Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

Abstract

The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728 -737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993-999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat-and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.Hepatitis C virus (HCV), 1 a member of the Flaviviridae family, is the major causative agent of non-A and non-B hepatitis (1). Persistent infection with HCV is an important cause of chronic liver disease around the world. Infections with this hepatotropic flavivirus are associated with inflammatory liver injury, progressive fibrosis, and, in the most severely affected patients, cirrhosis and hepatocellular carcinoma (2, 3). The HCV genome is a single-stranded RNA molecule 9.6 kb in length with positive-sense polarity (1, 5). The genome consists of a 5Ј-untranslated region, an open reading frame, and a 3Ј-untranslated region (1, 4, 5). The order of the gene products of the single open reading frame is NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein precursor can be processed by the host and virally encoded proteases to generate mature structural and nonstructural proteins required for virus replication and assembly (6 -8).Studies on HCV replication focusing on the inhibition of HCV replication may have therapeutic significance for chronic hepatitis and also could reduce the incidence of or even prevent hepatocellular carcinoma. NS5B is an RNA-dependent RNA polymerase ...

Regulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

“…This would help to explain how mutations in the ⌬1 loop, such as m26-30, and even the PV mutants were either incapable of or showed only a linear increase in de novo initiation activity with increasing enzyme concentrations. The residues in HCV NS5B identified to be critical for de novo initiation activity in this study, such as E18, L26-L30, and P495/ V499, have all been documented to be needed for HCV replication in cells, underlying their biological significance (8,10,32). The interdomain interactions may also be needed for productive RNA binding when longer RNA templates are used.…”

Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

“…We found that another mutant replicon, MA/K211A, reduced the number of G418-resistant colonies compared with the wild type and the other mutants. Because K211A of NS5Bt is close to the pocket of catalytic activity and did not affect binding to nucleolin, K211 may contribute to the structural integrity of the pocket or the heat-stable property of RdRp as reported previously (36).…”

“…1A). The wild-type replicon was represented by MA, in which S232 of NS5A was altered to I, because this mutant replicon can efficiently replicate in Huh7 cells (36,56). In the replicon MA/cm211, each of the amino acids at positions 208 to 214 of NS5B was changed to alanine, whereas in the replicons MA/ W208A, K209A, S210A, K211A, K212A, C213A, and P214A, each individual amino acid residue was changed to alanine.…”

“…According to the crystal models of NS5B, the former stretch is in the palm and the latter stretch is in the bottom of the thumb domain. We focused on identifying residues in aa 208 to 214 that are essential for nucleolin binding and HCV replication, as the CM mutant of aa 500 to 506 was defective in RdRp activity in vitro and HCV replication in vivo (36,48,49). We found that the W208 residue was critical for both nucleolin binding and HCV replication.…”

“…HCV NS5B has been reported to interact with NS3, NS4A, NS4B, NS5A, and NS5B itself (9,48,57,65). Using an HCV subgenomic replicon, we previously reported the critical role of the interaction between NS5A and NS5B and the oligomerization of NS5B itself in HCV replication (36,56). NS3 and NS4B have been shown to be positive and negative regulators, respectively, of NS5B in the replication complex (46).…”