HCV functional genomics: Protein interactions with NS3 and their role in viral replication Khu Yee Ling B. Sc. (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 I Acknowledgements I am indebted to my supervisor, Dr Goh Phuay Yee, for her patience and guidance. Thanks are also due to my committee members, Drs Alan Porter and Thomas Dick for their invaluable advices. I am also grateful to Dr Goh Phuay Yee for the dimerization mutants (Y267S, M288T and T266A) and Dr Tan Yee Joo for useful discussion and help with using the FPLC machine. The presence of wonderful lab members in the CAVR group, both past and present, has made my stay in the institute a memorable experience. I thank them for their friendship and gossip sessions, which were highly useful for de-stressing. Besides the excellent sequencing services provided by Dr Alice Tay, our prophet and guru of all things big and small, I would also like to thank her for all the stimulating conversations we have shared. Closest to my heart, I would like to thank my parents, especially mum, who encourages me, believes in me and been my greatest fan, always. My husband, one of the most important men in my life, thanks for being there whenever I needed you and Yong Teng, the other man in my life, who brought out the patience in me I never knew I have. I Table of Contents ACKNOWLEDGEMENTS I TABLE OF CONTENTS II LIST OF FIGURES IV LIST OF TABLES VII LIST OF PUBLICATIONS VIII LIST OF ABBREVIATIONS IX SUMMARY XI 1. INTRODUCTION 1.1 Medical Importance of HCV 1.2 Molecular biology of HCV 1.2.1 Structural Proteins 1.2.2 Non-structural Proteins 1.3 HCV protein-protein interaction 1.4 Aims and Objectives 11 2. MATERIALS AND METHODS 12 2.1 Construction of Plasmids 12 2.2 Yeast two-hybrid screens 2.2.1 NS3 NS3 interaction 2.2.2 NS3 Host interaction 12 12 13 2.3 Generation of mutations in NS3 helicase 14 2.4 NS3 helicase and helicase mutants expression, purification and analytical gel filtration 16 2.5 Helicase activity assay 17 2.6 In vitro binding assay 18 2.7 FL-NS3, LMP7 expression and purification 18 II 2.8 In vitro protease activity assay 20 2.9 Proteasome activity assay 20 2.10 Immunoprecipitation(IP) 22 2.11 Western blot analysis 22 2.12 Tissue culture 23 3. RESULT 27 3.1 Characterization of NS3-NS3 interaction 27 3.1.1 Delineating the region of self-interaction in NS3 27 3.1.2 Expression and purification of recombinant NS3 helicase for gel filtration analysis 32 3.1.3 Analytical gel filtration of dimerization mutants 37 3.1.4 Correlation between dimer formation and helicase activity 41 3.2 Characterization of NS3-LMP7 interaction 3.2.1 Screening for NS3 host interacting partner 3.2.2 Delineating the region of interaction between NS3 and LMP7. 3.2.3 Expression and purification of recombinant NS3 and LMP7 for in vitro assays 3.2.4 Effect of LMP7 on NS3 protease activity 3.2.5 Effect of NS3 on proteasome activity 45 45 47 4. DISCUSSION 67 4.1 NS3 NS3 interaction 67 4.2 NS3 LMP7 interaction 70 50 53 55 5. CONCLUSION 76 6. REFERENCES 77 III List of Figures FIGURE 1-2. HCV GENOME AND ENCODED VIRAL PROTEINS . FIGURE 2-1. SCHEME SHOWING THE GENERATION OF RANDOM MUTANTS THAT DISRUPT HELICASE INTERACTION. . 15 FIGURE 3-1. A MINIMAL DOMAIN OF NS3 REQUIRED FOR INTERACTION DEFINED BY YEAST-TWO HYBRID ASSAY . 28 FIGURE 3-2. IMMUNOPRECIPITATION BETWEEN FLAG-TAGGED NS3 AND MYC-TAGGED NS3. . 29 FIGURE 3-3. THE NS3 HELICASE INTERACTS IN AN N-TO-N ORIENTATION 30 FIGURE 3-4. MINIMAL REGION FOUND TO INTERACT WITH HELICASE DOMAIN AND ITSELF. 31 FIGURE 3-5. RECOMBIANT NS3 HELICASE EXPRESSION. . 33 FIGURE 3-6. PURIFICATION OF NS3 HELICASE BY FPLC. 33 FIGURE 3-7. GEL FILTRATION OF WILD-TYPE HELICASE. . 34 FIGURE 3-8. GEL FILTRATION OF HELICASE MUTANTS . 36 FIGURE 3-9. POSITIONS OF SOME OF THE MUTANTS THAT DISRUPTED INTERACTION BETWEEN TWO MINIMAL REGIONS . 38 FIGURE 3-10. GEL FILTRATION OF DIMERIZATION MUTANTS. . 40 FIGURE 3-11. HELICASE ASSAYS OF WILD-TYPE HELICASE, MUTANTS Y267 AND AAA. 41 FIGURE 3-12. DIMERIZATION MUTANTS SHOWS REDUCTION IN HELICASE ACTIVITY. 42 FIGURE 3-13. INHIBITION OF HELICASE ACTIVITIES BY THE ADDITIONS OF MUTANT PROTEINS. 44 FIGURE 3-14. RECOMBINANT GST-NS3 AND GST EXPRESSION. . 46 FIGURE 3-15. NS3-LMP7 INTERACTION SHOWN BY IN VITRO BINDING ASSAY 46 IV FIGURE 3-16. LMP7 INTERACTS WITH THE PROTEASE DOMAIN OF NS3. 48 FIGURE 3-17. NS3 INTERACTS WITH THE PROSEQUENCE OF LMP7. . 49 FIGURE 3-18. PURIFICATION OF RECOMBINANT GST-NS3 . 51 FIGURE 3-19. PURIFICATION OF RECOMBINANT LMP7 . 52 FIGURE 3-20. IN VITRO BINDING OF PURIFIED LMP7 TO GST NS3 AND PROTEASE ACTIVITY OF PURIFIED NS3 54 FIGURE 3-21. NS3 BINDS TO THE IMMUNOPROTEASOME COMPLEX. . 56 FIGURE 3-22. CHYMOTRYPSIN-LIKE ACTIVITY OPTIMIZATION IN HELA CELLS USING SUBSTRATE LLVY-AMC . 59 FIGURE 3-23. NS3 DID NOT AFFECT IMMUNOPROTEASOME CHYMOTRYPSIN-LIKE ACTIVITY IN HELA CELLS 59 FIGURE 3-24. TRYPSIN-LIKE ACTIVITY OPTIMIZATION IN HELA CELLS USING SUBSTRATE LRR-AMC 60 FIGURE 3-25. NS3 REDUCES IMMUNOPROTEASOME TRYPSIN-LIKE ACTIVITY IN HELA CELLS. 60 FIGURE 3-26. POST ACIDIC ACTIVITY OPTIMIZATION IN HELA CELLS USING SUBSTRATE LLE-AMC . . 61 FIGURE 3-27. NS3 DID NOT AFFECT IMMUNOPROTEASOME POST ACIDIC ACTIVITY IN HELA CELLS. 61 FIGURE 3-28. CHYMOTRYPSIN-LIKE ACTIVITY OPTIMIZATION IN HUH-7 CELLS USING SUBSTRATE LLVY-AMC . 62 FIGURE 3-29. NS3 DID NOT AFFECT IMMUNOPROTEASOME CHYMOTRYPSIN-LIKE ACTIVITY IN HUH-7 CELLS. 62 FIGURE 3-30. TRYPSIN-LIKE ACTIVITY OPTIMIZATION IN HUH-7 CELLS USING SUBSTRATE LRR-AMC 63 V FIGURE 3-31. NS3 DID NOT AFFECT IMMUNOPROTEASOME TRYPSIN-LIKE ACTIVITY IN HUH-7 CELLS. 63 FIGURE 3-32. POST ACIDIC ACTIVITY OPTIMIZATION IN HUH-7 CELLS USING SUBSTRATE LLE-AMC . . 64 FIGURE 3-33. NS3 DID NOT AFFECT IMMUNOPROTEASOME POST ACIDIC ACTIVITY IN HUH-7 CELLS. 64 FIGURE 3-34. EXPRESSION OF NS3-NS5B VIRAL PROTEINS REDUCES THE LMP7IMMUNOPROPTEASOME ACTIVITY. . 66 VI List of Tables TABLE 2-1. VECTORS USED IN THIS STUDY 24 TABLE 2-2. PLASMIDS USED IN STUDYING NS3-NS3 INTERACTION 25 TABLE 2-3. PLASMIDS USED IN STUDYING NS3 LMP7 INTERACTION 26 VII List of Publications Lim, S. P., Y. L. Khu, W. Hong, A. Tay, A. E. Ting, S. G. Lim, and Y. H. Tan. 2001. Identification and molecular characterization of the complete genome of a Singapore isolate of hepatitis C virus: sequence comparison with other strains and phylogenetic analysis. Virus Genes. 23:89-95. Khu, Y. L., E. Koh, S. P. Lim, Y. H. Tan, S. Brenner, S. G. Lim, W. Hong, and P. Y. Goh. 2001. Mutations that affect dimer formation and helicase activity of the hepatitis C virus helicase. J. Virol. 75:205-214. Khu, Y. L., Y. J. Tan, S. G. Lim, W. Hong, and P. Y. Goh. 2004. Hepatitis C virus nonstructural protein NS3 interacts with LMP7, a component of immunoproteasome, and affects its proteasome activity. Biochem. J. 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Hepatol. 15:382-386. 92 [...]... polymerase 68 5B 3’UTR (U/UC) The HCV ORF is flanked by 5’ and 3’ UTR The structural proteins (shaded) are located in the N terminus with the nonstructural proteins residing in the remainder of the genome Figure 1-1 HCV Genome and encoded viral proteins RNA Binding nucleocapsid Putative function MW kDa 5’UTR IRES Structural Proteins 1.2.2 Non-structural Proteins The NS proteins of HCV encode enzymes or regulatory... for studying HCV infection (Mercer et al., 2001) The generation of HCV- replicon systems, where the expression of the HCV NS proteins drives the self -replication of subgenomic HCV RNAs (Lohamnn et al., 1999; Blight et al., 2000), will also undoubtedly help accelerate our understanding of HCV replication and propagation 8 1.3 HCV protein- protein interaction Viral proteins are known to interact with one... (HCV) is one of the major causes of liver diseases worldwide The non-structural protein 3 (NS3) of HCV, which is both a protease as well as a helicase, plays important roles in the processing of the viral polyprotein and the replication of viral RNA This thesis attempts to answer several questions with regards to viral and host interacting proteins of NS3, which may eventually assist in the understanding... important for minus strand synthesis (Friebe et al., 2002) 3 1.2.1 Structural Proteins The HCV polyprotein is cleaved co- and post-translationally at several sites by both viral encoded and host cellular proteases into mature viral proteins About 10 distinct viral proteins have been identified which include at least three structural, Core, E1 and E2 (and p7), six non-structural (NS) proteins, NS2, NS3, NS4A... understanding these mechanisms of evasion from host immune surveillance, and will be useful for the development of anti -HCV therapies This thesis aims to identify both viral and host interacting partners to NS3, a pivotal player in HCV replication, in the hope of providing new insights into understanding function and effect of these interactions Yeast-two hybrid screens were set up to identify HCV proteins... host proteins that interact with NS3, using a spleen cDNA library The functions of these interactions were investigated and discussed in two parts The first section describes the characterization of NS3 self-interaction, while the second part covers the interaction between NS3 and host proteins, in particular, between NS3 and LMP7 11 2 Materials and Methods 2.1 Construction of Plasmids The NS3 coding... interaction between NS3 and a cellular protein, LMP7 NS3 was found to bind strongly with itself and the minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region Random mutations in this minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using yeast two-hybrid... on protein synthesis machineries in the host for viral protein translation, and other cellular components for their replication HCV proteins were reported to associate with several host proteins E2 binds the putative cellular receptors, CD81 and the low-density lipoprotein receptor (LDLR) (Pileri et al., 1998; Agnello et al., 1999; Wunschmann et al., 2000), which may act as receptors for HCV entry into... signal peptidases The core protein is strongly basic in nature and interacts with viral RNA to form the nucleocapsid (Hussy et al., 1996a) This highly conserved protein is very immunogenic and is used frequently for antibody detection in patient sera (Hosein et al., 1991) Glycoproteins E1 and E2 are the viral envelope proteins (Hussy, 1996b) These two proteins form heterodimers and dimerization is suggested... motifs of NS3 are highly conserved in the Flaviviridae family and among different HCV genotypes (Miller and Purcell, 1990) Productive replication was also abrogated in vivo when NS3 is mutated at the active sites, making this protein an attractive target for drug discovery (Kolykhalov et al., 2000) Besides the obvious role of NS3 in viral replication, this protein may also play a role in regulating cell . I HCV functional genomics: Protein interactions with NS3 and their role in viral replication Khu Yee Ling B. Sc. (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE. with regards to viral and host interacting proteins of NS3, which may eventually assist in the understanding of the mechanism of HCV replication and pathogenesis. Yeast two- hybrid assays and. XI 1. INTRODUCTION 1 1.1 Medical Importance of HCV 1 1.2 Molecular biology of HCV 3 1.2.1 Structural Proteins 4 1.2.2 Non-structural Proteins 6 1.3 HCV protein- protein interaction 9 1.4 Aims and