| Summary: | 博士 === 國立臺灣大學 === 微生物學研究所 === 88 === Hepatitis C virus (HCV) is the major etiology agent of posttransfusion and sporadic, community acquired non-A, non-B hepatitis. It plays a major role in the development of chronic hepatitis and hepatocellular carcinoma. The viral particle possesses a positive-sense RNA genome that contains a large open reading frame encoding a polyprotein about 3000 amino acid residues. Generation of mature proteins from the polyprotein precursor is mediated by cellular and viral proteases.
Amino acid sequence analysis revealed highly conserved function motifs within the NS5 and NS3 proteins of HCV. To elucidate biological functions of NS5 and NS3 proteins, the cDNA fragments of NS5B, NS5A and NS3 were generated by reverse transcriptase-nested polymerase chain reaction from sera of HCV patients and expressed in both prokaryotic and eukaryotic systems.
HCV NS5B protein possesses the conserved -GDD- motif and the viral RNA-dependent RNA polymerase. It is generally believed that the NS5B protein binds to the 3'' terminus of the viral genome to initiate viral replication. In this study, electrophoretic mobility shift assay was performed to analyze the RNA binding activity of the NS5B protein. The results demonstrated that HCV NS5B protein was capable of interacting with a 3'' viral genomic RNA with or without the 98-nucleotide at sequences extreme 3'' terminus. Both of the 3'' viral RNAs contain 295 nucleotides of the 3''coding region of the HCV polyprotein. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA, but not by the viral 5'' noncoding region and very poorly by the 3*''conserved 98-nucleotide tail. A 3'' coding region with conserved stem-loop structures rather than the 3'' noncoding region of the HCV genome is critical for the specific binding of NS5B. In addition, the binding activity was not observed with the HCV NS5A protein. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1 from amino acid residues 83 to 194 and RBD2 from 196 to 298. Interestingly, the conserved motifs of RNA dependent RNA polymerase for putative RNA-binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2.
HCV NS3 protein possesses NTPase and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase were not well-characterized. In this study, NS3 proteins with point mutations were analyzed for their activities on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV crosslinking experiments demonstrated that the lysine residue in the 1230-AX4GKS-1237 motif was directly involved in ATP binding, whereas an NS3 mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutations in the arginine-rich motif and was also affected by a substitution of the lysine residue in the AX4GKS motif. Previously, Arg-1490 in the arginine-rich motif of the HCV NS3 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the g-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg-1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.
To further elucidate possible interactions among the nonstructural proteins of HCV to form a replicative complex, co-immunoprecipitation assay was performed. The results indicated that the HCV NS3, NS5A and NS5B proteins interacted with each other, either directly or indirectly. Although the roles of cellular factors involved in the complex formation and HCV replication remained to be analyzed, the present studies have provided the information on the molecular mechanisms of the nonstructural proteins involved in the replication of HCV.
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