phenotypes of PV 5 3 , CVB4 2 8 0 , and FMDV 2 X 1 . In the study of CVB3, specific virulence of the virus towards cardiomyocytes is of particular interest due to its clinical consequences. Early studies on this subject were focused on the interaction between capsid proteins and the viral receptor. Zhang et al. 3 5 8 derived an attenuated Paul Cheung Page 34 strain of CVB3 from a highly cardiovirulent parental strain in cell culture. Characterization of this attenuated strain using a chimeric virus system demonstrated that sites other than the CVB3 5'-UTR were responsible for the attenuation. The search for determinants in the coding region of the viral genome was performed by passaging the attenuated CVB3 strain in the heart of SCID (Severe combined immunodeficiency disease) mice and eventually a revertant with wild type cardiovirulence was isolated 6 0 . Sequence analysis and comparison of the revertant virus with the wild-type cardiovirulent CVB3/Nancy strains suggested that amino acid 155 in the VP1 region might play a role in attenuation By comparing the near-atomic-structures of the CVB3 virion of different isolates, Couderc et al and Dunn et a l 8 5 ' 1 0 2 found that tissue tropism determinants of PV and CVB3 could be mapped to amino acids exposed on the surface of the virion, on sites of the VP1 and VP2 subunits which are known to form an important neutralizing antigenic domain of the PV virion 2 5 6 . The CVB3/AS (cardiovirulent) and CVB3/CO (non-cardiovirulent) strains differ from each other in the major surface protrusion region of VP3 1 0 2 . However, neither of these capsid surface differences appears to affect the tropism of the cardiovirulent strain CVB3/20 when its capsid proteins are replaced by those of either CVB3/CO or CVB3/AS 1 0 2 . Therefore the relationship between the capsid protein sequences and cardiovirulence remains at best a strong correlation. Besides CVB3 virion proteins, the CVB3 5'-UTR can also determine cardiovirulence. Current knowledge with respect to the determinants of CVB3 cardiovirulence resemble findings in the poliovirus system: the primary determinant of PV neurovirulence is localized in the 5'-UTR, while variations within the capsid protein sequence may also influence the full expression of the overall tissue tropism53'85. Paul Cheung Page 35 1.8 Expression and replication of viral genome Expression of the CVB3 genome occurs by the mechanism of internal ribosomal entry. This is also known as the cap-independent mechanism of translation initiation, as the viral RNA does not possess a methylated 5' guanosine structure which is present in eukaryotic mRNAs. Similarities between the prokaryotic translation system and the viral internal ribosomal entry mechanism have been discussed in several recent publications 3 4 8 2 4 7 ' 2 7 0 ' 2 7 2 ' 3 0 6 , Recent work in our laboratory has defined the structures of the IRES in the 5'-UTR of CVB3 (Figure 9) 3 4 9 . By mutational analysis and in vitro translation using a bi-cistronic plasmid containing a CVB3 PI reporter gene, the minimum IRES-sequence has been determined to be located within the 5'-UTR stem-loops F, G and H 3 4 9 . Further analysis by mutations of the full-length CVB3 cDNA and transfection of HeLa cells has defined the 5' and 3' boundaries of the IRES as spanning nts 309-432 and 639-670, respectively 2 0 7 . In addition, a 46-nt polypyrimidine tract was found to be critical for viral translation and infectivity 2 0 7 . An interesting property of the IRES mechanism is the continuum of translation initiation during the G2/M phase of the cell cycle. This is opposite to the expression of host mRNAs by the cap-dependent method 4 7 . As well, recent studies on HCV using a bicistronic RNA with the HCV IRES at the intercistronic space demonstrated that in synchronized cultures of stably transfected cells, the IRES activity varied in relation to the cell cycle: the IRES activity was the greatest during the M phase and the lowest during the quiescent (Go) phase 1 4 3 . It appears that HCV translation is regulated by the cellular IRES binding proteins that vary in abundance during Paul Cheung Page 36 Figure 9. The C V B 3 5' UTR secondary structures and IRES homology A schematic of R N A secondary structures in the 5 'UTR of C V B 3 (Yang et ai, 1997). The predicted base-pairing model between the pyrimidine-rich tract sequence of the viral 5 'UTR and the 3' terminal sequence of human 18S rRNA is indicated. Complementary nucleotide interactions are represented by dots. The ranges of the numbers indicate 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. Dots between two sequences denote base pairing. (Adapted from Yang, et al., 2003) A: 10-34 B: 40-81 C: 104-180 D: 188-208 E: 209-481 F: 484-514 G: 519-560 H: 581-624 1: 625-641 J: 646-659 K: 688-741 H 5 5 0 A CA A G 563 A A A 4nJCAUUUUAUUCCU J I • •••••• « • AAAAUGUGAAA" C C ' 650 - 5 ' u AUG o< 742 °> .V end of human 18S rRNA Paul Cheung Page 37 the cell cycle 1 4 3 . Further, the variation of IRES activity during different cell cycles suggests that the IRES mechanism is a tightly regulated function of the host and requires specific cytoplasmic conditions for maximal efficiency. Thus, investigating host proteins that promote such conditions is important to understand how the expression of viral genes can be intervened. 1.8-1 Classification of picornaviral IRES Extensive work has been performed to classify IRES elements based on their functional roles and / or their sequence homologies. A summary of the three types of IRES is presented in Table 2. Current data support the classification of all picornaviral IRES into three major categories. Results of classification based on IRES sequence homology 4 9 agrees well with the results that are based on the functionality of IRES in specific environments 5 1 . Borman et al. performed transfection studies and analyzed the activities of six different picornaviral IRES in six different cell lines 5 1 . From those analyses, it was proposed that type I IRES include those of the enteroviruses and the rhinoviruses. These IRES direct internal initiation efficiently only in specific human and mouse cell lines 5 1 . They are inefficient in driving translation initiation in rabbit reticulocyte lysates (RRL) unless HeLa cell lysates are added. In vitro translation reactions with type I IRES are sensitive to changes in concentrations of KC1 and MgCh in the reaction. Also, the in vitro activity of type I IRES can be enhanced by addition of the picornaviral 2A protease 202>359. In contrast, type II IRES are generally efficient in most human and mouse cell lines 5 1 . Type II IRES include those of the cardiovirus, apthoviruses, and the non-picornaviral HCV. Unlike the type I IRES, they are neither sensitive to the fluctuation of salt concentrations nor the presence of protease 2A 2 5 2 ' 3 6 0 . Finally, type III IRES appear incapable of directing efficient internal translation initiation in most cell lines and their in vitro Paul Cheung Page 38 Table 2. Classification of three types of IRES Adapted from B.N. Fields et. al. (ed)., Fields Virology (Lippincott-Raven Publishers, Philadelphia, Pa., 1996 and from The International Committee on Taxonomy of Viruses. http://www.ncbi.nlm.nih.gov/ICTVdb/ Representative I R E S virus/genus I R E S length Efficiency Other variables Type I Enterovirus (CVB3) Rhinovirus 30-150nt Highly restricted upstream in R R L and in authentic A U G other human or mouse cell lines Sensitive to KC1, M g C l 2 , Stimulated by 2AP r o Type II Cardiovirus Apthovirus Can extend Generally efficient Insensitive to beyond the in most cell lines KC1, M g C l 2 , and authentic A U G 2AP T O Type III Hepatovirus ( H A V ) Can extend Not efficient in Mutations in beyond the natural cell lines IRES may help authentic A U G efficiency. Paul Cheung Page 39 activity cannot be rescued by the addition of HeLa cell lysates. So far, the only type III IRES reported is that of the HAV. The extremely low activity in vitro 5 1 of the HAV IRES is also correlated to the poor replication of HAV seen in cell culture environments 2 0 0 . The unusually low efficiency of this type of IRES in cell lines can be alleviated by accumulating mutations in the IRES during repeated culture-adaptations 9 4 , implicating the importance of RNA structure in mediating efficient interaction with the translation apparatus. The general RNA sequences of both the type II and type III IRES appear to extend up to, 117 1 AH 1 ft \ 78^ include, or even continue beyond the authentic start site for translation ' ' ' . In contrast, the type I IRES (enteroviruses) typically lies between 30 and 150 nt upstream of translation initiation 48>191>247. Experiments conducted in our laboratory showed that a type I IRES exists in the CVB3 5'-UTR: in vitro translation of the CVB3 PI gene is restricted in the RRL system, but such a restriction can be relieved by the addition of HeLa cell extracts 3 4 9'. Thus, trans-acting factors that are specific for the CVB3 IRES must be abundant in HeLa cell extracts but not in the RRL cytoplasmic environment. To date, studies from other RNA viruses have revealed several HeLa cell factors that participate in viral translation and or replication via interactions with the UTRs, such as the PTB and La protein. The 5'-UTR binding locations of these proteins appear to be dependent on individual viruses 5'136'228>294. The protein interactions with picornaviral UTRs are further discussed in the section 1.9 1.8-2 Cap-independent translation initiation of eukaryotic mRNA Internal ribosome binding is not restricted to uncapped picornaviral RNA as IRES also exist in viruses whose RNAs are capped at the 5' end. These viruses include the HCV 2 5 0 and the pestivirus 2 1 1 . Interestingly, increasing number of reports have indicated that a certain number of cellular mRNAs also employ the internal initiation mechanism for translation. To date, such Paul Cheung Page 40 mRNAs include the immunoglobulin heavy-chain binding protein (Bip) 1 5 9 , 2 1 3 ; eIF4G 1 1 3 , death-associated-protein-5 (DAP5, also called p97 or NAT1) 1 3 8 , fibroblast growth factor-2 (FGF-2) 1 3 ' 3 2 5, the proto-oncogene product c-Myc 2 4 4 , ornithine decarboxylase (ODC) 2 1 9 , Rbm3 (a cold stress response mRNA) 7 7 and vascular endothelial growth factor (VEGF) 1 4 5 . A summary of genes that are translated by the IRES mechanism is listed in Table 3. The 5'-UTRs of these host genes have short IRES: Bip includes a 220 nt IRES 2 1 3 and eukaryotic translation initiation factor 4G (eIF4G) has a 101 nt IRES 1 1 3 . Note that the presence of a 50-100 nt 5'-UTR is 9^19^9 . • > commonly observed in host mRNAs ' , but the IRES is a specific sequence element that, if present, can facilitate cap-independent translation initiation of the respective mRNA. Interestingly, translation of these host proteins may also be stimulated or inhibited by proteins (such as La autoantigen and the PTB) that play specific roles in the translation of picornaviral RNA 1 7 2 ' 1 7 3 . One important structural distinction between host mRNA IRESes and picornaviral IRESes is the lack of a polypyrimidine tract in the 5'-UTR of host mRNAs 1 1 6 . However, this difference may not be critical for the functionality of mRNA IRESes because the activity of the cardiovirus IRES is also independent of such sequences 2 7 3 . Recently, an 9-nt segment in the 5'-UTR of the homeodomain protein Gtx has been reported to function as an IRES, and multiple copies of this 9-nt sequence increase its activity synergistically 1 6 . This 9-nt sequence is 100% complementary to the 18S rRNA at nts 1132-1124. Ten linked copies of this 9nt sequence can increase IRES activity up to 570-fold in neuro-2a cells. The level of this Gtx IRES activity is up to 63-fold greater than that obtained by using the well characterized EMCV IRES when tested in the same assay system. The synergism of the linked 9-nt copies is reduced when spacingTable 3 Paul Cheung Page 41 between the linked copies are increased, suggesting that higher order RNA structures may play determinant roles in the IRES activity, in a way similar to how a transcriptional enhancer element functions 7 6 . Paul Cheung Page 42 Table 3 Summary of host genes that are translated by the IRES mechanism, (adapted from Kozaket.al.,2001 1 8 9) Gene Changes in translation activity (by the respective IRES) vs controls Reference Apaf-1 8-10 fold increase 84,236 Bip 2.6-15 fold increase, Synergism with La and PTB 160,172,173 DAP 5 10 fold increase 138 FGF-2 5-35 fold depending on cell type 13,325 eIF4G 5-42 fold increase 113 Gtx 7-570 fold increase depending on IRES repeats 76 C-myc 14-70 fold increase 308,309 VEGF 4-60 fold increase 145 XIAP 150 fold increase Possible synergism with La protein 140-142 Paul Cheung Page 43 1.8-3 Apoptosis and IRES mediated translation initiation Apoptosis induction is an intriguing aspect of a CVB3 infection because it appears to be correlated to the process of viral gene expression. It is now accepted that coxsackievirus and other picornaviruses can induce apoptosis in vitro and in vivo 5 7 . Although previous investigation has found that this is in part due to the proteolytic cleavage of eIF4G (and its isoforms) by caspase 3 3 5 6 ' 3 5 7 , the relationship between cap-independent translation initiation and the process of apoptosis was further demonstrated in three recently published papers concerning c-Myc, MKK6 and DAP5. The c-Myc is a cellular proto-oncogene involved in very disparate cellular processes including proliferation, transformation and apoptosis 1 0 3 . The authors demonstrated that the c-Myc protein is still expressed by the cap-independent mechanism when more than 90% of cells are apoptotic 308>309. The presence of the protein MKK6, which is a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), appears to increase the IRES-mediated translation of c-Myc. Thus, it is proposed that IRES-mediated translation initiation of the c-Myc during apoptosis is activated by the p38 MAPK pathway . These data suggest that cap-independent translation initiation is an important and highly regulated alternative mechanism of protein expression. In a related paper, a cellular IRES that functions specifically during apoptosis is observed in the translation initiation of DAP5 1 3 8 . DAP5 is a 97 kDa protein which is homologous to eIF4G. It is cleaved during Fas-induced apoptosis, yielding an 86 kDa isoform. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60-70%, the translation rate of DAP5 protein was selectively maintained. The authenticity of IRES-mediated translation initiation of DAP5 has been further confirmed by using a bicistronic plasmid construct Paul Cheung Page 44 containing the 5'-UTR of DAP5 in the intercistronic space 1 3 8 . The experiments demonstrated that the translation initiation of DAP5 can depend on the cleavage status of the DAP5 protein in the cellular environment: the DAP5/p86 apoptotic form is more potent than the normal DAP5/p97 in stimulating IRES-mediated translation. Thus, a positive feedback mechanism is proposed for the translation of DAP5 during apoptosis 1 3 8 . Reflecting on our previous discussions, the process of CVB3 induced apoptosis and translation of the viral genome are probably interlinked and highly regulated. The cellular proteins that specifically bind to the IRES of CVB3 and mediate the regulation process remain to be discovered. /. 8-4 Transcription of Viral RNA Successful replication of the CVB3 virus involves synthesis of the viral capsid proteins (translation) and the transcription of the viral RNA (genome replication). The packaging of the genomic RNA into virions occurs late in an infection cycle and is prior to the release of progeny virions into the intercellular environment. At the present time, there is very limited knowledge of the mechanism and requirements of the process by which the genomic RNA is transcribed. 1.8-4a Tertiary RNA structure of the 3'-UTR and CVB3 replication In the CVB3 system, data from a mutational study strongly implicate a functional role of the 3'-UTR in viral replication. The CVB3 3'-UTR is a 99 nt long sequence starting from nt 7300 to 7399, followed by a poly-(A) tail of variable length 2 2 9 ' 3 3 0 . Work by Melchers et al. demonstrated that formation of tertiary structure from the secondary stem-loops in the 3'-UTR is important in viral replication 2 2 9 . This tertiary RNA structure in the 3'-UTR is maintained by interactions between two of the three stem-loops that are present in the 3'-UTR (Figure 10). Failure to form such tertiary structure results in the phenotype of reduced replication at non-physiological temperature while the translation of viral RNA is unaffected. The mechanism of Paul Cheung Page 45 how the tertiary structure mediates viral replication has not yet been discovered. Similar findings of the 3'-UTR in determining viral replication have been reported for Coxsackievirus A9 (CVA9) 2 3 5 . Analysis of a CVA9 mutant containing a single-base mutation in the 3'-UTR indicates that faulty tertiary RNA structures are related to replication defects. The mechanism of how the tertiary structure interaction can mediate viral replication remains elusive. 1.8-4b Requirement of host proteins in picornaviral replication A common strategy of replication is proposed for all picornaviruses 8. Thus, findings in the PV system may help our understanding of the CVB3 replication process. In the PV system, a cloverleaf structure in the proximal 5'-UTR region of the poliovirus forms a ribonucleoprotein (RNP) complex with host proteins and the polio 3CD protease / polymerase " ' . The importance of other 5'-UTR regions in viral replication of the PV system has been reported 5 0 . The formation of the RNP complex in the 5'-UTR of PV appears to be essential for positive sense RNA replication by serving as a double stranded RNA primer 2. The efficiency of RNA replication in turn determines the specific tissue tropism and pathogenicity of the virus 7 9- 2 4 2 . During replication, the RNP may involve interactions between stem loop structures in the 3' and 5' ends of the viral genome 9 ' 1 0 . Several daughter strands may be replicated at the same time from one template 2 1 . The time of replicating each RNA molecule is estimated at about 45 seconds 2 7 and is believed to take place on smooth membranes 41>58>59>196. Crude cellular extracts from HeLa cells, which also include cellular membranes, can support the replication of Paul Cheung Page 46 09 s o u 58 U CU •»-< tg u ' « a ex e •— 09 c« •a s C8 at u s -t-u s ^ .2 u ii 01 3 3 m CQ > U cu 6 ' T3 2 3 | | CU T 3 U .9 3 3 cu ctS tu t+- CU o > O <U .3 "3 § I 3 ,P 60 cm CU CJ r* cm 33 CU r- X .9 H 3 g 3 X cu _ , 1? > CU !* X XI -a 9 .2 x r3 CJ 3 2 ^ o < ^ a. Cj "3 £ cu <U C to i> Co •3 £ -3 cj a S3 .9 o ; 8 cu tu 3 cm t j a. co O k-o 2 2 m -3 2 s cm ON OO ' 3 E •3 A -a o o r- cj C 3 -a c o CJ CJ c/3 E 3 3 cm cm rj 2. .9 >i -a . tu •3 fi S 1 ca -Q X 3^  cm O 3- « ° 2 2 2 cu § . 9 « CO 2 3. •4-1 tN cu 0 \ 0) ^ 3 .S3 3 "3 W cu <C tN "C cu m > r» -3 cm 3 3 0 cu t j t3 1 x S & S 3 C ^ H >, ° X C -2 cu so 3 tu cd t-u "H •3 33 § 'I N o> m « "3 cm 3 td & E S 3 1 f 2 .9 tu .2 * x g>| 1 * 1 c3 cm tN ,5 X 3 33 cu S « i~ cu CJ ^ cu 3 X .2 2 % u to CJ cm u. 3 O is ao r-CJ C a . "3 O - 3 o o —< cu "« X 3 cm cu -3 U CJ o -to 3 § CT" -3 CU 3 cm .3 cm .to > 3 ' CU 00 •S t*H ~ I O t ->, - J tJ 2 3 .5 _JJ > CJ • c+-3 ~ O cj cm .2 is o . -s CJ _ X E 3 tu 2 o c t CD poliovirus in vitro . Interestingly, fractionation of the extract will inhibit replication, and such inhibition can be overcome by addition of the soluble fraction from the extract 3 0 . Similarly, injection of a HeLa cell cytoplasmic extract into the oocytes of X. laevis is a pre-requisite for poliovirus replication in this otherwise non-permissive environment n i . Therefore, it is believed that soluble host proteins in the cytoplasmic environment are critical to the process of RNA-dependent RNA transcription. Using UV cross-linking techniques, three proteins have been observed to bind the PV 3'-UTR RNA: pi05, p68 and p45 3 2 8 . The 105 kDa protein appears to be nucleolin, which translocates into the cytoplasm upon infection. The functional significance of this interaction is unclear. The other two proteins remain unidentified. Together with data that implicate the tertiary structures of CVB3 3'-UTR in regulating replication, it is probable that the efficiency of the protein-3'-UTR RNA binding process may be a crucial determinant in viral replication. In the CVB3 system, there is currently no data reported for the host proteins that bind to the 3'-UTR. 1.9 Functional roles of host protein-CVB3 UTR interactions As the infectious CVB3 virus particle carries no enzymatic molecules, host proteins and translational machineries will have to play an integral role in viral translation and or transcription. As discussed in the above sections, specific interactions between the host proteins and the CVB3 UTR sequences are most likely important in achieving the function of viral RNA translation and transcription. Although there is currently no data available with respect to the CVB3 system, studies in other RNA viruses have provided valuable data to understand the significance of interactions between host proteins and viral UTRs. Paul Cheung Page 48 1.9-1 Participation of eukaryotic translation initiation factors Despite fundamental differences in the mechanisms of translation initiation that are utilized by the eukaryotic mRNA and picornaviral RNA, both processes are thought to be dependent on the well studied eukaryotic translation-initiation factors (elF). Translation of EMCV RNA, for example, requires tRNAm e t, 40S ribosome, eIF2 and 3, and eIF4F 2 7 1 . These eukaryotic translation factors are often modified by viral proteases during an infection, most likely in order to favor viral translation. At the present time, little is known about protein requirements in the translation of CVB3 RNA. The translation initiation factors of interest can be summarized by the subunits that form the well studied eIF4F complex which includes eIF4A, B, E and G 1 2°. Binding of the eIF4F complex to the host mRNA is facilitated by the eIF4E subunit which recognizes the cap-structure . The eIF4B is an mRNA binding protein ' which also stimulates the helicase activity of the eIF4A 2 6 2 . Finally, eIF4G serves as the linker between eIF4A, B, E, and the 40S ribosomal subunit in the RNP-translation initiation complex 1 9 4 ' 2 0 4 ' 2 0 9 - 2 1 0 A schematic representation of the translation initiation process in eukaryotic mRNA is presented in Figure 11a. eIF4G is also an important molecule in the translation initiation of picornaviral RNA. eIF4G is a 175 kDa protein with multiple domains that bind other proteins and RNA molecules (Figure 8). Enterovirus proteases or FMDV leader proteases commonly sever the carboxyl-terminal domain, thus inhibiting interaction of eIF4E with the eIF4G complex 3 3 ' 1 2 4 ' 1 9 4 - 2 9 1 Since there is no cap structure on picornaviral RNA, the function of eIF4E should not be necessary in the translation initiation of picornaviral RNA. Severing the eIF4E binding domain of the eIF4G has been suggested to directly cause inhibition of host protein synthesis during an infection 124,194 Paul Cheung Page 49 Figure 11a. Summary of steps in Cap-dependent translation initiation. Typical capped m R N A interact with eIF4E (step 1). Secondary m R N A structures are unwound by eIF4A. Formation of eIF4F complex occurs between the eIF4G, 4A, 4B, 4E with eIF4G as the central docking module (step 2). Pre-initiation complex includes the eIF3 unit which can bind eIF4G. Interaction with the 60S ribosomal subunit (not shown) forms the final 80S complex which scans the m R N A for the A U G start codon (step 3). (Adapted from Gingras, A. C , B. Raught, and N. Sonenberg. 1999. Annu. Rev. Biochem. 68:913-963). Eukaryotic mRNA Step 1/ mRNA interaction with eIF4E and elF4B Methyl-7-guanidine cap structure / Stem loop structures | ~ | | A U G eIF4E Step 2/ interaction with elF4G and eIF4A (Forming the eIF4F complex) eIF4B A U G Pre-initiation complex (43S complex) eIF4A eIF4E eIF4G ] Q — [ eIF4B j " A U G Met-tRNA eIF4F complex 0 43S complex Step 3/ Translation Initiation by ribosomal scanning eIF4E eIF4AJ^ eIF3 \ _ J ~ | M e t - t R N A Paul Cheung Page 50 Binding of the central domain of eIF4G to the IRES of EMCV has been demonstrated 1 8 8 . Of note, eIF4A also exhibits binding to the EMCV IRES at a location close to the eIF4G binding site, indicating the importance of co-operation among the eIF4F subunits in mediating translation initiation of the picornaviruses. A schematic representation of the eIF4 factors in mediating picornaviral RNA translation via the internal ribosomal entry mechanism is presented in Figure l i b . In addition to eIF4G, recent reports have indicated the role of the poly-(A) tail and poly-(A) tail binding protein (PABP) in enhancing the efficiency of translation initiation by interacting with the translation machinery 52>87.151>234>311 ft has been suggested that circularization of the EMCV RNA can occur by interaction of the 5'-UTR-IRES-eIF4G complex with the PABP-3'-UTR complex 2 9 0 . However, the contribution of the PABP protein to the model of circularized translation initiation in picornaviral system requires further investigation, as the eIF4G cleavage by viral proteases can also separate the PABP binding site (which is also located in the carboxyl-terminal domain) of eIF4G from its RNA binding domain. For the coxsackievirus system, interaction of eIF4 factors and the PABP proteins with the CVB3 RNA has not been verified. /. 9-2 Functional roles of the La autoantieen and PTB Besides the above mentioned translation initiation factors and other possible RNA binding proteins 4 6 ' 1 4 6 - 1 4 7 5 two host proteins appear to play functional roles in the cap-independent translation of picornaviruses. The PTB and La autoantigen were among the first proteins characterized to bind to the picornaviral 5'-UTR and to stimulate the translation of a variety of RNA viruses 3 2 . Of note, interactions of these proteins with the viral RNA do not require Paul Cheung Page 51 Figure l i b . Current model of picornaviruses' IRES-mediated translation initiation. Without a cap structure, the viral R N A requires the participation of other U T R binding proteins, such as La, P T B , PCBP and eIF4G (step 2). This protein-viral R N A complex can then interact with the ribosomal subunit, directly over its IRES location, possibly involving sequence complementation with the 18S rRNA (step 3). In type I IRESes (CVB3), the spacing between the authentic A U G and the IRES may be important in translation initiation, whereas the authentic A U G in type II and III IRES is often located inside the IRES sequence. The ribosomal scanning mechanism is not at play. Other less well characterized host proteins that bind to the 5' U T R are not demonstrated. STEP 1/ Picornaviral mRNA enters cellular environment V P g \ Stem loop structures < I IRES A U G STEP 2/ Interaction with proteins such as La autoantigen. PCBP. and PTB <• -• elF4G 1 PTB l-l PCBP L a ^ — TRFS A U G PCBP Paul Cheung Page 52 modifications by viral proteases. Thus, their natural presence in a cellular environment may be important to the initial stages of CVB3 translation and / or replication, before the successful expression of the viral proteases. The La autoantigen is first discovered as a target of autoimmune recognition in patients with systemic lupus erythematosus and Sjorgren's syndrome 6 9 ' 7 0 . It is a 408 amino acid protein that participates in a variety of RNA transcription activities including maturation of RNA pol III 191 1 99 9S"^ transcripts ' ' . La protein contains an RNA recognition motif (RRM) within amino acids 1-194. The protein domain involved in the stimulation of translation was mapped to amino acids 1-293 8 8 . Dimerization of La protein occurs in the physiological cellular environment which enhances its translation efficiency 8 8 . The domain spanning amino acids 293-348 has been shown to facilitate dimerization, suggesting that the function of dimerization can be independent of RNA interaction. During infection by PV, La protein is cleaved by the protease 3C p r o and is transported from the nucleus to the cytoplasm 3 0 ' . The cleaved La protein still contains amino acids 1-358 and thus the cleaved protein remains functional in RNA binding, translation stimulation and dimerization 3 0 1 . La protein is capable of interacting with the RNA of a variety of viruses, including the HCV 5 , EMCV 1 7 4 , sindbis virus 2 5 9 , rubella virus 2 1 6 , human parainfluenza virus-3 9 6 , HIV 1 2 , and PV 3 1 2 . The La protein has also been reported to bind a variety of viral and non-viral RNAs, with a generally stimulatory effect on translation 89'96>153>266'313'320. Pertinent to the study of CVB3, binding of La to the HCV RNA stimulates translation activity in a dose-dependent manner 1 5 3 , while an optimal stoichiometric cooperation of La with PTB can enhance the activity of the EMCV IRES 1 7 4 . These data strongly suggest synergism among protein molecules and viral RNAs to form an optimal UTR-protein complex in order to mediate IRES-based translation Paul Cheung Page 53 initiation. Differences in UTR sequences among different enteroviruses may require a different set of host chaperone proteins for their specific systems of translation initiation 9 2 . The expression of La autoantigen may be tissue-specific and dependent on cellular conditions 6 3 . Hence, the availability of the La protein in CVB3 infected cells may also play a role in determining the rate of expression of viral proteins and consequently the occurrence of cellular injury. PTB is a cellular protein that normally functions as a splice regulator 2 0 3 - 2 6 7 . There are four RRMs in this protein. Like the La autoantigen, PTB can also exist as a dimer. Interaction of PTB with viral UTRs has been verified in poliovirus 131>135'136, EMCV 1 6 4 , and FMDV 1 8 7 , HCV 5 ' 1 5 4 , HIV 4 3 and Human T-cell leukemia virus -2 . In general, PTB is capable of interaction with type I and type II IRESes 3 2 . Interaction with PV occurs in multiple locations including sites that contain determinants of neurovirulence 1 2 9 . There are numerous observations of the stimulatory effects of the non-cleaved PTB in IRES-mediated translation. The activity is often observed in cooperation with secondary protein or RNA molecules 130>131>147>174>187>320 There is also a suggestion that the PTB activity may be virus-specific, as illustrated by the differential effect of PTB on the entero-/rhinovirus IRES elements (strong activity) 1 4 6 , 1 4 7 and the FMDV IRES (moderate activity) 3 2 9 . These observations further reinforce the assertion that an optimal complex of host proteins and viral UTRs specific to individual viruses are required for efficient translation initiation. As discussed earlier, the role of PTB in the pathogenesis of PV has been characterized as a possible switch between viral translation and genome replication 2 2 . The effect of PTB on the overall translation-stimulation decreases over time due to the cleavage of PTB by 3C p r o which increases with time, resembling a process that is governed by a negative feedback mechanism. Naturally, in a picornaviral system, the saturation of viral translation Paul Cheung Page 54 products (virion proteins and various proteases) signals the need for the virus to focus on genome replication and to proceed towards viral packaging / dissemination. Thus, the overall functions of the PTB in picornaviral pathogenesis may be in both the translation and transcription of the viral genome22. Whether this applies to the CVB3 system remains to be studied. 1.9-3 Role of protein-RNA interaction in stimulating RNA transcription During the replication of PV, the RNA replicative complex contains several host proteins in addition to the viral 3 CD protease/polymerase. These include the EF - la 1 3 4 , possibly poly(rC)-binding protein (known as PCBP or hnRNP E) 1 , 2 and Sam68 2 2 5 . Sam68 is a host protein involved in cellular mitosis and this protein is transported to the cytoplasm during infection by PV. However, there is no firm evidence suggesting a functional role of Sam68 interaction with the replicative complex. On the other hand, EF - la and hnRNP E appear to participate in positive strand RNA synthesis. Interestingly, EF - la interacts with the UTR of two positive strand RNA viruses: west nile virus 4 5 and turnip yellow mosaic virus 1 6 2 , although its exact role in RNA replication remains to be further investigated. Of interest, the hnRNP E binds to both the 5' end of the cloverleaf structure112 and the IRES of the PV RNA 4 6 . Because the PV 3 CD complex binds to a different location of the 5' cloverleaf structure and the hnRNP E is part of the complex, interaction of the hnRNP E at multiple sites in the PV 5'-UTR is thus proposed to serve as a bridge that brings together other protein complexes at distal regions of the 5'-UTR 4 6 . As discussed in the previous sections, proteins such as La and PTB either possess dimerization domains or bind to multiple sites of the PV 5'-UTR. Further work is needed to verify if these proteins also participate or affect the process of RNA transcription by the 3 CD complex in a manner similar to that of the hnRNP E protein. Interestingly, La protein interacts with HIV RNA in the TAR region (tat-binding site), an RNA sequence of the HIV that binds Paul Cheung Page 5 5 multiple cellular proteins and serves to regulate the switching between transcription and translation of the HIV RNA 7 2 ' 3 1 3 . 1.9-4 Characterization of other UTR binding proteins Using biochemical methods, such as UV cross-linking and mobility shift assays, several other cellular proteins have been reported to bind to viral IRES. These proteins include a 97 kDa protein 'unr' (for upstream of N-ras) and a 38 kDa protein (known as unrip, unr-interacting proteins), both of which can stimulate the activity of the rhinovirus IRES 1 4 6 . The unr-unrip complex can also correct aberrant translation initiation in PV 1 4 6 . Interestingly, recombinant unr acts synergistically with recombinant PTB to stimulate translation initiation by the rhinovirus IRES, but the same unr-PTB synergism can not be observed with the PV IRES I 4 6 . Thus, the observation highlights the co-operative nature of UTR-binding proteins and specific RNA sequences in effecting translation and or transcription. In summary, several important host proteins appear to play major roles in the regulation of picornaviral translation and transcription by interacting with the UTR sequences. There is very limited knowledge of other additional host proteins that participate in the processes. Besides the limited number of identified host proteins, one inadequacy of the current knowledge on protein - picornaviral UTR interactions is the lack of data on the specificity and affinity of the interactions. Upon cellular entry, the few copies of viral RNA will have to compete against the vast majority of cellular mRNA for interaction with RNA-binding proteins. In this respect, the affinity and specificity of interactions between the viral RNA and the host proteins are two critical factors in evaluating the functional importance of the observed interactions. A high specificity of interaction between the host and the viral RNA (UTR sequences) is required to ensure successful competition against host mRNA for protein translation. On the other hand, a Paul Cheung Page 56 high affinity of interaction will contribute to the stability of the RNA-protein complex to allow for the initiation of translation and or transcription events. From this logic, interactions that have low specificity and affinity are less likely to be significant in the pathogenesis of the virus. At present, most studies that characterize UTR binding proteins do not quantify or address the questions of specificity or affinity. 1.9-5 Research Focus As discussed above, the 5' and 3'-UTR of the CVB3 mediates the expression and replication of the viral genome. The efficiency of these two processes can determine the extent of cellular injuries and overall tissue tropism of the virus. Among all enteroviruses that cause myocarditis, CVB3 has evolved to be specifically virulent in cardiomyocytes. Several lines of studies strongly implicate that the 5'-UTR sequence of the virus contains determinants of cardiovirulence, although the mechanism is still a mystery. As well, the tertiary structures of viral 3'-UTR are critical to the replication of the virus. RNA sequences alone can not effect phenotypic observations. Rather, it is their interactions with host proteins that result in the processes of translation initiation (via the IRES mechanism) and genome transcription (via the host-viral protein RdRP complex). The significance of these processes have been studied for CVB3 and other related enteroviruses. However, the specific interactions of host proteins with the CVB3 5' and 3'-UTR sequences and their potential correlations with the virulence of the virus remain to be discovered. In particular, there is no studies that focus on the presence or absence of these UTR-interacting proteins in CVB3-susceptible and non-susceptible cell types. Further, these UTR-interacting proteins can potentially interact with various localized RNA structures of the UTRs to result the various phenotypic observations of tissue tropism and infectivity. Therefore, in my dissertation, I focused on the study of three major issues: Paul Cheung Page 57 1. Verification of specific interaction between host protein-viral UTR sequences. 2. Characterization of these interactions in the context of tropism, viral protein translation, and genome transcription. 3. Assessment of the significance of these protein - UTR interactions in the process of viral pathogenesis. Paul Cheung Page 58 CHAPTER II HYPOTHESIS AND SPECIFIC AIMS The central hypothesis of this work is that the interactions of host proteins and coxsackievirus B3 untranslated regions are specific and correlate with the viral tissue tropism, replication and translation initiation. The following specific aims are addressed experimentally: Aim 1. To demonstrate the presence of specific interactions between cellular proteins and CVB3 UTRs. Aim 2. To determine whether specific interaction(s) can correlate with the tissue tropism of CVB3 in the A/J mouse model. Aim 3. To determine the molecular weights of UTR-binding-HeLa cell proteins. Aim 4. To determine whether HeLa cell protein-UTR interaction(s) can correlate with the CVB3 mutations that inhibit viral replication. Aim 5. To determine the biochemical characteristics of the interaction between the 5' and 3'-UTR sequences and the La autoantigen. Paul Cheung Page 59 CHAPTER III RESEARCH QUESTIONS AND STRATEGY The questions relevant to the hypothesis and specific aims are: 1) How specific are the interactions between host proteins and viral UTR sequences? Are the mobility shift / UV cross-linking assays effective in determining the nature of the interactions? (e.g. the sensitivity in distinguishing different specificities and the resolution in the MWs of the binding proteins.) 2) By using the UV cross-linking technique, is it possible to identify a reasonable number of unique targets that correlate to the determinants of tissue tropism and the processes of viral translation and replication? 3) How can biochemical characterizations be used to infer functional and mechanistic information of a protein-UTR interaction? To answer these questions, I have conducted my research with the strategy described in Figure 12a. Results and discussion of the experiments are organized into two chapters: Identification of UTR Binding Proteins (Chapters IV) and Biochemical Characterization of the La Protein-UTR interactions (Chapter V). Details of the materials and methods utilized in these experiments are presented in Chapter VII. Paul Cheung Page 60 Figure 12a The overall strategy employed in my dissertation Cloning 5' and 3' U T R Determine specificity Investigate tissue tropism determinants ISH of viral RNA in various organs U V cross-linking with 5' UTR probes 5'UTR-HeLa cell protein interactions 3' UTR-HeLa cell protein interactions Proteins that are related to translation Proteins that are related to replication - • Determine factors that contribute to tissue tropism GST-La protein expression and purification 3' U T R mutant generation Biochemical characterization of La protein-CVB3 UTRs interactions Determine dissociation constant (Kd), and stoichiometry of interaction. PaulCheung Page 61 CHAPTER IV IDENTIFICATION OF SPECIFIC CVB3 UTR-BINDING PROTEINS Prior to my studies, there was no report on the subject of host protein - CVB3 UTR interactions. Also, the use of UV cross-linking and mobility shift assays in the detection of RNA-binding proteins had met with limitations in two aspects: the determination of specificity and the resolution of MW. Further, the different sequences of the 5' UTR appear to be differential in their functional significances. The proteins that bind to such sequences and possibly mediate the specific functions have not been investigated. Thus, in my studies, I began by sub-cloning the viral UTR sequences. The overall goal is to identify specific CVB3 UTR-binding proteins in HeLa cells and A/J mouse tissues, and to correlate some of these interactions with pathogenic attributes of the virus. 4.1 EXPERIMENTAL STRATEGIES Mobility shift assay and probe synthesis Mobility shift assays were conducted to demonstrate the presence of specific interactions between cellular proteins and CVB3 5' and 3'-UTRs (Aim 1). Proteins from non-infected HeLa cell lysates (nuclear and cytoplasmic) were prepared following procedures described in Chapter VII (Materials and methods). The concentration of proteins was determined by standard Bradford assays, and lysates were stored at -80 °C in 1 ml aliquots (~ 2.5 mg/ml). RNA molecules of the viral UTRs were synthesized from linearized plasmid templates by in vitro Paul Cheung Page 62 transcription. Plasmid templates carrying sequences of the CVB3 5'-UTR were created by PCR cloning. Detail of the primers and PCR conditions are listed in Chapter VII. In brief, the CVB3 5'-UTR sequences of nt 1-209, 210-529, 530-630 were sub-cloned, based on the 5'-UTR secondary structures (Figure 9). As discussed, extensive secondary stem-loop structures have been predicted for the 5'-UTR. Secondary RNA structures are often domains of recognition in protein interactions. As a result of this consideration, sequence nt 1-209 was sub-cloned to preserve the first four stem loops (Figure 9) and nt 210-529 was sub-cloned to preserve the major stem loops E and F. On the other hand, the functional significance of the sequence nt 529-630 was considered more important than the preservation of complete stem loop structures. Previous mutational studies revealed that this is the minimal sequence required for the CVB3 IRES activity 3 4 9'. Thus, this exact sequence was sub-cloned. DNA fragments of these 5'-UTR sequences were produced by PCR reactions and directly cloned into T/A vector (PCR 2.1, Invitrogen). Positive (sense) and negative (antisense) sequences of the 5'-UTR RNA were produced from linearized recombinant T/A vectors by in vitro transcription reactions, with the appropriate restriction and transcription enzymes (Figure 12b). In a similar fashion, the 3'-UTR of the CVB3 was sub-cloned into the T/A vector. The sequence nt 7299-7399+ 33 bases of adenosine [poly-(A) tail, artificially included into the PCR primer)] was sub-cloned (total 133bp). Again, sense and antisense RNA sequences were produced from this recombinant vector. Figure 12b illustrates the cloning scheme for the 5'-UTR and Figure 12-c illustrates the cloning scheme for the 3'-UTR. Paul Cheung Page 63 Figure 12b. Sub-cloning of the CVB3 5' UTR. Secondary stem-loop structures of the 5' U T R is shown, with nucleotide sequences indicated by numbers (not drawn to scale). D N A fragments of the three 5' U T R regions (nt 1-209, 210-529 and 530-630) were generated by P C R and inserted into the T / A vectors*. Orientations of inserts with respect to the transcription promoters were determined by D N A sequencing. Restriction enzyme sites utilized for linearization of the plasmids are also indicted. Pau l Cheung Page 64 Figure 12c. Sub-cloning of the CVB3 3' UTR. Secondary structure of the 3' U T R is shown, with nucleotide sequences indicated by numbers (not to scale). P C R products of the respective 3' U T R sequences are ligated into P C R II vectors (Invitrogen). Orientation of the inserts with respect to the transcription promoters was determined by D N A sequencing. Restriction enzyme sites utilized for linearization of the plasmids are indicted. Mutations (mt) was indicated at the specific nucleotide sequences. nt. 7350 Stem loop ^— Z W nt. 7299 Stem loop Y Stem loop X nt. 7399 —I A A A A A A . Not I T7 Sail T3 Wild type WT EcoR T7 BamH if SP6 Poly(A) deleted 3'UTR dA Mt @ 7352/3 ot I 44 Sal I T7 i  r; T3 Mutant Y Double Mts Not I U U Sail T7 H_J: Mutant X Y Not I T7 Mt @ 7391/2 U Sal 1 T3 Mutant X T7 otl Deletion 7391- 5all T3 Mutant dX Paul Cheung Page 65 In the mobility shift assays, a radiolabeled RNA transcript (probe) of each UTR sequence was mixed with HeLa cell protein lysates in reaction buffer (Chapter VII), and incubated at 37°C for 10 min. The host proteins in the HeLa cell lysate that bound to the UTR were expected to form protein-radiolabeled-RNA complexes. To determine the specificity of interactions, non-labeled RNA of the same sequence to each of the corresponding probes was added at high molar excess ratios (20-250x) prior to addition of the respective probe. In addition, as a negative control, yeast tRNA was added to the reaction at the same molar excess ratios as the non-labeled RNA. Yeast tRNA was chosen because its secondary structures resemble those predicted for the CVB3 5' and 3'-UTRs, and interaction of yeast tRNA with HeLa cell proteins should not be physiologically significant. The reaction products were analyzed by a non-denaturing poly-acrylamide gel electrophoresis (PAGE). The gel was fixed, dried and examined by autoradiography. Thus the distribution of the probes in its free form (fast migrating) or in protein complexes (slow mobility, 'mobility shift') could be distinguished. The presence of specific interaction would be concluded when the mobility shift of the probe was dependent on the amount of non-labeled competitor RNA of the same sequence but not the yeast tRNA. Figure 12d summarizes the strategy applied in the mobility shift experiments. Paul Cheung Page 66 Figure 12d Determination of specific interaction. Radiolabeled R N A s (probes) and protein lysate were allowed to interact (1). Interactions resulted in RNA-protein complex formation that exhibited a slower electrophoretic mobility (2) than the free non-interacting probes (3). Non-denaturing gels and autoradiography were used to analyze the interactions. Addition of non-labeled R N A of the same sequence served as specific competitor, while yeast tRNA was used as negative control (4). Probe Protein lysate Complex formation (3) Mobility shift (with various amount of homologous competitor) Mobility shift (with various amount of Yeast tRNA competitor) (4) (2) Free probe (no interaction) 3 e 3 i a n ore ~ 3 •o sr o -i Autoradiograph of radioactivity (Mobility shift/ complex formation is rescued only by homologous RNA) Paul Cheung Page 67 Correlation between protein-UTR interactions and viral infectivity in A/J mouse organs. To determine whether specific protein-UTR interactions correlate with tissue tropism of CVB3 in the A/J mouse model (Aim 2), I investigated correlations between the following two observations: 1) the in situ hybridization (ISH) of viral genome in organs of A/J mouse; 2) the respective patterns of protein-5'-UTR interactions in the organs. The A/J mouse was chosen because it has been a well established model from which much data of CVB3-induced myocarditis was derived. Various A/J mouse organs also display significantly different but often interesting patterns of susceptibility to the CVB3 virus. The patterns of protein interactions between organ proteins of the A/J mouse and the 5'-UTR was a focus of my study because: 1) CVB3 5'-UTR has been demonstrated to stimulate viral translation by the IRES mechanism. 2) cardiovirulent determinants have been mapped to the 5'-UTR. The background assumption was that the efficiency of CVB3 translation initiation was dependent on protein-UTR interactions, and the presence of these interactions in different cellular environments can lead to the specific patterns of CVB3 tissue tropism in A/J mice. Thus, the goal of this investigation is to identify targets that are important in the regulation of the CVB3 translation initiation and at the same time relevant to the development of disease in specific cellular environments. A schematic representation of the experimental design is shown in Figure 12e Paul Cheung Page 68 Figure 12e. Correlation between viral infectivity and protein-RNA interaction. The pattern of viral load in each organ of infected A / J mice is detected by in situ hybridization. Corresponding lysate of the organ from uninfected mouse is tested for interaction with the 3 probes of the 5' U T R via U V cross-linking method. Possible correlation of viral titre and protein interactions were thus investigated. C V B 3 Infection 4-week old 10-week old Liver Spleen Brain Pancreas Kidney Tissue sectioning ISH of viral genome One representative image per organ per age group 4-week old 10-week old Non-infected A / J mice Nt 1-249 249-529 530-630 U V Cross-link profile Non-infected Lysate vs three pairs of 5' U T R probes (sense and antisense) One profile per organ per age group Paul Cheung Page 69 In situ hybridization of viral genome. To acquire data on viral infectivity in various mouse organs, adolescent (4-week-old) and adult (10-week-old) A/J mice were infected by intraperitoneal injection of lxl0 5 plaque forming unit (pfu) of myocarditic CVB3 (Kandolf) and euthanized on day 4 post infection (p.i.). The heart, liver, kidney, pancreas, brain and spleen were fixed and frozen in 4% paraformaldehyde. Organ lysates were obtained by a standard method (Chapter VII) and 8 pm sections of the fixed organs were used for ISH. Viral negative sense probe sequence was labeled with digoxigenin-1 l-uridine-5'-triphosphate DIG-11-UTP and signals were detected with BCIP /NBT substrate / Carmalum (SIGMA). Representative images of the sections under light microscopy were captured. UV cross-linking between radiolabeled 5'-UTR RNA and its binding proteins. To determine the MW of UTR binding proteins, the method of UV cross-linking was applied. Radiolabeled sense and antisense 5'-UTR RNA of sequences nt 1-209, 210-529 and 530-630 were synthesized by in vitro transcription. Protein lysates from the respective mouse organs were incubated with the radiolabeled probes under conditions as described in the mobility shift assay. After the interaction, the reaction was exposed to UV light which induced chemical bond formation between UTR binding proteins and probe RNA, thus covalently linking radioactive RNA to the UTR-binding proteins. Following UV exposure, unbound probe and non-interacting regions of the probe were digested by RNase A so that subsequent analysis of the reaction by denaturing SDS-PAGE would separate proteins according to their native MWs. A schematic representation of the UV cross-linking procedure is presented in Figure 12f. Each SDS PAGE was fixed, dried and analyzed by autoradiography. One profile of protein-UTR Paul Cheung Page 70 interaction for each lysate was therefore generated. Specifically, I searched for differences that could correlate with the infectivity (ISH of viral genome) in the respective organs. Paul Cheung Page 71 Figure 12f. UV cross-linking methodology. Radiolabeled R N A o f the U T R (probe) were incubated w i t h protein lysates under standard conditions (1). Interactions are covalent ly l inked by U V exposure (2). Excess and non-interacting probes o f the probe is digested by R N a s e A (3). Thus, on ly U T R - b i n d i n g proteins are labeled. Protein m i x is analyzed by S D S P A G E and autoradiograph (4). Mo lecu l a r weights were estimated by a concurrently loaded marker. • Reaction . * * Marker (4) Autoradiograph of radioactivity (Only U T R binding proteins are visualized by autoradiography) Paul Cheung Page 72 Competitive UV cross-linking. Competitive UV cross-linking was performed to determine the specificity of UTR binding protein. Clones from aim 1 were used to produce 5'-UTR RNA transcripts. Both radiolabeled (probe) and non-labeled RNA (competitor) of the respective sequences were produced by in vitro transcription. The pattern of UV cross-linking between radiolabeled probe and HeLa cell proteins were compared to the reactions in which non-labeled RNA (of the same sequence) were added as competitors. Non-labeled competitor RNA was added to the reactions at various molar excess ratios prior to the addition of the radiolabeled probe. The non-labeled RNA would thus compete specifically against the radiolabeled probe for interactions with the same set of UTR-binding proteins, (note: It was experimentally determined that the order of the addition of non-labeled/ labeled probe to the reactions had no observable effect on the final protein complex formation). For interactions that are specific and not random or weak RNA associations, significant reduction in the radioactivity of the protein complex would be expected with increasing amounts of non-labeled competitor. Thus, this method can differentiate the specific and non-specific 5'-UTR-protein interactions that are detected in the normal UV cross-linking experiments. Construction of the 3 '-UTR mutants. As mentioned earlier, mutations at the 3'-UTR inhibit viral replication. This may be due to the disruption of protein-RNA interactions which play important roles in translation and / or transcription. To confirm this speculation, protein-RNA interaction assays using the wild type and mutated 3'-UTR probes were performed (Aim 4). The 3'-UTR mutants were constructed based on previous reports regarding the crucial nucleotides in the 3'-UTR which maintain the Paul Cheung Page 73 wild type tertiary structures (between stem loops Y and X, Figure 10). Mutations in those sequences led to disruption of tertiary structures and subsequently, inefficient replications 2 2 9 . The primers and conditions with which the cloning was performed are listed in Table 7 (Chapter VII). In addition to the tertiary structural mutations, I have created a new 3'-UTR clone to investigate the functionality of the viral poly-(A) tail sequence. In this clone, the wild type 3'-UTR sequence was preserved without the poly-(A) tail. In summary, six different CVB3 3'-UTR sequences were generated: 1) wild type with poly-(A) tail, Clone WT. 2) wild type without poly-(A) tail, Clone dA. 3) mutation in stem loop X (nt 7391/2), Clone X. 4) mutation in stem loop Y (nt 7352/3), Clone Y. 5) compensatory mutations (replication efficiency was partially rescued), Clone XY. 6) deletion mutation of loop X (nt 7391-6), Clone dX. Figure 12c illustrates the strategy of constructing the 3'-UTR mutants. The plasmid templates of these mutants were used in the preparation of RNA probes for the UV cross-linking assays. Paul Cheung Page 74 4.2 RESULTS Specificity of HeLa protein-CVB3 UTR interactions (aiml). We measured mobility shifts of the 32P-labeled 5'-UTR probes in the presence of HeLa cell proteins and various competitor RNAs. Data for the mobility shift assays are presented in Figure 13a-d. In each of these figures, the integrity and mobility of the free probe is shown in lane 1. Formation of protein-probe complex (Co) results in changes of the electrophoretic mobility of the probes under non-denaturing conditions. The specificity of the complex formation was tested by adding various amounts of non-labeled RNA of the same sequence (competitor RNA). Competitor RNAs at the indicated molar excesses were effective in inhibiting protein-probe interactions: In all the experiments (Figure 13a-d), competitor RNAs reduced the amount of protein-probe complex (Co) (lane 2-4), and progressively restored the normal mobility of the labeled RNA probes in a dose dependent manner. This contrasted to the findings where yeast tRNA was used: at the same molar excess ratios, the mobility and amount of protein-probe complex remained unaffected by the yeast tRNA. This suggests that the interactions between the HeLa cell proteins and the CVB3 UTR regions are specific. In the case of the 5'-UTR nt 530-630 (Figure 13c), adding competitor RNA at high molar excess (lane 5) resulted in the detection of two major bands of protein-complexes. Similar band-like patterns were also observed in the case of the 3'-UTR sense probe (Figure 13d, left panel). A possible cause of this aggregation like phenomenon may be related to mode of interactions: While some protein-UTR interactions are reversible and competitive, specific Paul Cheung Page 75 UTR-interacting proteins may exhibit a mechanism of co-operative binding to the RNA molecules, such that when the presence of additional competitor RNA enhances the formation of the specific protein-RNA complex, leading to observation of major bands. Paul Cheung Page 76 Figure 13a. Mobility shift of labeled 5' UTR probe nt 1-209. The probe (P) alone is shown in lane 1. Significant mobility shifts of the protein-probe complexes are observed in lane 2, indicated by (Co). Such protein-probe interactions could be competed when identical non-labeled R N A was added to the incubation, at 20x, 50x and lOOx molar excess (lane 3, 4 and 5 respectively). Yeast t R N A molecules was added in lane 6, 7 and 8 as non-specific competitors, at lOx, 50x and lOOx mass excess ratios. Complex formation was competed for only by the use of homologous non-labeled R N A . Competitor RNA 1 2 3 4 5 6 7 8 Lanes Paul Cheung Page 77 Figure 13b. Mobility shift of labeled 5' UTR probe nt 210-529. The probe (P) alone is shown i n lane 1. Significant mobi l i ty shifts o f the protein-probe complexes are observed in lane 2, indicated by ( C o ) . Such protein-probe interactions cou ld be competed when identical non-labeled R N A were added to the incubation, at 20x, 50x and lOOx molar excess (lane 3, 4 and 5 respectively). Yeas t t R N A molecules was added i n lane 6, 7 and 8 as non-specific competitors, at lOx , 50x and lOOx mass excess ratios. C o m p l e x formation was competed for on ly by the use o f homologous non-labeled R N A . Competitor RNA 1 2 3 4 5 6 7 8 Lanes P a u l C h e u n g Page 78 Figure 13c. Mobility shift of labeled 5' UTR probe nt 530-630 (IRES). The probe (P) alone is shown in lane 1. Significant mobi l i ty shifts o f the protein-probe complexes are observed i n lane 2, indicated by (Co) . Such protein-probe interactions cou ld be competed when identical non-labeled R N A were added to the incubation, at 20x, 50x and 1 OOx molar excess (lane 3, 4 and 5 respectively). Yeas t t R N A molecules was added i n lane 6, 7 and 8 as non-specific competitors, at lOx, 50x and lOOx mass excess ratios. C o m p l e x formation was competed for only by the use o f homologous non-labeled R N A . Competitor RNA 1 2 3 4 5 6 7 8 Lanes P a u l C h e u n g Page 79 CU u a a- Z S 2 e U o © o o IT) O 09 09 CU W at s-cs oo 09 <u e es I / 03 t OH co cd O eg CU M ca o -»-> a Z S S o U 00 09 09 cu u H cu s-09 CU s cs \ / 03 t CU W ) B 3 O JS U CS a. Tissue tropism of CVB3 in A/J mice by ISH (Aim 2). The specific infectivity of the virus in each mouse tissue is revealed by the ISH observations, which is then compared to the pattern of interaction between the 5'-UTR and the respective protein lysates. In situ hybridization of viral genome in CVB3 infected A/J mice. In this experiment, I performed in situ hybridization of the viral genome to determine the extent of infection in different organs of the immunocompetent A/J mice. The antisense (negative) strand of the viral RNA genome (nt 888-7399), labeled with digoxigenin-11-5'-triphosphate (DIG-11-UTP), was used as the RNA probe. Thus ISH positivity on day 4 p.i. is a strong indicator of successful viral entry, translation and replication. Figure 14 summarizes typical images of ISH observation of the various 4- and 10-week-old A/J organs during the acute phase of infection (4 days p.i.). Positive sense viral RNA genome in the tissues was hybridized to the DIG-11-UTP labeled probes and stained by Sigma BCIP / NBT (blue black) in the tissue sections. I have chosen day 4 p.i. to perform ISH because CVB3 viral replication in systemic organs, including that of the heart and liver, typically reaches a maximum. In general, CVB3 infection of A/J mice begins to exhibit increased viral replication in most organs at day 2 p.i.. Specific immune mediated viral clearance begins at day 4 and beyond 6 4 , 6 6 , 6 8 . Paul Cheung Page 81 Age dependent susceptibility of the heart. CVB3 infection of the heart muscles was random and dispersed, without specific localized sites of replication. From experience, the atrial tissue demonstrated limited CVB3 infection when compared to ventricular cardiomyocytes. My tissue sections thus were obtained from the left ventricular free wall. From the ISH observations, young mice appeared more susceptible to CVB3 than older mice, and such contrast is illustrated by the representative images in Figure 14a. Contrasting susceptibility of the pancreatic cell types. Pattern of CVB3 infection of the pancreas tissue was intriguing (Figure 14b). High levels of viral replication and cellular injury was observed in the exocrine cells. However, the neighboring endocrine acinar and ductal cells were contrastingly resistant to viral replication and appeared healthy. Occasional interstitial and endocrine cells contained some viral genome. It is unclear if the resistance of the endocrine cells was due to the lack of the CVB3 receptor or the presence of intracellular inhibitors of the viral replication. Both the 4-week and 10-week-old mice appear to be similarly susceptible to infection in the pancreas. Increased susceptibility of the liver with age. Liver cells of the A/J mice exhibited a generally susceptible phenotype to the CVB3. The pattern of infection appeared random and non-localized (Figure 14c). The level of genomic viral RNA was lower than that observed for the cardiomyocytes. Overall, cellular injury and viral replication in the livers of older A/J mice were similar to that in the younger A/J mice, in contrast to the decreased viral replication with age that was observed for the cardiomyocytes. Paul Cheung Page 82 CVB3 infectivitv in the brain, spleen and kidney. In general, CVB3 injury in the brain and spleen was not as severe as in the heart, pancreas, or liver. However, it is interesting to observe that in the brain and spleen, CVB3 replication appears to localize in specific regions of the tissue (Figure 14d-e). There was no age dependence of tissue injury or significantly different levels of viral titre in these organs. However, the reduced level of viral genome in the spleen of A/J mice on day 4 pi may be a result of rapid viral clearance by the immune cells in the organ. Finally, ISH observation of the kidney was in contrast to all the other organs. Almost no virus genome could be detected in the kidney cells (Figure 14f). The tissue also appeared healthy and was highly resistant to the CVB3 replication regardless of age. Paul Cheung Page 86 Profiles of the protein-5'UTR interactions (Aim 2) UV cross-linking reactions were performed using protein lysates of the various AJ] mouse tissues and the radiolabeled probes of the 5'-UTR regions (nt 1-209, nt 210-529, and nt 530-630) . The pattern of protein-UTR interaction of each protein lysate is presented in one autoradiograph, in the format described in Figure 15. Each experiment was repeated at least 3 times to verify the reproducibility of the pattern and estimations of the molecular weights. Lanes CO V) 0 o Q . s o <D M — o c o o 0) Autoradiograph of SDS PAGE Figure 15. Layout of each profile of tissue protein-5'-UTR interaction. Labeled probes of the 5'-UTR regions were added to tissue lysates. The sense and antisense sequences of each 5'-UTR region were paired up (lane 1,2 for nt 1-209; lane 3,4 for nt 210-529; Paul Cheung Page 90 and lane 5,6 for nt 530-630). MWs of the protein-UTR complex were determined by a concurrently loaded MW marker. UV cross-linking analysis was performed for protein lysates from each of the A/J mice organs. Two autoradiographs were generated per organ: using 4-week-old lysate and 10-week-old lysate. Thus, a total of 12 autoradiographs were produced. The results are presented in Figure 16. A comprehensive list of identified protein-UTR complexes in each profile is presented in Table 5. In the following section, I shall summarize important observations from these profiles of interaction. Intense interactions in the IRES region (nt 530-630). The 100 nt long IRES region displayed the most intense protein-RNA interaction in each of the profile (lanes 5 and 6 in each panel of Figure 16). The intensity of bands was generally stronger in comparison to protein-RNA interactions within other regions (nt 1-209, lanes 1 and 2; nt 210-529, lanes 3 and 4). The prominence of bands of the IRES-interacting proteins indicates abundance of these factors, or the capability of these factors to bind multiple copies of the IRES. The patterns of the interacting bands were not as discrete as others, possibly due to complex aggregations among the various UTR-binding proteins. This may reflect the functional importance of the IRES, in which multiple molecules such as the ribosomal proteins, rRNAs and the eIF4 factors interact and form the translation initiation complexes. This interpretation is in line with our previous observation that the short 100 nt IRES core sequence is essential for the initiation of viral translation 3 4 9 . Paul Cheung Page 91 Four unique interactions with the sense sequence of nt 210-529. There are four UTR binding proteins that are consistently observed in all profiles, in a configuration that resembles two sets of doublets, with molecular weights 29 kDa / 31 kDa (Marked A in all of Figure 16) and 43 kDa / 45 kDa (Marked B). In the 10-week pancreas, the 29 kDa / 31 kDa band is not very distinctively resolved under standard exposure time, but using longer exposure the doublets at the anticipated position could be observed (data not shown). Differential intensity of such interactions among the tissue extracts reflects the differential expression / availability of the UTR-binding proteins in the tissues, since the overall protein- and probe-quantities in each UV cross-linking experiment and the time of autoradiograph exposure were standardized. Lack of UTR-binding proteins in the pancreas. The intensity of protein-CVB3 RNA interactions in the pancreas appeared weaker than in other organs, despite the use of identical experimental conditions. Particularly in the antisense region of nt 1-209 and 210-529 (lane 2 and 4, Figure 16c), I have observed little significant protein interaction. This is in contrast to other profiles. This weakness in band intensity can be due to two major reasons: 1) Proteases in the pancreatic lysate began digestion of the UTR-binding protein during the incubation with hot probes. 2) Nucleases in the pancreatic lysate began digesting the radiolabeled UTR probes during the incubation with proteins. I would also like to emphasize that it was not possible to isolate the endocrine and exocrine cell types by the methods that were available at the time of experiments. Thus, the Paul Cheung Page 92 lysate was in fact a combined lysate from the endocrine and exocrine cell types which were, from my in situ hybridization data, contrasting in susceptibility. This short-coming in my experiment is recognized and possible resolution to this issue is discussed in the future directions (Chapter VII). The p28 interaction correlates with kidney's resistance. Perhaps the most intriguing result from this set of UV cross-linking experiments is the intense 28 kDa protein interaction with the antisense sequence of nt 250-529 (marked C in the respective profiles in Figure 16a). As illustrated by in situ hybridizations, kidney is the only organ that remains consistently resistant to CVB3 infection. To compare protein-probe interactions of the lysates in this UTR probe, Figure 17 displays a summary of lane 4 of Figure 16 from the brain, pancreas, kidney and heart. Both the 4-week and 10-week profiles of the kidney demonstrated the p28 interaction at very similar intensities. In addition, the p28 band was detected in the 10-week-old heart profile, but not in the 4-week-old profile. This p28 interaction in the heart corresponds strongly to ISH observation wherein older mice have less viral infection in the heart as compared to younger mice. Moreover, no such p28 interaction was observed in the severely infected pancreas. Minimal p28 interaction was also observed in the liver and brain, which can be correlated to the overall lower level of infection in these organs (Figure 14). Thus, the intense p28 interaction with the kidney lysates correlates well with the ability of different organs to limit viral titer during a CVB3 challenge. Paul Cheung Page 93 Figure 16a. Profile of kidney protein interaction with CVB3 5'-UTR. Protein lysate of the A / J mouse kidney was U V cross-linked to the three 5 ' -UTR probes (nt 1-209, nt 210-529 and nt 530-630). Protein interactions with the sense (s) and antisense (s) probes are observed as bands. The UTR-binding proteins are observed as bands. The doublets labelled A and B are consistently observed in all profiles. The strong 28 kDa interaction (C) correlates with the kidney's resistance to C V B 3 infection at both ages. 4-week-old kidney N t . 1-209 Nt. 210-529 Nt. 530-630 M W 10-week-old kidney Nt. 1-209 Nt. 210-529 Nt. 530-630 s a.s. s a.s. s a.s. (kDa) Paul Cheung Page 94 Figure 16b. Profile of heart protein interaction with C V B 3 5'-UTR. Protein lysate of the A / J mouse heart was U V cross-linked to the three 5 ' -UTR probes (nt 1-209, nt 210-529 and nt 530-630). Protein interactions with the sense (s) and antisense (s) probes are observed as bands. The UTR-binding proteins are observed as bands. The doublets labelled A and B are consistently observed in all profiles. The 28 kDa interaction (C) in the 10-week-old heart correlates with the age-dependence susceptibility of the organ as revealed by the in situ hybridization observation. 4-week-old heart 10-week-old heart Nt. 1-209 Nt. 210-529 Nt. 530-630 M W s a.s. s a.s. s a.s. (kDa) Paul Cheung Page 95 Figure 16c. Profile of pancreas protein interaction with CVB3 5'-UTR. Protein lysate of the A / J mouse pancreas was U V cross-linked to the three 5 ' -UTR probes (nt 1-209, nt 210-529 and nt 530-630). Protein interactions with the sense (s) and antisense (s) probes are observed as bands. The UTR-binding proteins are observed as bands. The doublets labelled A and B are consistently observed in all profiles. The absence of the p28 interaction in lane 4 is correlated to the intense viral genome detection by the in situ hybridization, supporting the postulation that p28 may be a potential inhibitor of infection. 4-week-old pancreas Nt. 1-209 Nt. 210-529 Nt. 530-630 M W 200 _97_ 68 43 29 18.4 14.3 a.s. a.s. a.s. (kDa) Nt. 1-209 10-week-old pancreas Nt. 210-529 a.s. a.s. a.s. (kDa) Paul Cheung Page 96 Figure 16d. Profile of brain protein interaction with CVB3 5'-UTR. Protein lysate of the A/J mouse brain was U V cross-linked to the three 5 ' -UTR probes (nt 1-209, nt 210-529 and nt 530-630). Protein interactions with the sense (s) and antisense (s) probes are observed as bands. The UTR-binding proteins are observed as bands. The doublets labelled A and B are consistently observed in all profiles. However, the p28 was weakly detectable in lane 4. 4-week-old brain Nt. 1-209 Nt. 210-529 Nt. 530-630 a.s. a.s. a.s. (kDa) 10-week-old brain Nt. 1-209 Nt. 210-529 Nt. 530-630 M W 200 _97_ ..68. 43 29 18.4 14.3 a.s. a.s. a.s. (kDa) Paul Cheung Page 97 Figure 16e. Profile of spleen protein interaction with CVB3 5'-UTR. Protein lysate of the A / J mouse spleen was U V cross-linked to the three 5 ' -UTR probes (nt 1-209, nt 210-529 and nt 530-630). Protein interactions with the sense (s) and antisense (s) probes are observed as bands. The UTR-binding proteins are observed as bands. The doublets labelled A and B are consistently observed in all profiles. However the p28 was not observed in lane 4. Nt. 1-209 4-week-old spleen Nt. 210-529 Nt. 530-630 M W 200 a.s. a.s. a.s. (kDa) Nt. 1-209 10-week-old spleen Nt. 210-529 Nt. 530-630 B / / a.s. a.s. a.s. (kDa) Paul Cheung Page 98 Figure 16f. Profile of liver protein interaction with CVB3 5'-UTR. Protein lysate of the A / J mouse liver was U V cross-linked to the three 5 ' -UTR probes (nt 1-209, nt 210-529 and nt 530-630). Protein interactions with the sense (s) and antisense (s) probes are observed as bands. The UTR-binding proteins are observed as bands. The doublets labelled A and B are consistently observed in all profiles. The 28kDa interaction (C) is also observed and such correlates to the reduced viral load as observed by the in situ hybridization. 4-week-old liver Nt. 1-209 Nt. 210-529 Nt. 530-630 M W Pau l Cheung Page 99 0 O 1 « , cj c — .2 -c tS .SP 2 ^ CJ 0) 'S 2 CJ -o •a O I i 1 b xi o £ -a X C 3 CJ , „ 3 s cj C Cu i -Sa ?— CJ a CJ 0 o £ ^ -2 •S © c <*> * 1 N CJ cj .9 >> _ CJ fN -a «? 3 O cj i-H J= N c e -g <U CJ C c cj _ ^ 5 ° cj o c CJ CJ H 3 8 •C o X e a -b O O •r vs !s CJ 5 C3 S | o •=  U CJ • 2 c o ,CJ c oo CN OH CU S on a U. H •a <u s -CJ > cn CJ a -5 CJ o fc <a o .S u > t CS CJ S e T3 05 CJ 1-u B CS ft. 03 2 ... CJ CJ is JL cj CJ e CJ CJ CJ CJ e CJ CJ T ... CJ CJ St JL cj cj CJ CJ CJ CJ JL CJ CJ O In order to quantify the descriptive correlation (ISH and p28 protein) as much as possible, observation CVB3 viral genome by ISH staining of the CVB3 genome in the kidney, heart and pancreas is subjectively scored from 1 to 5 (1 being the lowest level of infection and 5 the highest). The scoring was averaged from myself and two fellow laboratory technicians, and the results are listed in Table 4.0 below. Table 4. Summary of the correlation between ISH observations and the p28 interactions. The detection of CVB3 genome by the ISH observation and the intensity of the p28 interaction were listed. The amount of viral titre is subjectively scored from 1 to 5 and represented by the number of'+' in the table. No observable viral genome was detected from the kidney. Organ and age Viral infectivity by ISH Intensity of p28 interaction 10-week kidney lysate No virus genome observed STRONG 4-week kidney lysate No virus genome observed STRONG 10-week Heart lysate + + + MODERATE 4-week heart lysate + + + + + WEAK 10-week-old Liver lysates ++ MODERATE 10-week-old + + + + + NONE Pancreas lysates Paul Cheung Page 101 Unique p43 interaction with the antisense sequence in the brain. From Figure 17 and Figure 16, a strong 43 kDa protein from the brain lysate is observed to bind to the antisense nt 210-529 (lane 4). Although this may not be correlated to a specific pattern of infectivity, it appeared to be only strongly observed in the brain lysate. A modest 43 kDa interaction has been observed in the 10-week-old heart and the 10-week-old liver. The intensity of the p43 interaction may indicate possible abundance of this specific factor in the brain lysate. Whether this interaction plays a specific role in the replication of the virus and correlates with the apparent localized infection of the virus in the organ requires further investigation. Other important protein interactions In comparing the pattern of protein-UTR interactions from the 10-week-old heart to the 4-week-old heart, there is a major difference in the protein interactions with the anti-sense sequence of the nt 529-630 (lane 6, Figure 16b). The ~200, ~100kDa protein bands were observed in the 10-week-old profile while the intense 43 kDa protein band was only weakly observed in the 4-week-old profile. These bands correlate with the differences in susceptibility of the two age groups. Thus, these bands of proteins are interesting targets that are worth further identifications and studies. To summarize other UTR-binding proteins, Table 5a and 5b presents the molecular weights of the protein complexes that are observed in each of the profiles. Paul Cheung Page 102 Table 5a Summary of cellular protein interactions with viral 5'-UTR sequences for multiple organs of the 4-week-old animals. Organ/ polarity of RNA probe nt 1-249 Protein (kDa) binding to nt 250-530 nt 530-630 Heart (+ve) (-ve) 29,38,45 68 29,31,43,45 43 Under U 28,38 28,43 Pancreas (+ve) (-ve) 18,43,57 29,31 (both weak), 43,45 Under 29 38,43 35 Liver (+ve) (-ve) 22,29,38,45,68,170 68, 80,170, 29,31,43,45 28,43 (weak) Under 18 28,29,43 29,43 Brain (+ve) (-ve) 22,29,38,43,85 57,68 29,31,43,45 43 under 14 29,43,97 Spleen (+ve) (-ve) 22,29,38,43 Under 22 29,31,43,45 14,22,29 22,28,29 Kidney (+ve) (-ve) 29,38,43 68,200 29,31,43,45 28 Under 28 29 28 Paul Cheung Page 103 Table 5b. Summary of cellular protein interactions with viral 5'-UTR sequences for multiple organs of the 10-week-old animals. Organ/ polarity of R N A nt 1-249 Protein (kDa) binding to nt 250-530 nt 530-630 Heart (+ve) (-ve) 29,35,43 70 29,31,43,45 28,-> 43 Under 18, 28,35 28,43,97,200 Pancreas (+ve) (-ve) 22,29,35,43 170 29,31,43,45 Under 29 ,29 29 Liver (+ve) (-ve) 28,35,43,85,170 68, 170 29,31,43,45 28,43 Under 18 28,29 29,43 Brain (+ve) (-ve) 22,29,35,57,85 57,170 29,31,43,45 28,43,57 Under 18 28,35,43,57 28,35,43,57,85 Spleen (+ve) (-ve) 22,28,35 29,31,43,45 Under 18 28,35 weak signals Kidney (+ve) (-ve) 28,29,43 57,170 29,31,43,45 28 Under 29 29 29 Paul Cheung Page 104 HeLa cellproteinS'-UTR interactions (Aim 3). Identification of 5'-UTR binding HeLa cell proteins was achieved by competitive UV cross-linking assays. Specifically, the sense sequences of the 5'-UTR were tested because of their functional significances in tissue tropism and viral translation initiation. Non-labeled homologous RNA of each probe sequence was used as competitors to assess the specificity of the protein-UTR complex formation observed in the autoradiograph. In each of Figure 18a-c, Lane 1 illustrates probe-protein complexes without competitor. Lane 2 is a negative control which includes samples that were not treated with UV radiation. No cross-linking took place and thus no protein-RNA complex could be detected after the RNase A treatment. This is a negative control for the protein-probe complexes that were observed in other lanes. Homologous competitor of the corresponding probe sequence was added in lanes 3, 4, and 5 at the indicated molar ratios relative to the probe. As in the mobility shift experiments, specific interactions were concluded when the increase in band intensity corresponded to the decrease in competitor ratios. Specific interactions. Figure 18a demonstrates a total of five proteins that specifically interacted with the probe nt 1-209. The MWs of these proteins were 200, 80, 62, 52, and 45 kDa. Similarly, a total of six proteins were observed to bind the nt 210-529 sequence (Figure 18b). These proteins had MWs of 200, 100, 57, 52, 45, and 39 kDa. Four major proteins were observed to bind the IRES (Figure 18c). The MWs of these proteins are 170, 95, 57, and 52 kDa. All these proteins appeared to specifically interact with the core IRES sequence. Paul Cheung Page 105 Figure 18a HeLa cell protein interactions with the 5' U T R nt 1-209. HeLa cell lysate is U V cross-linked to the radiolabeled R N A , in the presence of non-labeled R N A at the indicated molar excess ratios. U T R -binding proteins are visualized as bands, and their respective M W estimate is indicated on the right. Lane 2 is a negative control of interaction without U V treatment. Non-specific protein interactions are marked NS 1. Paul Cheung Page 106 Figure 18b HeLa cell protein interactions with the 5' U T R nt 210-529. HeLa cell lysate is U V cross-linked to the radiolabeled R N A , in the presence of non-labeled R N A at the indicated molar excess ratios. U T R -binding proteins are visualized as bands, and their respective M W estimate is indicated on the right. Lane 2 is a negative control of interaction without U V treatment. Non-specific protein interactions are marked NS2. Molar ratio of unlabeled RNA to probe 1 2 3 4 5 Lanes Paul Cheung Page 107 Figure 18c HeLa cell protein interactions with the 5 ' -UTR nt 530-640. HeLa cell lysate is U V cross-linked to the radiolabeled R N A , in the presence of non-labeled R N A at the indicated molar excess ratios. U T R -binding proteins are visualized as bands, and their respective M W estimate is indicated on the right. Lane 2 is a negative control of interaction without U V treatment. Molar ratio of unlabeled RNA to probe 1 2 3 4 5 Lanes Paul Cheung Page 108 Some of these 5'-UTR binding proteins appeared to be differently susceptible to competition. For example, the 200 kDa protein band (lane 1, Figure 18a and b) was heavily competed by homologous RNA at the 5x (lane 3) and lx (lane 4) molar excess ratios. It was restored only at the l/50x molar excess (lane 5), while in contrast, the 45 kDa band was heavily competed at 5x excess but could be detected at lx molar excess ratio (lane 4). These differences in susceptibility to competition reflected the differential binding specificities or differential quantities of the respective proteins towards the probe sequence. Non-specific interactions. Several HeLa cell protein-5'-UTR interactions were concluded to be non-specific. These include the complexes (NS1) in the nt 1-209 experiment (Figure 18a) in the MW range below 38 kDa and the 65/ 85 kDa bands in the nt 210-529 experiment (marked NS2). These proteins were not reported as specific interactions for two reasons: 1) the intensity of these bands did not vary significantly in response to the changes in the amount of homologous RNA competitor (lane 3-5); 2) the band intensities were weak and the bands were poorly defined. Non-specific or very low affinity associations of labeled RNA to protein can occur, and due to the non-specific nature of these interactions, increasing the amount of homologous RNA competitor would not have resulted in competition for probe binding. A summary of protein molecular weights that bound to the three 5'-UTR sequences is presented in Table 6. Of interest, the MW of 170 kDa, 80 kDa and 45 kDa resemble eIF4G, Paul Cheung Page 109 eIF4B, and eIF4A respectively (bold type in the table). Functional roles of the eIF4A and B in resolving RNA secondary structures were discussed in Section 1.6.1. Proteins with MWs that corresponds to PTB (57 kDa) and La autoantigen (52 kDa) were also observed. Of note, the 52 KDa protein was observed to bind all three probes. Table 6. Summary of Protein-RNA interactions between CVB3 5'-UTR sequences and the HeLa cell Lysate. Among these, the p200, p57, p52, p45 can bind to more than one 5'-UTR probe. Each of them may be the same protein binding to multiple sites in the 5'-UTR or different proteins that exhibit the same MW. 5'-UTR sequence Molecular weight Comment Nt 1-209 200, 80, 62, 52, 45 Non-specific bands not included in list. Possible ID: eIF4B (80 kDa), eIF4A (45 kDa); La protein (52 kDa) Nt210-529 200, 100, 57, 52, 45 This 5'-UTR region contains cardiovirulence determinants. Possible ID: eIF4A (45 kDa); PTB (57 KDa); La protein (52 kDa) Nt 529-630 170, 95, 57, and 52 Possible ID: eIF4G (175 kDa); DAP 5 (95 kDa); PTB (57 KDa); La protein (52 kDa) Paul Cheung Page 110 HeLa cell protein-3 '-UTR interactions (Aim 4). I have tested six different 3'-UTR clones for their ability to interact with the HeLa cell proteins. As before, UV cross-linking experiments were performed. The pattern of protein-RNA interaction with each probe was compared side by side in Figure 19. Data for the sense and antisense sequence was presented in the left and right panel, respectively. Significant complex formation in the range of 70-100kDa (Cl) and 36-45kDa (C2) were observed for both the sense and antisense probes (Figure 19a and b). The intense signals were observed with smear-like band patterns. Repeated experiments with reduced amount of probe and / or protein lysate did not improve the resolution of bands in these areas. This smear but intense signal suggests possible aggregations of the 3'-UTR-binding proteins in those ranges of MWs. In general, the poly-(A) intact probes of the sense and antisense sequences demonstrated a visibly different pattern of protein-RNA interactions (comparing lanes 1, 3-6 of the left and right panel). For example, a 32 kDa (marked Q) protein complex was noted to bind to the antisense sequences specifically (comparing Figure 19a and 19b). The corresponding area of the sense sequence did not show any comparable complex. There were fewer complexes observed in the C l and C2 region of the antisense probes than in the sense probe. On the other hand, a common feature of the two probes was the interaction with a 52 kDa protein (marked L). This MW resembles that of the La autoantigen. Paul Cheung Page 111 ft g H -b CM in cu to ^ S <! 'c? "1 s ca ^ A <N co 3 C 8 CJ f s i co CD O JO o fN 4 F" E s 3 -2 -g •E E £M X CO 'I T3 3 3 X) "5 V, cu cu y N S CA a"> eu 05 CD V cd 2 co C 3 5 B, 00 3 3 CO to (N J3 . O U 3 1 P fN c/5 < < cu a . p cd 3 J s S a cu CO 3 m -—- 55 tN 3 V> i^T cu 3 X § F 1) "505 • a n cj H J= U 5 ro ; 3 E X g § Q. cd cu Mi. co £ .CT* 3 ' § fN O 3 cu '-3 co B U .2 "53 ® Oi -3 " F 5 D * -a 1? P J2 05 jS 3 O • | ' | 3 cu "tt 3 — -a .9 J - 1 e ~r 1* CO S. g 8 > « D —1 CO cu a 1 CD ro ^ 3 ju - ' •3 cu A § "3 5 .x i I I S 3 E I = v I .3 '53 8 s-a. cs tN ro cu co 3 J J E a. H S ro . « ro 33 '—1 O CQ -i f S .3 D. co *~" 3 cu .Sf ^ u X cu •3 3 D a, c « e cu 3 5 ° a. u CO TO -*-t O 5, Qi H D I Q-cu o i co cu cj 5 3 a-cu CJ JO o pi f— b I si x •3 X X "3 X "3 X •3 cu CJ e cu 3 a" CJ C/5 ca Unique protein interactions with the mutated 3 '-UTR sequences. When comparing the patterns of the protein interaction of the wild-type 3'-UTR sense sequence to that of the mutated 3'-UTR sense sequences (Figure 19a, lane 1 vs lane 3-6), no significant differences were observed. The extent of intense complex formations (area Cl and C2) were highly similar, and the 52 kDa band appeared to be similar. Thus, mutations in the stem loop X and Y, which led to tertiary structural instability of the 3'-UTR, appeared not essential for inhibiting protein interactions with the sense 3'-UTR sequence. When inspecting complex formation with the antisense probes (Figure 19b), the majority of the protein-RNA interactions were similar between the wild type probe (lane 1) and the stem-loop mutants (lane 3-6). However, for the mutants, two interaction bands (labeled AS1 and AS2, Figure 19b) appeared exclusively with the antisense mutants (lane 3-6). Such complexes were estimated to be approximately 22 and 24 kDa in size. The interactions were not as intense as other protein bands, suggesting either a low abundance of such proteins in the lysate or the low efficiency in the formation of such complexes. These two interactions were unique to the mutated 3'-UTR antisense probes, including that of the complementary mutant. Since the wild-type tertiary structure of the 3'-UTR is partially restored in the complementary mutant 2 2 9 , this observation strongly implies that the two additional interactions occurred by recognition of the mutated RNA sequences at nt 7352/3 and or nt 7391/2. The interactions of these two proteins with the mutated sequences, however, do correlate with the defective replication which is due to the mutations. In other words, the interaction of AS1 and AS2 can be related to the observation of impaired viral replication. Paul Cheung Page 113 Significance of the poly-(A) tail in mediating protein interaction. By comparing lane 1 to lane 2 in each of Figure 19a and 19b, we have observed that the poly-(A) tail of the sense 3'-UTR and (poly(U) for the antisense 3'-UTR) were crucial for complex formations. In the sense sequence of the poly-(A) deleted probe (lane 2, Figure 19a), the 52 kDa band was discretely observed, while smear -like bands were detected in the ranges of 85- to 90 kDa and also from 15- to 45 kDa. In the antisense probe, only the 52 kDa and the 75 kDa were strongly distinguished, while bands in the 35- and 90 kDa range were weakly observed. In general, these poly-(A)/poly-(U) deleted probes demonstrated highly reduced protein binding capabilities. Paul Cheung Page 114 4.3 DISCUSSION Specific interactions between host proteins and CVB3 UTR RNA probes were shown by the use of mobility shift assays and UV cross-linking experiments. Data from Aim 1 demonstrates the feasibility of detecting protein-RNA complexes by in vitro methods. In the following sections, I shall focus on the discussion of the findings that are significant and related to the pathogenesis of CVB3 infection. Interactions of A/J mouse proteins with the viral 5'-UTR. In my study, the differential susceptibility of A/J mouse tissues has been further established by data from the ISH experiments. Three major observations are: 1) Endocrine and exocrine pancreas cells had contrasting susceptibilities to CVB3; 2) Kidney cell types were resistant to infection despite previously reported high-level of receptor expression; 3) Cardiomyocytes had an age-dependent susceptibility to infection. Currently, these phenotypic observations are not adequately explained by any known molecular mechanisms. To investigate these issues, I have focused on the identification of host proteins that may be responsible for viral translation or transcription via interactions with the CVB3 5'-UTR. However, a draw-back in these UV cross-linking experiments of the A/J organ tissues was the variability in the content of the lysates, as organs contain variable amounts of fibrous material, DNA / RNA, lipid and salt. As a result of these variations, resolution of protein-UTR complex bands by the standard SDS PAGE was less than ideal. The nature of autoradiograph imaging has limited the ability to perform quantitative comparisons of the UTR Paul Cheung Page 115 binding proteins. Thus, my data analysis is focused on the identification of unique proteins that relate to the susceptibility of organs to CVB3 infection. In all the profiles of tissue protein-UTR interactions, a high degree of sequence specificity was observed. This is concluded from the significant differences in the number, intensity and MW of the proteins-UTR complexes formed by each of the 5'-UTR sense and antisense probes (comparing lane 1 to lane 2, lane 3 to lane 4 and lane 5 to lane 6 of each profile, Figure 16). Since the antisense sequence of the CVB3 5'-UTR does not serve as an mRNA template for translation, my initial intent was to exploit the results using the antisense probe as controls to verify the specificity of bands detected by the UV cross-linking method. To this purpose, the patterns of protein-probe interactions of the sense and anti-sense were indeed contrasted and thus demonstrated the probe-sequence-dependent nature of the protein-RNA interaction. The same can be concluded when comparing the protein interactions of the three regions of the 5'-UTR (nt. 1-209, nt. 210-529 and nt. 530-630). Common-UTR binding proteins in cell lysates of A/J mouse tissues. Despite these sequence-dependent differences in protein-UTR interactions, the overall profiles of interaction among all the A/J organ-lysates appeared similar (comparing the complete profile of UV cross-linking from one lysate to another). This overall similarity in the patterns of protein-probe interactions suggests that some UTR-binding proteins are universally present in all cell lysates. For example, four proteins, the 29 kDa / 31 kDa (Marked A) and 43 kDa / 45 kDa (Marked B; lane 3 in each panel of Figure 16), were found to bind to the sense sequence of nt 250-529, which were commonly observed in all profiles. As discussed in the Introduction, translation initiation of the viral or host RNA requires the eIF4 family factors (and other host Paul Cheung Page 116 proteins). These factors are universally present in all cell types, and considering the role of the CVB3 5'-UTR in translation initiation, those UTR-binding proteins that are commonly observed in all profiles are possibly involved in the process of viral RNA translation initiation. In light of the extensive secondary 5'-UTR structure in the sequence of nt 210-529 (Figure 9), I further speculate that the above mentioned four proteins may be involved in the stabilization of higher-order RNA structures. In this respect, the eukaryotic translational initiation factor 4A (eIF4A) can be a potential candidate. The eIF4A is a 45 kDa RNA dependent helicase protein. The eIF4A molecule functions by ATP hydrolysis to unwind secondary structures within the 5' leader of host mRNA, thereby facilitating the 40S ribosomal binding and scanning for the initiation codon. The affinity towards single strand RNA of the ATP bound eIF4A can vary significantly depending on phosphorylation status 2 6 3 , 2 8 6 . Thus, interaction of this protein to the CVB3 UTR sequence may be observed as closely migrating doublets of the phosphorylated/ dephosphorylated forms. Of note, a 45 kDa protein complex is also observed to bind this 210-529 sequence in the experiments using the HeLa cell protein lysate (Table 6). Whether the eIF4A is indeed one of the four proteins that bind to the nt 210-529 region remains to be verified by other methods, such as immunoprecipitation and / or the use of recombinant proteins. These proteins are also interesting because the sequence of nt 210-529 is currently regarded as important in the determination of the cardiovirulence of the virus (see Introduction). The UTR-protein complexes detected in my UV cross-linking experiments using the A/J mouse organ lysates are documented in Table 5. These proteins were not identified further than their apparent MWs due to technical limitations. At the time when the study was conducted, expertise and equipment for large scale protein purification and characterization was limited. This was compounded by the fact that the method of detection was dependent on radioactive Paul Cheung Page 117 labeled RNA. Although this method, in contrast to other fluorescence or biotin-labeling techniques, has the advantage of being highly sensitive and preserving the chemical properties of the UTR sequence, it required the use of dedicated equipment and bench space to limit environmental contamination. To further identify these proteins, I have proposed the use of several recently emerging proteomics technologies, and some preliminary data is discussed in Chapter VI. At the present time, in order to remain concise in the discussion of the key results, I will focus on the potentially important p28 protein interaction. The 28kDa protein interaction with antisense nt 210-529. The objective of the UV cross-linking experiments with the A/J mouse organ lysate was to discover if specific protein interactions may be responsible for the tissue susceptibility as revealed by the ISH observations. To this end, the 28 kDa protein-UTR complex that is observed in the profiles of kidney, liver and 10-week-old heart is unique and correlates well with the organs' resistance to the virus. The 28 kDa protein-RNA interaction occurs only in the 5'-UTR of nt 210-529, specifically to the antisense sequence (not observed in the sense sequence of this region). Considering the general rule of nucleotide base-pairings, It is expected that the sense and antisense probes would form similar (but mirror-imaged) secondary structures. Thus, the absence of such interaction in the corresponding sense sequence of nt 210-529 suggests that the p28 likely recognizes and binds to a specific single strand RNA sequence (e.g. loop regions), as opposed to secondary structures, of the antisense probe. As noted, CVB3 is a positive sense single-stranded RNA virus. Progeny RNA (sense RNA) is synthesized by transcription that is initiated from the 5'-UTR of the antisense template. If the p28 interaction does contribute to Paul Cheung Page 118 resistance to viral replication in the kidneys, the mechanism could be due to the sequestration of the viral antisense template by p28, preventing effective production of progeny viral RNA: a transcription block at this location of the antisense template may prevent the genomic RNA synthesis beyond the 5'-UTR. During acute myocardial infection, the antisense strands of CVB3 are synthesized rapidly and reach a high concentration in a very short period of time. Thus, a large amount of p28 is possibly required to inhibit viral transcription. To this end, the high level of p28 expression in the kidney correlates to its high resistance to CVB3 replication. The moderate to low level of p28 interactions in the liver and 10-week-old heart also correlate with their respective viral titre. Further investigation on the cellular concentration, identity and function of the p28 is required to verify my postulate. Data from these further work may provide very important information on the molecular determinants of CVB3 infectivity and tissue tropism. HeLa cell protein vs 5'-UTR. In this study, I have demonstrated a number of proteins bound to the three different 5'-UTR sequences. Some of these UTR-binding proteins may be identical proteins that have multiple binding sites in the 5'-UTR; some of them may be different proteins with similar MWs. Since there is no previous report on protein-RNA interactions of the CVB3,1 would compare my findings to other close relatives of CVB3. Dildine and Semler 9 9 reported that HeLa cell proteins that interact with the poliovirus 5'-UTR can be cross-competed by the CVB3 5'-UTR, suggesting a conservation of both the RNA stem loop structures as well as the basic function of the RNA-protein interaction in the viral life cycles, especially in the IRES-directed translation initiation. Further work has identified a Paul Cheung Page 119 number of known / unknown picornaviral-UTR binding-proteins 1 3 2 such as the hnRNP, PTB, La autoantigen and PCBP2 that bind to the IRES, with MW 68, 57, 52 and 39 kDa, respectively. Notably, the PTB can inhibit the activity of another host protein GAPDH 3 5 2 , while GAPDH is also capable of IRES interaction 3 5 2 . This observation is contrasted to the synergistic effect between the PTB and the La autoantigen in stimulating the EMCV IRES activity 1 7 4 . From current knowledge, it appears that specific picornaviral UTR sequences may interact with a similar set of host proteins, but each viral UTR-protein interaction can occur by a unique mechanism to achieve optimal translation initiation in their respective target cellular environments. In my experiments, some of the CVB3 5'-UTR-binding proteins have MWs that are identical to the above mentioned five polio-UTR interacting proteins (Table 6). In addition, I have also observed similarities in MWs between the CVB3 5'-UTR-binding proteins and the eIF4 family of translation initiation factors. These host proteins include the eIF4A (45 kDa), eIF4B (80 kDa), and eIF4G (170-220kDa). The three eIF4 proteins are constituent molecules of the 220kDa eIF4F, which is also critical in the translation initiation of eukaryotic mRNA. The eIF4F family and CVB3 S'-UTR. The large 170 kDa protein complex bound to the CVB3 IRES sequence is similar to the MW of the well-characterized eIF4G (Figure 18). The eIF4G is occasionally detected as a 220 kDa protein when it combines with other elF factors to form the eIF4F complex. The integral eIF4F is crucial for the host cap-dependent mRNA translation initiation 1 1 6 . Incidentally, UV cross-linking experiments detected a -200 kDa complex for 5'-UTR probes nt 1-209 and 210-529. The normal cellular functions of eIF4A and eIF4B are to resolve complex secondary structures of the mRNA prior to binding of eIF4G and ribosome. The binding of these factors to Paul Cheung Page 120 nt 1-209 and nt 210-529 of the CVB3 5'-UTR would not be surprising since extensive stem-loop structures have been predicted within these sequences (Figure 9) and the eIF4A and eIF4B could resolve the secondary structures prior to internal ribosomal entry and translation initiation. Pertinent to viral pathogenesis, eIF4G alone has been reported to bind the IRES of the EMCV; the eIF4E binding domain of the eIF4G is cleaved by CVB3 protease 2A p r o during active infection, leading to the inhibition of host mRNA translation while viral uncapped RNA remains unaffected (Chapter I). As a side note, a protein of MW that is similar to the eIF4E (25 kDa) is not observed in the UV cross-linking experiments with the HeLa cell lysates (Table 6). Reflecting on these findings, I propose that eIF4 family of proteins may also bind the IRES of CVB3 and play important roles in the translation initiation of the viral RNA, while eIF4A and eIF4B can be involved in the resolution of secondary structures in the 5'-UTR and facilitate the binding of eIF4G and ribosomes. Significance of the Poly-(A) tail in mediating protein-interaction. The existence of the poly-(A) tail sequence (in the sense 3'-UTR probes) or the poly-(U) tail (in the antisense 3'-UTR probes) was found critical to protein interactions with the 3'-UTR. This suggests two possible roles of the poly-(A) tail in the interactions: 1) the poly-(A) tail directly interact with the observed complexes; 2) the poly-(A) tail maintains a stable secondary structure that favours protein binding to the 3'-UTR. Based on the predicted stem loop X formation by base-pairing between the poly-(A) (or poly-(U) of the negative sense) and nt 7380-4 (Figure 10), deletion of the poly-(A) tail could disrupt base-pairing in the X-stem loop and thus lead to changes in the 3'-UTR secondary structures. In contrast, mutations in other 3'-UTR mutants did not result in changes in secondary structures 2 2 9 as mutations in the loop region only Paul Cheung Page 121 affect tertiary interactions (Figure 10). In other words, the absence of the poly-(A) or -(U) sequence resulted in altered secondary structure which in turn reduced the ability of the 3'-UTR in binding host proteins. This is a possible explanation for why the 3'-UTR mutants exhibit protein interaction patterns that are identical to the wild type, while the patterns of the poly-(A) or poly-(U)-deleted mutants were dramatically different. Further identification of the 3'-UTR binding proteins will thus be important to the understanding of the mechanism of poly-(A) tail on the viral pathogenesis. Protein interactions unique to the replication defective mutants. A major objective in the investigation of 3'-UTR mutants is to identify protein-UTR interactions that correlate with the inhibition of viral replication. To this end, the interactions between 22 / 24 kDa (AS1 and AS2) and the antisense mutant 3'-UTR probes were identified. The antisense sequence of the 3'-UTR is a template for the synthesis of the positive sense viral genome. As mentioned in the results, these two proteins are likely recognizing the primary (linear) RNA sequence on the 3'-UTR at the mutation site, since no 22 / 24 kDa proteins were observed binding to the sense mutant 3'-UTRs. While the low level of the 22 / 24 kDa protein complexes are suggestive of inefficient complex formation, it may also be due to a lack of these proteins in the HeLa cell lysate. Since, these two bands were not observed in the wild type probe (lane 1), it is likely that such interactions are specific. It is important to note that the complementary mutations of the stem loops also exhibit interactions with these two proteins, which was not unexpected. Melchers et al. 2 2 9 demonstrated that in a cell culture environment, different complementary mutations in the 3'-UTR can lead to contrasting growth characteristics, from as low as 5% to non-wild type efficiency. The instability of the tertiary structures in the 3'-Paul Cheung Page 122 UTR complementary mutants was evident from the temperature sensitive phenotypes of the mutants 2 2 9 . Hence, the tertiary structural stability of the 3'-UTR mutant RNA is possibly not sufficiently restored to prevent the interactions of the 22 and 24 kDa proteins. The 22 and 24 kDa interactions correlate well with the inhibitory function of the mutations and thus raise important issues for further studies. Investigating these proteins, along with the p28 kidney protein that was observed to bind the antisense sequence of nt 210-529, may greatly advance our understanding of the participation of host proteins in the CVB3 viral replication process. Paul Cheung Page 123 CHAPTER V Characterization of the La autoantigen - CVB3 UTR interactions. Data from studies described in Chapter IV indicated that a 52 kDa protein bound to the 3'-UTR and all three regions of the 5'-UTR. In light of current knowledge of picornaviral systems, including literature that shows a stimulatory effect of La protein in translation initiation from an IRES, it is worth investigating if the La protein may bind to multiple regions in the 5' and 3'-UTRs, and whether the La protein is processed by CVB3 proteases in a fashion similar to that is observed in the poliovirus system. 5.1 EXPERIMENTAL STRATEGIES A flow chart of the strategy employed in this series of experiments is presented in Figure 20. In addition to techniques described in Chapter IV, several other techniques were used. These include the expression and purification of the GST-fusion La protein in an E.coli system. The La autoantigen has been studied in other virus systems. The plasmid vector pGEX-La was a gift from Dr. Y. K., Kim / S. K. Jang, Pohang University of Science and Technology, Korea. Using this plasmid, recombinant La protein was produced and purified. Detailed modifications to the original purification procedure 1 7 4 are described in Chapter VII. Each batch of La protein production was performed with 100 ml of LB medium. A typical yield of 3.5 mg La protein was achieved. The functionality of this recombinant protein had been proven in a study of the IRES of the EMCV system 1 7 4 . The schematic structure of the pGEX-La plasmid and the process of protein purification is presented in Figure 21 and Figure 22, respectively. Paul Cheung Page 124 Figure 20. Strategy for characterizing the interaction between La protein and the CVB3 5'- and 3'-UTR. The recombinant G S T - L a protein was expressed in E.coli. Purified L a protein and G S T - L a fusion protein were used in competitive U V cross-linking assays to determine specific interaction. A saturation curve was generated by increasing the amount of R N A probe to further confirm the specificity of interactions. The corresponding data was transformed into a Scatchard plot to estimate the apparent dissociation constant (Kd). The concurrent / exclusive binding mode of the La protein with the 5'- and 3 ' -UTR was determined by two-stages U V cross-linking. Radiolabeled 5' or 3' U T R probes U V cross-linking with 5'; U T R probes •> La binds 5' UTR? + U V cross-linking with 3' U T R probes La binds 3' UTR? Purified L a protein saturation plot Scatchard plot Dual stage U V C \ Affinity of La-3'UTR interaction Can a La protein bind both 5' and 3' UTRs at the same time? Pau l Cheung Page 125 Figure 21. Structure of the pGEX-La protein plasmid. Expression of the GST-fusion protein is mediated by the T7 promoter. The fusion protein can be cleaved by thrombin to separate the La protein from the GST domain. Thrombin T7 , cleavaee site domain ~ | La protein sequence pGex-La Vector T o p i Amr^ rf*ctctdtiPA I J C I V X Paul Cheung Page 126 Figure 22. Strategy for the expression of recombinant La protein. E.Coli BL21 is induced to produce the recombinant protein, which was then purified by the G S T affinity method. Either the -80 kDa GST-fusion L a protein is eluted by 1M glutathione or the 52 kDa L a protein is obtained from the matrix by thrombin protease digestion. The purity of the protein was verified by SDS P A G E and coomassie blue staining. Lyze bacteria culture after the IPTG induction and growth @ 26 °C for 20 hrs GST-fused La protein GS4B beads Bacterial Lysate Repeat wash with PBS (purify) On bead cleavage to release La (52 kDa) by thrombin Mix bacterial lysate with Glutathione sepharose 4B (GS4B, green). GST-interacts with GS4B while other proteins (blue) is removed by washing Elution for GST-La (80 kDa) Elute the GST-La protein with 1M glutathione: La protein domain GST domain 5 units of thrombin is added to the beads and the supernatant. Paul Cheung Page 127 Quantification of the La-UTR complex The purified recombinant La protein was used in competitive UV cross-linking assays to determine if the La protein interacts specifically with the 5' and 3'-UTRs. In addition, I have developed the use of scintillation counting to quantify the amount of Cherenkov radiation emitted by the La-UTR complex in 70% ethanol. In brief, the UV cross-linked La protein-probe complex in each reaction was analyzed on the SDS PAGE as described in Chapter IV. The SDS PAGE was dried on Whatman paper and the band of protein-probe complex was physically cut out from the dried acrylamide gel. The band was immersed in 5 ml of 70% ethanol (covering the entire strip) in a transparent scintillation count vial. The activity of each band was recorded as counts per minute (CPM). Final CPM counts were adjusted for radioactive decay ([a-32P]-UTP, Vi life of 14.3 days) based on the day of each experiment. This approach is advantageous over the use of autoradiography in demonstrating the specificity of interaction because the data is numerically quantified, reproducible, and does not depend on uncertainties such as exposure time and image processing, etc. The method is also necessary because the scintillation count (cpm) allows calculation of the extent of complex formation in the saturation experiments, which were designed to biochemically characterize the La-UTR interactions. Paul Cheung Page 128 Estimation of the apparent Kd and stoichiometry of La-UTR interaction The apparent dissociation constants of interaction between the La protein and the 5' or 3'-UTR labeled RNA were determined by saturation experiments and the subsequent scatchard plot transformation of data. The two quantities allow a biochemical assessment of the physiological significance of the La-UTR interactions. The equilibrium assumption is summarized by the following equation: Ka La protein + RNA probe Protein-RNA complex Kd And using this equilibrium assumption, the interaction between La and the UTRs were characterized. Details of the method is described in Section 7.10. Similar Scatchard plot analyses have been used in other single substrate-interaction studies such as the poliovirus receptor-virion interactions 1 2. By transforming the data of the saturation curve into a standard scatchard plot, I have determined the apparent dissociation constant (Kd) of the La protein- 3'-UTR interaction. All data was organized, processed and plotted using Microsoft Excel. Paul Cheung Page 129 5.2 RESULTS Purification of the La autoantigen. The pGEX-La plasmid was successfully transformed into BL21DE3 (pLys) (Novagen). I have observed that induction of the transformed bacteria with IPTG at O.D. <0.6 will produce the maximum yield of La protein. Induction with IPTG beyond 0.5 mM (final concentration) did not result in significant gain in protein yield. There had been suggestion of using <0.3% glucose to stimulate expression of the recombinant protein. I have not observed significant improvement with this procedure. Purity of the La protein was verified by Coomassie Blue staining (Figure 23). The purity of the recombinant protein was verified by the absence of other protein bands and was suitable for use in subsequent interaction experiments. Each batch of La protein was aliquoted and stored in -80°C freezer for a maximum of one week to avoid protein degradation. Paul Cheung Page 130 Figure 23. Coomassie blue staining of the La protein preparation. Recombinant expression of the La autoantigen in BL21 DE3 (pLys) and purification. Protein lysate of the IPTG induced bacteria is shown in lane 4. Over expression of the recombinant GST-La protein (-78 kDa) is observed among other proteins. Lane 3 is the final supernatant to check against protein loss during washes of glutathione-sepharose beads. Lane 1 and 2 are the purified protein products. Recombinant GST-la (~78kDa) is eluted with 1M glutathione pH7.0 (lane 2). Purified La is obtained by 5 units thrombin on-beads-cleavage for 3 hrs at 4'C. The 10% SDS PAGE is stained by Coomassie blue. Lane MW(kDa) 2 3 4 GST-La (78 kDa) La (52 kDa) 200+ kDa 5 kDa Paul Cheung Page 131 Interaction of the 5'-UTR with the La protein. Purified La autoantigen was incubated with radiolabeled 5'-UTR probes and UV cross-linked. The results using probe sequences nt 1-209, nt 210-529, and 530-630 were shown in Figure 24a, b, and c, respectively. Interaction between labeled probe and La protein was observed by the 52 kDa band of complex on the autoradiograph (lane 3, all of Figure 24) . Homologous RNA and yeast tRNA were used as competitors to test for the specificity of interactions. Weak interaction of La protein and nt 1-209. Initial experiments did not detect complex formation between the La protein and the probe nt 1-209: No significant signal was observed in the autoradiograph. By trying different interaction procedures, I could observe the 52 kDa protein-probe complex when the labeled probes of nt 1-209 were preheated to 95 °C for 2 min prior to reaction with the La protein at 37 °C. Hence, it is likely that complex formation between the La protein and the sequence nt 1-209 was sub-optimal at 37 °C. Pre-heating of the RNA at 95 °C would have denatured secondary and tertiary structures. When added to the reaction, the temperature transition from 95°C to 37°C should have allowed formation of intermediate RNA structures that favored the binding of the La protein. Despite the requirement of pre-heating, the interaction between La and nt 1-209 appeared specific. In lane 1 (Figure 24a), the use of GST-La fusion protein resulted in a very weak interaction at the corresponding MW range (80 kDa). The use of 2x molar excess homologous non-labeled competitor (lane 2) resulted in a reduction in the amount of labeled complex. The use of non-labeled competitor of the nt 210-529, or nt 529-630, (lane 4, 5 respectively) also resulted in the reduction in complex intensity. Of note, intensity of the La Paul Cheung Page 132 protein-probe complex in lane 4, in which non labeled nt 210-529 was added, is much weaker than that in lane 2. Thus the competition effect by the non-labeled RNA of nt 1-209 (self) was weaker than that of the nt 210-529, and as a result the intensity of the protein-probe complex in lane 4 was lower. In other words, affinity of the La protein towards nt 210-529 is considered to be stronger than that of the self-competitor (nt 1-209). Similarly, competitor of the IRES core sequence (lane 5) also appeared to have a stronger competition effect than the self competitor. The sequence specific nature of the La protein - nt 1-209 interaction is confirmed by the use of vector RNA at 2x molar excess, which did not result in reduction in band intensity (lane 6). Strong interactions of La protein with the nt 210-529 and the IRES. Strong La protein interactions with the probe nt 210-529 and the IRES core sequence were observed (lane 3, Figure 24b and c). For these probe sequences, no pre-heating was necessary and standard interaction conditions were applied. In Figure 24b, the intensity of the probe-protein complex in the presence of self competitor (lane 2) was much weaker than in lane 4 and 5. The band intensity in lane 5 was in turn weaker than that in lane 4. Strong competition from a homologous RNA resulted in less labeled probe being bound to the La protein, and hence a lower band intensity would be the consequence of a more effective competitor. Therefore, the data suggests that the La protein bound most strongly to the nt 210-529 (lane 2), less so to the 530-630 region (Figure 24b, lane 5), and the weakest to the nt 1-209 region (Figure 24b, lane In Figure 24c, this hierarchy of La-5'-UTR affinity was confirmed by the use of nt 529-630 as probe: the intensity of the IRES-La complex (Figure 24c, lane 5), in which non-labeled nt 210-529 was used as a competitor, was weaker than in lane 2, in which the IRES self Paul Cheung Page 133 competitor was added. The nt 210-529 had a stronger affinity towards La protein than the IRES. Together with the observation in Figure 24a, the sequence nt 210-529 consistently demonstrated the strongest La-binding activity than other 5'-UTR sequences. In Figure 24b and c, the GST-fusion La protein appears to result in moderate complex formation (lane 1). The GST domain on the recombinant protein can reduce the ability of the La protein in RNA interaction, as the band intensity was noticeably lower than the corresponding La-probe complex. Paul Cheung Page 134 - a cu •a -3 ca cu i i cu ca CJ £ "8 s <p on 1° cu c o U o ro CJ ca CO cj , O cu c 00 T3 e -3 CJ »-o ca IS 3" CM cu CO O U CJ CJ CO CO CJ a X CU ON e CM CJ X) O l-D. cj > o ca O •5 C3 C cu 00 S3 u '5 _ca o B cu ca • £ cu cu O r>< a X H P IT; o B Q CM 2 3 T 3 ca cu j a cu i r --|. ex oo c 06 U CU cs S CU e a OS - J Of CM o OX) e CS -r CN CU > P CU 3 s -g * & cu VO CO ' JO CM o cu CO C —' p C^ — i ^ •a & § o CJ CO 3 CJ J*i ca cu CJ > CJ g T3 ca n -—' - c «8 £ JO O co .co f— -° , O VO cu CM cfi CO M 1 n. I CJ 02! H P io OX a cu CO CU S CM Vector RNA Nt 529-630 Nt210-529 No Comp. Nt 1-209 (self) j _ ce CS cu 0 V 1 2 a c S £ U CM C 3 cu J3 U "s a. T3 CJ T 3 -a a to CJ CJ so O CJ a. J CO W -J e T3 CJ cn S2 o U o' CN CJ C 3 CJ O a CJ oJ) T3 CJ CJ ~* 3 f N a" io CJ I/) o CJ o T3 CJ -t—i o CJ ON fN IT) fN CJ > C J a o '•5 — S. <N x ~ t U TO is S.2 En k « 2 73 S 93 C > D <u i -CJ c CJ SP o CS ra —J •o IS 2P 8 9 > f N U cu 5 * , — , © vo - i a <^  « 8 J 3 C ** u **« -5 » 2 tsjj a. o CJ 3 CJ JS H c e <* CJ ^ "3. 1 S |a C cj ca .o v—' "C o c a .2 § CJ fc •a TT fN CJ S-CCS -1 I H a -o CJ CJ > •a CJ I CJ CA X ) o CA 6X ' C •»• 3 U. e_ vo x JD a E o o a: f -b VO M OS ft. fN ID CA CU S C8 -1 fN §§8 Vector RNA Nt 529-630 Nt 1-209 No Comp. Nt210-529 (self) « y S - X o <u •S l-a o S E U fN OJJ e 3 I) SJ ft. TJ CD T3 -a CO CD tu CD VO CD . g '53 o Cu J O CO s — uJ C CO •a CD CO o <3 •Sh CO 00 | o o • U o m vo i o ro ro CD C CD O . E o CD c CO C »-CD <J = 3 CD 3 fN cr i n CD CO O CD O 60 "3 " -3 co co CD CD X CD CD CD J O o JS o E Cu <N ' to CD o ro v© i © ro CD > a co O •3 CD CD CD M O 3 CD C CD 42 O U Ou -3 op 6 1 O fN u CD CD co "3 < CD IB S ' § b rv O - a 3 CS §g o a cs CO 3 •3 CD CD _ CD •S H _ g '33 3 J> .2 § OX) s e •— M CO E j>> CD CD • 3 g o U 3 co U *3 8 CD CO xi . [2, ^3 rN 41 ox) 'C f*" 3 CD > •3 CD CD CO X) O co X _CD s. E o CD Qi t— Cfl CU S 3 —1 t-ro ai ex cs fi-ve IT) ro rN Vector RNA Nt210-529 Nt 1-209 No Comp. Nt 530-630 (self) u- 8 5 CS 3} O V •S fc-a o | a ©X) 3 cu 3 SB CL fe ft JS D Specific La protein - 3'-UTR interaction - hyperbolic saturation curve. Using the scintillation counting method, I demonstrated specific interaction between the La protein and the 3'-UTR by the saturation experiments. A fixed amount of La protein (3.27 x IO"7 M) was interacted with increasing amount of labeled 3'-UTR wild type probe (Table 8, Appendix section 7.10). Resulting radioactivity count of the complex increased until reaching a saturation level (Figure 25). The presence of specific protein-RNA interaction (as opposed to random / weak RNA association) was implied by the observation of the hyperbolic curve. The maximum saturation value in this experiment was estimated to be about 9600 cpm (marked by the line S). Of note, the cpm count of protein-probe complex was generally much lower than the specific activity of the labeled probe (~108 cpm / ug). This is because the procedure of RNase A digestion degraded non-interacting sequences of the RNA to nucleotides which were rapidly run off the SDS PAGE. The resulting cpm count of the complex reflected only the radioactivity of the sequence of the 3'-UTR RNA that was bound to the La protein. Paul Cheung Page 138 C P M of cut out band — —• to <Di O <Di © © o o o o o o o t o U l o o o o o © © o © o ID. o o ID. o o o (Dl o o ON o © © ~4 © © © - J <D1 © © 00 00 VO VO © U i © O i © © © © © © © © © © © © 3 CT n -I ft -o M CTQ n w oo+aoo'o 80-300? io-aooi -I io-aos'i Z.0-300X £0"30S'Z Z.0-300'£ i O " 3 0 J £ Z.0-300> iO-30S> £ 0 - 3 0 0 ? 4 Z.0-30SS iO-300'9 = iO-30S9 Z.O-300'i iO-30S'Z. Z.0-300'8 J Z.0-30S8 Z.0-300'6 4 iO-30S'6 4 90-300'I J Specific La protein - 3'-UTR interaction - hyperbolic saturation curve. Using the scintillation counting method, I demonstrated specific interaction between the La protein and the 3'-UTR by the saturation experiments. A fixed amount of La protein (3.27 x 10"7 M) was interacted with increasing amount of labeled 3'-UTR wild type probe (Table 8). Resulting radioactivity count of the complex increased until reaching a saturation level (Figure 25). The presence of specific protein-RNA interaction (as opposed to random / weak RNA association) was implied by the observation of the hyperbolic curve. The maximum saturation value in this experiment was estimated to be about 9600 cpm (marked by the line S). Of note, the cpm count of protein-probe complex was generally much lower than the specific activity of the labeled probe (~108 cpm / ug). This is because the procedure of RNase A digestion degraded non-interacting sequences of the RNA to nucleotides which were rapidly run off the SDS PAGE. The resulting cpm count of the complex reflected only the radioactivity of the sequence of the 3'-UTR RNA that was bound to the La protein. Stoichiometry of interaction (1:1) and the 90% saturation quantity. From the saturation curves (Figure 25), two important observations are noted. First, the curve indicates that at 4,800 cpm (50% of the maximum 9,600 cpm), the corresponding molar ratio of RNA to protein was approximately 0.5 (point B). This value is the ratio of RNA and protein at the start of reaction (not at equilibrium). A ratio of 0.5 indicates that for every 2 molecules of protein in the reaction, one 3'-UTR probe was present at the start of reaction. The 4800 cpm is the 50% saturation point of the maximum amount of La protein-complex achievable (which was limited at 3.27 x 10"7 M) under that particular experimental conditions. Together, the data indicates that 1.64 x 10"7 M of the La protein (50%) was saturated Paul Cheung Page 140 by 1.6 x 10 7 M of probe RNA (from Figure 25): this is consistent with an interaction stoichiometry of 1:1. This observation suggests a simple one to one molecular interaction between the La protein and the probe RNA, thus supports the use of the Scatchard plot transformation to estimate the value of the apparent Kd. Second, at the 90% saturation point (0.9 x 9,600 cpm), the initial concentration of the 3'-UTR probe in the reaction was 680 nM (6.8xl0~7 M) (Figure 25, point A). This concentration of probe corresponded to -11 pmole (1.1 xlO"11 mole) of the 3'-UTR RNA. Therefore, under the experimental conditions, 11 pmole RNA was required to saturate 90% of the available La protein. I defined this 11 pmole as the 90%-pre-blocking quantity. In the subsequent competition experiments, this amount of competitor non-labeled RNA was added to reaction to simulate a 90% saturation effect. By the same approach, the 50%-blocking quantity was determined to be 2.5 pmole (2.5xl0"12 mole) at the 50% cpm point (Figure 25, Point B). Mutations in the 3'-UTR do not affect interaction with the La protein. To determine if interaction between the La protein and the 3'-UTR mutant exists, competitive UV cross-linking assays were performed. The La protein was first incubated and UV cross-linked with a non-labeled RNA prior to adding the radiolabled wild-type-3'-UTR probe. As both radiolabeled and non-labeled RNA are chemically identical, UV cross-linking non-labeled competitors at the 90%-blocking-quantity would thus significantly inhibit the subsequent complex formation with radiolabeled probes. Thus, if the pre-blocking non-labeled RNA competed specifically against the 3'-UTR probe for interaction, the amount of radioactive complex would be significantly reduced. Paul Cheung Page 141 The reactions were carried out under standard conditions (5.2 pmole, 245 ng of La protein), pre-incubated with different non-labeled 3'-UTR RNA or vector RNA at the 90%-pre-blocking quantity, followed by UV cross-linking with 11 pmole radiolabeled wild type 3'-UTR RNA. Radioactivity of the corresponding band was determined from two independent experiments and plotted on the bar chart (Figure 26). A representative autoradiograph of the complex formation was included at the bottom of the bar chart to demonstrate the actual band patterns. Intensity of the La protein-3'-UTR complex under no competition was demonstrated in lane 1, with an averaged cpm count of-9,600. The use of non-labeled self RNA (wild-type-3'-UTR) at the 50%-blocking quantity resulted in a corresponding decrease in cpm count of the complex to -5,600 cpm (lane 2). When using the 90%-blocking quantity of cold self RNA, the complex count was reduced to -2,150 cpm (lane 3). Thus, the use of self RNA as competitor at the 50%- and 90%-blocking quantity demonstrated the specific nature of the La-3'-UTR interaction. This was contrasted to the use of non-labeled vector RNA (100 nt) at the 90%-blocking quantity, which did not result in noticeable changes of the cpm count of the complex (lane 10). Therefore the sequence specific nature of the interaction between the 3'-UTR wild type sequence and the La protein has been clearly demonstrated. The mutations in the 3'-UTR RNA did not affect the La protein-binding. When using the 90%-pre-blocking quantity of the 5 different non-labeled mutant 3'-UTR RNAs (Figure 26, lane 4-9), the resultant cpm counts were essentially identical to that observed when the non-labeled wild type RNA was used as competitor (lane 3). Thus, the corresponding 3'-UTR mutations as well as the poly-(A) deletion did not affect the ability of the 3'-UTR to interact with the La protein. This interpretation is consistent with the observation of the 52 kDa protein band in my previous 3'-UTR UV cross-linking experiments (Chapter I V ) . Paul Cheung Page 142 Figure 26. Two-stage UV cross-linking of La protein to 3'-UTR RNAs. Radiolabeled RNA probes of the 3'-UTR clones were interacted with the La protein. In lane 1, the wild type probe interacted with 5.2 pmole of La protein without competitor. CPM of the complex in this lane indicates maximum complex formation under non-competed conditions. In lane 2 and 3, wild-type non-labeled RNA were added at the indicated pre-blocking quantity. These non-labeled RNAs were UV cross-linked (1st UV crosslink) to the protein prior to the incubation and UV cross- linking (2nd UV cross-link) of the radiolabeled probe. The corresponding decrease in complex observation (reduction in yield) indicates specific competition. RNAs from the mutated 3'-UTR sequences (Chapter 4) (lane 4-8) also demonstrates similar competition, suggesting that the mutations did not affect the La protein interaction. Lane 9 is a non-labeled vector RNA control that did not compete for La protein binding. Radioactivity count of the respective complexes was averaged from two independent experiments. Paul Cheung Page 143 Each La protein molecule can only bind to either one of the 5'/3'-UTR at a time. To investigate whether the La protein could bind both the 5'-UTR and the 3'-UTR concurrently, I performed the experiment with the use of three different 5'-UTR RNAs at the 90%-blocking quantity. The results are shown in Figure 27. In lane 1 the non-competed La protein-3'-UTR interaction was measured with a radioactivity of ~ 12000 cpm. This is slightly higher than the maximum radioactivity as observed in the saturation experiment, possibly a result of a different batch of radiolabeled probes with higher specific activity (more 3 2P labels incorporated per molecule of RNA during transcription). Using 3'-UTR wild type non-labeled RNA at the 90% and 50% pre-blocking quantity (lane 2 and 3, respectively), I demonstrated the pattern of competitive complex formation under an exclusive binding scenario. This was because the cold 3'-UTR RNA was chemically identical to the radiolabeled 3'-UTR RNA and thus must have occupied the same binding site on the La protein as the labeled probe. In lane 4 to 6, pre-blocking by the 5'-UTR non-labeled RNAs also resulted in significant reduction of the cpm count of complex. In fact, the radioactivity counts appeared lower than that of the 3'-UTR wild-type competitor at the 90%-pre-blocking quantity (lane 2). This observation (competitions from the 5'-UTR non-labeled RNA) would not be expected if there were two different binding sites on the La protein for the 5' and 3'-UTR RNA. Paul Cheung Page 145 Figure 27 Two-stages UV cross-linking of La protein to 3' and 5'-UTR RNA. Radiolabeled RNA probes of the 3'-UTR Wild type RNA were interacted with the La protein. In lane 1, the wild type interacted with 5.2pmole of La protein without competitor. CPM of the complex in this lane indicated maximum complex formation under non-competed conditions. In lane 2 and 3, homologous (WT) non-labeled RNA were added at the indicated pre-blocking quantity. The corresponding decrease in complex observation (reduce in cpm count) indicated specific competition. In lanes 4- 6, non-labeled 5'-UTR RNAs were UV cross-linked to the La protein prior to interaction with the 3'-UTR probe. Competition from the 5'-UTR RNA appeared stronger than that presented by the homologous 3'-UTR RNA. Overall, the result suggests that the La protein can only interact with one UTR molecule at the time, and thus the model of concurrent 5' and 3'-UTR binding was not supported. Lane 9 is a control: non-labeled vector RNA did not compete for La protein binding. Radioactivity count of the respective complexes was averaged from three independent experiments. Paul Cheung Page 146 An important observation during my experiment was the apparently stronger competition effects of the 5'-UTR non-labeled RNAs than the 3'-UTR self RNA at the 90%-pre-blocking quantity. During the experiments, in order to obtain a more visually distinct band pattern of the La-3'-UTR probe under competitions (lane 4-6), the autoradiograph in Figure 27 was exposed with -50% longer time than the one presented in Figure 26. The data seem to suggest that the same quantity of the pre-blocking 5'-UTR RNA can result in more reduction in the protein-probe complex formation, than the 3'-UTR competitor RNAs. Hence the 5'-UTR RNA may have stronger affinity towards the La protein than the 3'-UTR. Further experiments to quantify the difference may be useful in understanding the functional significance of the interactions. Apparent dissociation constants of the La - UTR interactions In order to compare the affinity of the 5'-UTR and the 3'-UTR towards the La protein, I have determined the apparent Kd between the La protein and the 3'-UTR by transforming data of the 3'-UTR saturation curve (Figure 25) into the variables described by the standard scatchard plot (Figure 28). Standard equations of the transformation was programmed into a Microsoft excel work sheet and the results of each steps of the calculations were listed in Section 7.11, Table 9. In the scatchard plot (Figure 28), the quantity of [Protein Bound] / [RNA total] [Protein Free] was plotted against [Protein Bound] / [RNA total] (all items in concentrations, M). Paul Cheung Page 148 Figure 28. 3'-TJTR Scatchard plot Data from the La-3'-UTR saturation experiment (Figure 25) was transformed by the scatchard plot method (Table 8). The quantity of [ Protein Bound ] / ( [ RNA total ] x [ Protein Free ] ) was plotted against [ Protein Bound ] / [ RNA total ] . A best fit line and its respective linear equation was generated by Microsoft Excel to minimize its variance from each data point. The dissociation constant of interaction was deduced from the slope (figure printed on the next page). Paul Cheung Page 149 The apparent dissociation constant (Kd) and stoichiometry of interaction were deduced from the scatchard plots. The Kd is calculated from the slope of the curve (Kd= -1/slope) while the stoichiometry of interaction is reported by the X-intercept. In other words, the scatchard plot derived these two quantities by calculations involving all the data points in each of the experiment. From the slope of the best fit lines the Kd for the 3'-UTR was estimated to be 46 nM (4.6xl0"8 M, Figure 28). In general an experimental Kd value of 1-100 nM reflects a moderate to strong interaction. Whether the interaction is physiologically significant will depend on the concentration of the reactants in a cellular environment. Paul Cheung Page 151 5.3 DISCUSSION The La autoantigen was initially identified in patients suffering from autoimmune disorders such as systemic 2 2 3 and Sjogren's syndrome 1 . It is now understood that the La autoantigen mainly functions in the synthesis, termination and release of polymerase III transcripts. In normal cellular environments, La has also been reported to interact with the U-rich 5' regions of ribosomal mRNAs in vitro and to stimulate their in vivo translation in a Xenopus tissue culture system 8 9 ' 2 6 6 . A uridine-rich sequence is also present in the CVB3 IRES sequence (Figure 9). In our experiments, by comparing cross-competition from the 5'-UTR sequences, the La autoantigen appears to bind most strongly to the sequence nt 210-529. The presence of a major tRNA-like stem loop in this region (nt 210-481, Figure 9) is consistent with the tRNA processing function of the protein. This sequence, although not as critical as the core IRES in determining translation initiation of the virus 3 4 9 , may have served as an important site for the recognition of the CVB3 RNA by the La autoantigen. Role of secondary structures in the interaction of La with the 5'-UTR probes. Interaction of La with the three 5'-UTR probes were demonstrated by UV cross-linking between labeled probes and purified La protein (Figure 23). Although the La autoantigen appears to bind to all three probes of the 5'-UTR, we have not observed a common primary RNA sequence within the three 5'-UTR probes. Thus, interaction of the La protein with CVB3 5'-UTR is likely dependent on RNA secondary structures. This postulation is also supported by the observation that pre-heating the sequence nt 1-209 was required for detecting the probe-La complex formation with the nt 1-209. Heating the probe at 95 °C prior to the 10-minute Paul Cheung Page 152 incubation at 37 °C would have allowed the probe RNA to transit from a denatured state (at 95 °C) to the native state (37°C). Thus, the transition may have provided intermediate secondary structure(s) that are optimal to the binding of the La protein. In a cellular environment, factors such as eIF4A and eIF4B normally help resolve and modify the high-order RNA structures, thereby facilitating efficient protein binding for translation initiation. My experiment on UV cross-linking of HeLa cell proteins (lysate) has shown that the proteins binding to nt 1-209 are similar in size to eIF4A and eIF4B (Table 6). However, when using the purified recombinant La protein in UV cross-linking with the 5'-UTR probes, no such elF factors were present. In other words, the apparent low affinity of this sequence towards the La protein as observed by our experiment is likely due to the lack of other chaperone molecules that helps optimize the RNA structure for protein interaction. Functional significance of the La yroteinS'-UTR interaction. Reflecting on the findings that the La protein can associate with the 40S ribosomal subunits (Chapter I), my data suggests that the La autoantigen - CVB3 5'-UTR interaction stimulates internal translation initiation of the CVB3 genome by forming a pre-translation initiation complex. This hypothesis is supported by the observation that the La autoantigen binds to the IRES of EMCV and stimulates translation initiation, and that La protein interacts with multiple sites on the hepatitis C virus RNA 5. My findings on the La autoantigen and 5'-UTR binding were accepted for publication in the Virus Research 8 0 . At the same time when my work was being published, Ray et.al. 2 8 1 also reported a stimulatory effect of the La protein in a dose dependent manner on CVB3 IRES, although the mechanism of stimulation was not investigated. Paul Cheung Page 153 This stimulatory effect of the La protein has thus verified the functional significance of the protein-CVB3 UTR interactions that I have observed. Interaction of La protein with the 3'-UTR did not depend on tertiary structures. In addition, data from the 3'-UTR UV cross-linking experiments (Chapter IV) suggest that a 52 kDa protein may bind to the 3'-UTR, a process that is independent of mutations and the poly-(A) sequence. Thus, the binding site and / or RNA structure that this 52 kDa protein recognizes is located within the actual 3'-UTR sequence. The 52 kDa La protein is subsequently confirmed to bind to the 5'- and 3'- UTR. The La-3'-UTR interaction is unlikely to depend on the 3'-UTR tertiary structures, because the 3'-UTR mutant RNAs, which do not possess the wild type tertiary structures, also competed for the interaction with the La protein. Using the saturation plot and the scatchard plot, I have determined the apparent dissociation constant. The results are discussed below. Affinity of the La protein - 3 '-UTR interaction. The La protein-3'-UTR interaction was characterized by the scatchard plot method. The apparent Kd value is estimated to be significant (46 nM). In consideration of the previously reported effect of the 3'-UTR on CVB3 replication, my data suggests the La-3'-UTR interaction may have functional implications in the overall process of viral replication. In this respect, the interaction of the La protein with the minus-strand of the vesicular stomatitis virus (VSV) RNA 777 778 _ _ has recently be reported to stimulate viral replication ' . This finding suggests the potential function of La protein in viral RNA transcription. The potential roles of La-3'-UTR interaction in CVB3 replication may be highlighted by two separate events: 1) transcription of viral RNA Paul Cheung Page 154 from a template; 2) expression of functional viral proteases and other mature non-structural proteins from the genome, and hence the amount of replication complex that is available to perform the RNA-dependent RNA transcription. The first event is directly related to the 3'-UTR sequence. As discussed in the introduction, the picornaviral 3'-UTR sequence serves as the anchoring point of the RNA-dependent RNA transcription complex. Whether the La-3'-UTR interaction may serve to facilitate template transcription remains unknown. Thus, as an extension of my current findings, it would be interesting to test for potential interactions between the La protein and the viral 3D polymerase. The second event is in fact highly dependent on the viral RNA translation initiation. As the viral replication complex is composed of viral and host proteins, sufficient expression of viral proteins would affect the rate of viral replication. At this point it is worth noting that host proteins such as PTB have been suggested to serve dual roles in the translation and RNA replication of the poliovirus (discussed in the Introduction). Considering my observation that the La protein can interact with both the CVB3 5' and 3'-UTR^ it is reasonable to hypothesize that the La-UTR interactions may serve in both the viral translation and transcription processes. Consequently, this raises the question of whether the CVB3 3'-UTR may also co-operate with the 5'-UTR in promoting viral translation and or replication. To date, there is no data that suggest the participation of the CVB3 3'-UTR in viral translation initiation. Findings by Melchers et.al. 2 2 9 seem to suggest that the transcription mediated by the CVB3 3'-UTR and the CVB3 translation were two discrete processes 2 2 9 . However, Melchers' experiments were designed to specifically discover determinants of viral replication, not translation initiation. Also, the lack of participation of the tertiary mutants at nt 7391/2 and 7352/3 in the CVB3 translation initiation does not exclude other sites within the 3'-UTR in regulating viral translation. At the same time, data from the HCV system seem to Paul Cheung Page 155 support a functional role of the 3'-UTR in directly stimulating viral translation 2 4 I n short, further investigation into the role of the 3'-UTR in promoting picornaviral translation initiation will reveal important mechanistic data on the subject. Circularization of CVB3 RNA. In the mammalian and yeast systems, the concept of circularized mRNA during translation has gained wide acceptance 3 3 4 . Synergism between eIF4G, poly-(A) and poly-(A) tail binding proteins in stimulating translation initiation has been a focus of many studies. Circularization of the poliovirus RNA was recently demonstrated by Herold and Andino 1 3 9 . In brief, long-range interactions between protein and PV UTRs complexes were shown to be necessary for RNA replication. The authors also demonstrated that the initiation of negative strand RNA synthesis requires a 3' poly-(A) tail, which also echoes my findings with respect to the importance of the CVB3 poly-(A) tail in recruiting host protein interactions. The PV 5' and 3'-UTR bridge is formed when the 5'-UTR RNP complex interacts with the poly-(A) binding protein bound to the 3' poly-(A) tail, essentially circularizing the viral genome and this process is required for the initiation of negative strand RNA synthesis. RNA circularization may be a general mechanism for replication of positive strand RNA viruses. Reflecting on these findings, my data does not directly support a model of circularized RNA for the CVB3 viral translation and/or replication process. In contrast to PTB protein which contains 4 RRMs and has been demonstrated to bind to multiple sites in the 5'-UTR of other viral RNAs (Introduction, chapter I), La protein only possesses one true RRM. Thus, it is likely that a La protein may not interact simultaneously with multiple RNA sites on the CVB3 5' and 3'-UTRs. Data from my dual-stage UV cross-linking experiments suggested that while the La protein may interact with either of the Paul Cheung Page 156 UTR sequences, only one of the UTR may occupy the protein at a time. Circularization of a CVB3 genomic RNA by one molecule of La protein is unlikely, although under the experimental conditions the possibility of protein dimerization could not be examined. At the same time, it is also important to realize that recent findings demonstrated the dimerization of La protein and the consequential enhancement of translation. Thus the question of whether a multiple La protein molecules interacting with the 5'-UTR and the 3'-UTR to form a circularized complex during viral translation and replication remains a possibility. Possible dimerization of La protein-UTR complexes. Protein-RNA UV cross-linking experiments have been used in other RNA studies. Throughout my investigation, I have experimented with different buffers and reaction conditions. Data presented in my results were optimized for the best band patterns, and the same patterns were reproducible in at least 3 independent experiments with different batches of radiolabeled UTR probes and protein extracts. During my trial experiments, I have noticed that the use of > lpg La protein in a reaction with total volume exceeding 30 pi at times can result in the detection of labeled protein complexes with MW of approximately 100+ kDa. These data were not included in my analysis because the detections were not always reproducible. However, since only one pure protein (recombinant La protein) was used in these reactions, the only logical explanation for observing the 100+ kDa bands were due to the dimerized La protein-probe complex. This is a reasonable preliminary evidence to indicate that La protein-UTR complexes may dimerize under specific conditions, possibly leading to the circularization of the CVB3 genome RNA. Relevant to the potential La protein-mediated RNA circularization is the observation that the dimerization of the La protein can stimulate viral translation, although the Paul Cheung Page 157 mechanism of such is still a mystery . Thus, my current observations provide a molecular basis for the synergism of dimerization: Multiple copies of the La protein may interact with several sites in the 5'-UTR and 3'-UTR; dimerization / multimerization of the La protein can result in circularized protein-RNA complexes which serve to stimulate translation initiation. To demonstrate the La protein facilitates CVB3 RNA circularization would be a significant step towards understanding the mechanism of CVB3 pathogenesis. The standard models of mRNA and RNA circularization in yeast / eukaryotic / other viral systems, rest on the interaction of eIF4G and PABP, bringing the 5' and 3' ends of the RNA closed together to form a loop. This model, however, can not be applied directly to the CVB3 or poliovirus system. In a CVB3 infection, the eIF4G carboxyl domain is cleaved by the viral protease. This cleavage will remove the domain of PABP interaction on the eIF4G and thus disables the interaction of the two and inhibits the circularization process. If circularization does occur with the CVB3 RNA, additional 5'-3' bridging molecules are thus required. To this end, the La protein can be the key alternative molecule in mediating RNA circularization. Circularization of the viral RNA would have two major theoretical advantages to the virus. Firstly, without the proper cap structure and with the extensive RNA secondary structure in the 5'-UTR, the CVB3 genome may not compete favorably against host mRNA for the standard translation apparatus. In this regard, the circularized complex confers improved efficiency to the viral translation by facilitating repeated rounds of protein expression, eliminating the need to recruit and re-assemble a new translation initiation complex at the 5' end. The advantage of this process may be most significant during early infection when very limited Paul Cheung Page 158 number of viral RNA must compete with the vast majority of capped mRNA for translation. Secondly, replication of the viral RNA occurs late in the viral cycle. In a HeLa cell culture environment this occurs typically in a two-hour time frame, from 7-9 hrs p.i. (cell lysis), when most host protein synthesis has been inhibited. It is conceivable that rapid viral RNA transcription is necessary to produce the progeny genome and allow packaging into the virions. A circularized complex, again, may assist and enhance the efficiency of this process. Although this is still conjecture, the La protein may be an important factor that helps the overall viral pathogenesis by facilitating efficient translation and transcription. Method developments and experimental limitations. Throughout my investigations, I have optimized and refined parameters related to the UV cross-linking experiments. Significant progress has been made to improve sensitivity, reproducibility and resolution of the technique in detecting protein interactions. Of note, the use of competitor RNAs in the HeLa cell experiment was successful to detect differential affinities of the protein-probe interactions (Chapter IV). This has led to two important developments in my subsequent studies. Firstly, given that a protein-RNA interaction is observed by the UV cross-linking technique, it is important to determine the specificity (e.g. by competition between yeast tRNA and self RNAs) and the affinity (e.g. by how the band intensity changes in response to the doses of competitor). These two variables would be important in characterizing the significance of each protein-UTR interaction. Secondly, the sensitivity of my current UV cross-linking procedure was suitable for revealing such differences. The development of a method to quantify the degree of complex formation in the UV cross-linking experiments would greatly improve my ability to characterize the interaction. Paul Cheung Page 159 To this end, I have modeled the La protein-UTR interaction by a two-molecule-interaction equilibrium. This model of interaction is supported by the saturation assay that each La protein appears to interact with one molecule of the 3'-UTR probe. Consequently, using the scatchard plot transformation, the Kd of the interaction was estimated and the value was concluded as an apparent value because of several reasons: 1) the concentration of the complex was estimated indirectly from the amount of radioactive protein-probe complex. Variables and uncertainties in the measurement of radioactivity thus impose a limitation in the actual estimate of the amount of protein complex. 2) the reaction was executed in an in vitro condition which does not necessarily reflect the physiological concentration of the reactants (e.g. La protein and viral RNA at different stages of the infection in a cytoplasmic environment). Thus, while the apparent Kd value does suggest a significant interaction between the protein and the RNA, the functional implications in the CVB3 pathogenesis remains to be verified. Importantly, my approach in the characterization of the La protein-UTR interactions has provided an example of how the specificity and affinity of other host proteins - CVB3 UTR interactions may be investigated. Finally, adaptation of the dual-stage UV cross-linking provided a means to extend the UV cross-linking technique to assess whether concurrent binding of La protein to the 5' and 3'-UTR was feasible. Although highly reproducible, I have observed that the dual stage UV cross-linking procedure imposed a reduced resolution of protein-complex on the SDS-PAGE: the bands in Figure 27 were not as discrete as those observed in Figure 18 (one stage UV cross linking between HeLa cell protein lysate and the 5'-UTR probes) and some level of protein Paul Cheung Page 160 degradation was observed. The cause of reduced resolution can not be definitively identified, but it may be related to prolonged protein-RNA incubations and manipulations: both the labeled probe and the recombinant La protein are susceptible to heat degradation; the small volume of reaction (necessitated by the optimized UV cross-linking procedure) could suffer changes in volume as a result of evaporation during the extended period of UV light exposure and or incubation, leading to changes in salt concentration and pH of the reaction. Paul Cheung Page 161 CHAPTER VI CONCLUSION AND FUTURE DIRECTIONS. In the course of my graduate program, I have had the opportunity to investigate the molecular aspects of protein-UTR interactions. Prior to the commencement of my studies, there were several interesting publications which described determinants of CVB3 tropism and viral replication. These determinants included specific nucleotide sequences that do not encode for functional proteins. The molecular effectors and mechanisms of CVB3 tissue tropism were unknown. Limited work was published in the investigation of protein-UTR interactions with other related viruses. My study made the first attempt to verify protein-CVB3 UTR interactions and identified targets that are worth further investigations. 6.1 Conclusions and scientific contributions. By using molecular techniques, such as PCR based cloning and mutation, mobility shift assay, UV cross-linking assay, recombinant protein expression and purification, in situ hybridization, and modeling of the protein-RNA interaction by a chemical equilibrium, I have investigated specific protein-CVB3 UTR interactions from the perspective of viral pathogenesis. The interactions of the La protein with the 5' and 3'-UTR is verified and further characterized. A summary of the important findings and contributions to scientific knowledge are listed below: 1. ISH indicates a strong resistance of the A/J mouse kidney towards CVB3 infection. In addition, the endocrine and exocrine pancreas cell types exhibit contrasting susceptibilities to CVB3 infection. Paul Cheung Page 162 2. A specific 28 kDa protein interaction with the CVB3 5'-UTR antisense sequence of the nt 210-529 correlates with the resistance to viral infection in the kidney, liver and the 10-week-old heart, as compared to other organs. 3. Fourteen HeLa cell proteins specifically bind the CVB3 5'-UTR sequences. Molecular weights of six of these proteins resemble that of the eIF4A, 4B, 4G, DAP5, La protein and PTB, respectively. 4. Specific interactions of certain HeLa cell protein with the 3'-UTR is dependent on the presence of the poly-(A) tail sequence. 5. Two HeLa cell proteins, 22 and 24 kDa in sizes, interact with the antisense sequence of the mutated 3'-UTRs and such interactions correlate with their respective inhibitory function on virus replication. 6. The La protein may interact with multiple sites of the CVB3 5'-UTR. Binding affinity is the highest in the nt 210-529, followed by the nt 530-630 and nt 1-209. 7. The La protein also interacts with the CVB3 3'-UTR. The interaction is not dependent on the 3'-UTR tertiary structures. 8. Apparent Kd of the La protein-3'-UTR interaction has been determined to be 46 nM. 9. Interaction of the La protein with either the CVB3 5'-UTR or the 3'-UTR is mutually exclusive. 10. Concurrent binding of both UTRs to the La protein is not supported by my observations. However, based on the ability of the protein to dimerize, the circularization of the CVB3 RNA by La protein remains a possibility. Paul Cheung Page 163 One of the significant contributions of my work is the discovery of many specific protein interactions with the CVB3 5' and 3'-UTR, some of which correlate to the CVB3 tissue tropism. A few of these interactions resemble known eukaryotic translation initiation factors, while other novel proteins require further sequence identification and biochemical characterization. My work on the La protein-CVB3 UTR interaction has provided an initial attempt to study protein-UTR interactions based on the UV cross-linking and the purified recombinant protein methods. 6.2 Strategies for future directions To further investigate the identities of the important UTR-binding proteins, I propose to perform experiments in the following directions: 1. To identify the amino acid sequence of the UTR-binding proteins that are potentially important in CVB3 susceptible and resistant cell types, by the methods of immunoprecipitation, recombinant protein expression, and other proteomics techniques. 2. To identify the protein binding sites on the 5' and 3'-UTR by foot-printing assays. To achieve the above directions, I have initiated experiments by three major techniques: the Laser capture microdissection (LCM), two dimensional polyacrylamide gel electrophoresis (2D PAGE) and Mass spectrometry (MS). Paul Cheung Page 164 To separate the endocrine and exocrine cell types by LCM An important ISH observation was the contrasting susceptibility of the endocrine and exocrine cell types. Currently, there is no satisfactory explanation for the difference in susceptibility of these two cell types. While receptor expression may account for such, it is also probable that specific cytoplasmic UTR-binding proteins that may either enhance or inhibit the CVB3 pathogenesis in the respective cell types. In this regard, investigation of the UTR binding proteins in the two respective cell types will be important to understand the mechanism of CVB3 tropism. To achieve this objective, the laser capture microdissection (LCM) is ideal for isolating these two pancreatic cell types. A flow chart describing the general LCM approach is listed in Figure 29. In brief, frozen tissue sections are placed under a normal optical microscope. Capturing of the specific cells involves the use of an adhesive plastic material (cap) which is placed between the lens and the frozen tissue section. When stimulated by a laser light, this cap extends to physically contact the laser-targeted cells. The cells then adhere to the cap for removal. Preliminary data of isolating the A/J mouse endocrine / exocrine cell types by the LCM method were demonstrated in Figure 30. Following isolation, the exocrine and endocrine cell types can be studied independently for their differences in UTR-binding proteins. Paul Cheung Page 165 Figure 29. General strategy for L C M procedure. Laser Capture Microdissection (LCM) Process. Image courtesy of Arcturus. The Laser Capture Microdissection Process 1. Place cap on tissue. 2. Pulse laser on target cells. 3. Remove cap with adhered target cells. 4. Extract molecules from target cells. Paul Cheung Page 166 Figure 30. L C M separation of endocrine and exocrine cells. A / J mouse pancreas was flash frozen, section at 8mm thickness, embedded in paraffin and stored at -80 °C. L C M was performed to remove the endocrine cell types. Before Capture Exocrine Endocrine Endocrine cells captured Exdocrine cells remaining Endocrine removed (adhered to cap) Endocrine removed (bubbles represent unsuccessful adhesion) Paul Cheung Page 167 To sequence identify UTR binding proteins by 2D PAGE and Mass Spectrometry (MS) The use of 2D PAGE enable protein isolation first by their isoelectric points (pi values) and then by MWs. This allows greater resolution of the numerous proteins in a lysate than the ID PAGE. My initial attempts of protein separation were highly reproducible (Figure 31) with good resolutions. Samples of 5'-UTR (IRES sequence) cross-linked HeLa cell lysate was compared to non-cross-linked lysates. Specific differences in the migration of proteins indicated proteins that are bound to the respective UTR sequence. Thus, proteins of interest can be subsequently identified: Protein on the 2D PAGE can be removed, trypsinized and the sequence identity can be analyzed by the Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) method. Of note, unique proteins such as the p28 from the kidney (which bound to the nt 210-529) and the 22/24 kDa doublets (which bound to the 3'-UTR sequence) can be specifically traced on the 2D gel by the corresponding molecular weights. Direct isolation and identification of these proteins would thus be possible. To verify UTR-protein interactions by standard molecular techniques Following the sequence identification from 2D PAGE/MS experiments, verification of the interaction would be necessary. This can be achieved by traditional molecular techniques such as immunoprecipitation and recombinant protein expressions. Also, literature search and analysis of the upstream/ downstream molecules of the identified proteins will provide important information with respect to the mechanism of how the interaction with the CVB3 UTRs can lead to specific pathogenic functions. Paul Cheung Page 168 To enhance the biochemical analysis of protein-RNA interaction by large format SDS-PAGE A short coming of my biochemical analysis of the La -UTR interaction was in the calculation of the apparent dissociation constant (apparent Kd). The amount of protein-UTR complex in my calculations was estimated by a conversion from radioactivity count. The conversion assumed a linear relationship between complex formation and radioactivity, and depends on the estimated cpm count of the saturation of the La protein by RNA probes. This assumption is largely valid based on the the principle of chemical equilibrium but remains an indirect measurement of the complex-concentration. Factors such as the efficiency of UV cross-linking and radioactivity detection can incur uncertainties in the calculation process. Thus, in order to derive a more realistic (non-apparent) Kd value, I propose to perform the experiments based on large format (30 cm+) ID SDS-PAGE. The increase in gel dimension would allow extended electrophoresis time to completely separate the labeled complex from RNase A-digested probe sequences, but retaining both in the gel. Throughout my investigation, small format SDS PAGE (12 cm) was used. While sufficient to resolve the protein-RNA complexes and MW of UTR binding proteins, RNase A-digested probes usually exit the gel rapidly. These RNase A digested probe sequences represent excess probe sequences that did not react with the protein. The large format gel would retain these RNase A-digested products in the lower part of the gel and thus allow quantification of the total radioactivity. By measuring radioactivities of the protein-UTR complex, the non-protein bound radioactive ribonucleotides and the initial activity at the beginning of the reaction, I can obtain a direct estimate of the ratio of complex formation in relation to the input quantities (amount of protein and specific radioactivity of the labeled probe). This can avoid assumptions that were made in my calculations. The approach Paul Cheung Page 170 should be technically feasible and requires only optimizations to ensure reproducibility without significant modification of the interaction procedures. 6.3 Final remarks In summary, by identifying novel UTR-protein interactions and their potential correlations with CVB3 infectivity over the course of my graduate studies, I have provided interesting and testable protein targets for further investigations. In the course of CVB3 infection, molecular mechanisms of the viral translation and transcription are fundamental processes that lead to a variety of pathologic observations and systemic illnesses. Further investigation of the protein-UTR interactions by the use of high throughput proteomics technologies will allow us to gain a much better understanding of how molecular events in the translation and transcription of the CVB3 genome can account for the CVB3 specific pathological phenotypes. The knowledge can significantly advance the design of CVB3 specific interventions. Paul Cheung Page 171 CHAPTER VII MATERIALS AND METHODS 7.1 Plasmid construction. PCR reactions were performed to amplify DNA fragments corresponding to the various 5' and 3'-UTRs. The respective primers are listed in Table 7. The amplified DNA fragments were cloned directly into the dual promoter T/A vector (Invitrogen) as per manufacturer's instruction. Cloned sequences and their orientations with respect to the RNA polymerase promoter on the vector were verified by DNA sequencing. 7.2 In vitro transcription. Prior to transcription, plasmid templates were linearized and purified by phenol/chloroform extraction and ethanol precipitation. RNAs of the three cloned sequences were synthesized by in vitro transcription using 1 pg of linearized plasmids and the appropriate RNA polymerase in the presence of [a-32P]UTP or [a-32P]CTP (3,000 Ci / mmol, Amersham) as per manufacturer's instruction. Non-radioactive labeled RNA was also synthesized, and served as specific competitors in the mobility shift and UV cross-linking experiments. RNA transcripts were purified by RNase-free DNase I digestion, phenol/chloroform extractions and ethanol precipitation at - 80 °C. The integrity and purity of RNA were verified either on a 1.5% formaldehyde-containing agarose gel (for non-radioactive transcripts) or autoradiography with 6 % SDS-PAGE ([ot- 3 2P] UTP labeled transcripts). Non-labeled transcripts were quantified by spectrophotometry and radioactive transcripts were adjusted to a specific activity of 1 x 107 - 1 x 108 cpm / pg. Paul Cheung Page 172 a _© u S s-lfl| c o W <* H & 93 3 93 H CO 03 . £ <u +-* C CJ CO 4) IH OH CJ 4> O 1) cr m co PH B, H Cn cj -4-» O i2 c o c o o I i -8 T3 CJ OH o PH CS 1-4 CJ O . s CJ a c rn OJ e OH Cn CJ C o U cj o a cu CT cj CC E H P I LO 00 KD ro I < E-H CJ E H C l < CJ o o E H CJ I (O I O E H U CJ CJ < U <: < < E H E H I LO O CM I 4-1 C u OO LO 00 I < E H E H O CJ U U CJ E H E H CJ •3 CJ I LO 00 I o < o E H E H CJ CJ U a u i LO CM LO I o <-H Csl 4-1 ro I E H U CJ <H E H U u E H <d I LO LO o ro <£> I o 00 LO 4 J C L O L O CJ CNJ L O Pi U P H co co O cu o a CD 3 CT CJ &Cj '3 "3 a JS ft! 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C J C J o C J EH ol C J C J C J < u C J C J EH EH EH EH EH EH EH EH EH EH EH EH EH H EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH C J < C J C J EH C J C J C J C J EH U CD g •H u X SO os ro os ro r -o Oi H I ro e « s S e o cu "3 Q cj EH EH EH < C J < C J < C J < EH EH U CD g • r H M a C J C J C J EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH EH C J < C J C J EH C J C J O C J EH U CD g •H U a 7.3 Preparation of HeLa cell extracts, HeLa cells (ATCC) were grown in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. Cells were harvested mechanically at approximately 95% confluency, washed 2x with PBS and incubated with lysis buffer (50 mM Tris-HCl pH 7.4, 150mM NaCl, 1% Triton, 0.1 % SDS, 0.5 % Na Deoxycholate, 10 mM EDTA) for 10 min. Cell lysates were dialyzed against buffer A [90 mM KOAc, 1.5 mM Mg(OAc)2, 1 mM dithiothreitol (DDT), 10 mM HEPES, pH 7.6] for 12 hrs at 4 °C. Protein concentration was determined by spectrophotometry using the Bradford protein assay kit (Bio-Rad). The concentration of the protein extracts was adjusted to 20 pg / pi. The extract was used directly for RNA-protein interaction assays or stored in aliquots at -80 °C until use. 7.4 Preparation of lysates from A/J mouse organs. 4- and 10-week-old non-infected A/J mice were sacrificed and internal organs (heart, kidney, brain, liver, spleen and pancreas) were flash frozen by immersion in liquid nitrogen. Protein extracts were prepared by the method of Correia et al.. Approximately 0.5 g of organ tissue is rapidly triturated with a pestle and mortar. One ml of the solubilization buffer [10 mM Hepes (pH 1.9), 10 mM KC1, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 % v/v leupeptin] is added to the powdered tissue. The mixture was then homogenized by 10 strokes in a Dounce manual tissue grinder with 20 pi of Nonidet P40 (NP-40), and left on ice for 15 min. The mixture was further homogenized by 5 strokes before centrifuging at 1000 x g for 5 min. The bottom fraction was washed three times with 1 ml of solubilization buffer with 0.5% NP-40. The remaining pellet was resuspended in 200 pi of storage buffer [20 mM Hepes (pH 7.9), 400 mM NaCl, 1.0 mM EDTA, Paul Cheung Page 176 1.0 mM EGTA, 1.0 mM DTT, 1.0 mM PMSF, 10% glycerol and 0.1% leupeptin]. The suspension was then incubated on ice for 30 min and centrifuged at 12000 x g for 5 min. The supernatant was then aliquoted in 50 pi and stored at -80 °C. The aliquots contained ~ 400 pg protein and were used directly for RNA-protein interaction assays or stored in aliquots at -80 °C until use. 7.5 RNA mobility shift assays. All RNA-protein interactions were carried out in a total volume of 20 pi. Five pi of binding buffer (2.5 mM KC1, 10 mM HEPES, 2 mM MgCl 2, 0.1 mM EDTA, 2 mM DTT, 5 D g heparin and 3.8 % glycerol), 500 ng (~5 x 106 cpm) of radioactive probe and 20 pg of HeLa cell extract were incubated for 15 min at 37 °C. In competition assays, either an unlabeled form of the probe RNA (self competitor) or yeast tRNA was incubated with cell extracts for 5 min prior to adding radioactive probes. Unlabeled yeast tRNA (Sigma) was used as a non-specific competitor because it was not homologous to the viral RNA sequences but contained similar stem-loop structures. Protein-RNA mixes were analyzed by electrophoresis through a 4 % non-denaturing polyacrylamide gel. After drying the gel, radioactivity was visualized by autoradiography. 7.6 UV cross-linking experiments. Reactions were carried out in a total volume of 20 pi containing 5 pi of binding buffer (see above). Unless otherwise indicated, 500 ng of labeled transcripts (5 x 106 cpm) was incubated with 10 pg of HeLa cell extracts or 10 pg organ lysates at 37 °C for 15 min. Competitor RNAs were added five min. before adding radioactive probes, at indicated molar Paul Cheung Page 177 excess ratios. The reaction mixtures were kept on ice and UV cross-linked (Stratagem 2400) for 25 min at a distance of 3 cm from the UV bulb. Total UV energy applied was 22.5U. After UV-irradiation, all samples were treated with 200 ng of RNase A at 37 °C for 20 min. As control, a reaction not treated with UV irradiation was digested with RNase A. Protein-probe complexes were then analyzed under reducing conditions by 9 % SDS-PAGE. The gels were fixed in a solution containing 15 % methanol and 20 % acetic acid for 10 min and dried on Whatman paper. The dried gel-paper was overlaied with autoradiograph film to image the protein-probe complex. MWs of probe-protein complexes were determined by comparing against simultaneously loaded protein markers. Additional modifications to the above core procedures were described in the respective results sections. 7.7 Virus, animal and tissue processing. CVB3 was generously provided by Dr. Reinhard Kandolf (University of Tubingen, Germany). The full length cDNA sequence of the virus is listed in section 7.11. Virus was propagated in HeLa cell monolayers in modified Eagle's medium (MEM) containing 10% fetal bovine serum (FBS) and 50 pg / ml gentamycin (Gibco, Grand Island, NY) at 37 °C in a humidified 5% CO2 atmosphere. When 70-90% of the cell monolayer showed viral cytopathic effects, flasks were frozen-thawed three times. Large cellular debris was removed by centrifugation at 2000 x g. Viral titers in the supernatant were determined by plaque assays. Dilutions of this supernatant were used for inoculation of mice. Inbred adolescent and young adult A/J [H-2a] mice (Jackson Laboratory, Bar Harbor, ME), 4 and 10 weeks of age, respectively, were infected with coxsackievirus B3 (105 pfu) or sham-infected with PBS intra-peritoneally, and euthanized by CO2 narcosis on day 4 post infection (p.i.). Fresh tissue was Paul Cheung Page 178 taken from heart, kidney, brain, liver, spleen and pancreas for fixation in 4% paraformaldehyde, paraffin-embedding, and histological sectioning for histopathological staining and in situ hybridization (ISH) of viral genome. Two 3-mm thick tissue slices of each organ were obtained per animal at sacrifice. Slices from the heart included left and right ventricular myocardium at the mid-cavity level of the ventricular free walls and ventricular septum. A portion of each tissue was archived for plaque assay, immediately frozen in liquid nitrogen and stored at - 80 °C. 7.8 In situ hybridization. CVB3 specific riboprobes were prepared by in vitro transcription of full length CVB3 cDNAs containing Sp6 and T7 promoters. These riboprobes were of the antisense sequence and labeled with digoxigenin-1 l-5'-triphosphate (DIG-11-UTP, Boehringer Mannheim, Laval, PQ). ISH was performed according to the procedures as previously described 7 0 . Briefly, tissue sections were formaldehyde-fixed, alcohol-dehydrated, xylene-cleared, paraffin-embedded, cut at 4-micron thickness, mounted on silanated slides and dried overnight at 60 °C to ensure proper continuous adhesion of the sections during processing. Slides were deparaffinized, rehydrated and permeabilized with proteinase K. The hybridization solution contained a digoxigenin-labeled, viral strand specific riboprobe (antisense strand, nt 888-7399), 50% deionized formamide, 10 mM Tris-HCl (pH7.4), 1 mM EDTA, rabbit liver t-RNA 200 pg/ml, 600 mM NaCl, 10 mM dithiothreitol, 0.02% polyvinylpyrrolidone, 0.02% ficoll, 0.02% bovine serum albumin, 0.1% SDS. Twenty pi of hybridization mixture was applied to each slide, then the siliconized coverslips were mounted and sealed with rubber cement. Hybridization was allowed to proceed at 42 °C for 24 h. Post-hybridization washing was followed by blocking with 2% lamb serum. Anti-digoxigenin-AP-Fab fragments (Boehringer Mannheim, Laval, PQ) were then Paul Cheung Page 179 applied to each slide (1:500 diluted, 150 ul per section) and incubated at 37 °C for 30 min. Sigma Fast BCIB/NBT buffered substrate tablet was used as colour substrate for the detection of alkaline phosphatase activity. Two tablets were dissolved in 300 ml of distilled water for the incubation of slides at 37 °C with very slow shaking. The colour usually developed in 24 h. Slides were subsequently counterstained with carmalum and examined quantitatively for reaction product by light microscopy. 7.9 Protein purification. I have modified the original pGEX-La protein purification procedures to achieve optimal purity and yield. Each 100ml of the transformed E. coli (BL21 DE3) cell was induced at mid-log phase by Isopropyl-(3-D-thiogalactopyranoside (IPTG, final concentration 0.1 mM) and allowed to grow at 26 °C for another 20 hrs. During purification, cell lysate / protein samples were kept on ice at all times to prevent thermal degradation of the La protein. Induced cells were collected by swing bucket centrifiigation at 4 °C at 3,500 rpm for 15 min. Higher centrifugation speeds results in cell pellets that appear resistant to sonication and produce frothy cell lysates. The 100ml culture was centrifuged in four equal volumes to ensure efficient precipitation. Each of the four cell pellets were resuspended in 5 ml phosphate saline buffer (PBS, pH7.0), exposed to two freeze-thaw (-80 °C to 4 °C) cycles, followed by five 1-min 50% output sonication (SONEX). A straw colored clear solution indicated optimal cell lysis. Cloudiness of the lysate may result from cells resistant to lysis and in this scenario, addition of 1% volume triton X-100 should clear the solution in about 20 min. The final lysate was centrifuged at 4,000 rpm for 5 min to remove cellular debris. Each of the 5 ml supernatant was treated with 150 ul 50% Paul Cheung Page 180 glutathione sepharose 4B beads {Pharmacia Biotech) at 4 °C for 1 to 3 hrs, in narrow diameter (1 cm) 15 ml Falcon tubes. The tubes were laid flat to maximize the interaction surface area between the beads and proteins. This resulted in noticeable increase in final yield. After incubation, the lysate was centrifuged at 3,000 rpm for 10 min and the pelleted beads were washed with 10 ml PBS. Five washes were repeated. Finally, 5 units of thrombin were added to the washed beads together with 100 to 200 pi PBS. This quantity of thrombin was insignificant and not detectable on SDS PAGE by coomassie blue staining. Cleavage of the La protein from the GST-coupled sepharose beads were allowed to occur at 4 °C for 2-5 hrs, followed by a centrifugation at 2500 rpm for 5 min. The supernatant was tested for protein concentration and purity by the Bradford assay and 10 % SDS-PAGE, respectively. To obtain the non-cleaved form of the GST-La recombinant protein, the washed beads were treated with the standard Glutathione elution buffer (Pharmacia). 7.10 Estimation of the apparent Kd and stoichiometry of La-UTR interaction The apparent dissociation constants of interaction between the La protein and the 5' or 3'-UTR labeled RNA were determined by saturation experiments and the subsequent scatchard plot transformation of data. The two quantities allow a biochemical assessment of the physiological significance of the La-UTR interactions. To characterize the interaction between the La protein and the UTR RNA, I have modeled the interaction by the following equilibrium equation: Ka La protein + RNA probe Protein-RNA complex Kd Paul Cheung Page 181 This equilibrium was studied by a simple saturation experiment: I have used a fixed amount of La protein in each of the reactions (initial concentration: 3.27 x 10"7 M) while the amount of radiolabeled RNA was increased until complex formation was saturated. The ratios of interaction was listed in Table 8. Saturation was observed in reactions where labeled-RNA probe was in large excess over protein. At such large probe-excess condition, the equilibrium of the protein-probe interaction was driven forward to form products and thus, it was reasonably accurate to assume that the final amount of protein-probe complex was the same as the initial amount of La protein used in the reactions. The cpm count of complex at saturation was estimated from graph (the plateau level). This maximum cpm count at saturation was equated to the initial concentration of La protein used in the reactions. Hence, the concentration of complex at other points in the same experiment was calculated from its radioactivity. The background radioactivity (-250 cpm) was subtracted from all values. The calculation was achieved with the assumption that the radioactivity recovered bears a linear relationship to the amount of protein-probe complex. The equation used is listed below: After calculating the concentration of complex of each reaction, I generated the corresponding scatchard plot according to the standard scatchard equation: (CPM of complex - background) [Bound complex] = X [ total La protein] (Max. CPM - background) Bound complex / total RNA - Ka (N -Bound complex ) Free proteins Total RNA Paul Cheung Page 182 Where N denotes the molecular ratio of interaction. The concentrations of the total RNA were given by the amount of probe added in each reaction. The concentrations of the bound complex were calculated by the above mentioned assumptions. The concentration of free proteins was calculated by subtracting the amount of bound complex from the amount of total protein. Data processing was programmed on a Microsoft excel work sheet and the calculated values were manually checked for accuracy. The values of [bound complex / (total RNA x free proteins)] were plotted on the Y axis and the values of [Bound complex / total RNA] were plotted on the X axis. From this plot, I calculated the apparent Kd of the interactions. The standard Scatchard plot for a one to one molecular interaction is a linear graph in the general form of Y = MX + C where M is the slope of the curve and corresponds to the (-Ka) value. Ka is the association constant which is the inverse of Kd. 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