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.
Highlights
Hepatitis C virus (HCV),1 a member of the Flaviviridae family, is the major causative agent of non-A and non-B hepa
The Five NS5B Residues Are Critical for hepatitis C virus (HCV) Replication in Vivo —Previously, we identified five amino acid residues in NS5B of a JK-1 isolate that are critical for RNA-dependent RNA polymerase (RdRP) (Figs. 1 and 2)
For enhanced HCV replication efficiency, we utilized an improved HCV RNA replicon system using an M1LE HCV replicon harboring the S232Iadapted mutation, and Huh-7-KV-C cells, a Huh-7 cell line cured from replicating HCV replicon by interferon treatment [39]
Summary
Hepatitis C virus (HCV), a member of the Flaviviridae family, is the major causative agent of non-A and non-B hepa-. A cell culture system was developed based on subgenomic selectable replicons consisting of the HCV 5Ј-nontranslated region directing translation segment of the neomycin phosphotransferase gene, internal ribosomal entry site of the encephalomyocarditis virus, HCV NS proteins NS3 to NS5B, and HCV 3Ј-nontranslated region. After transfection these RNAs into Huh-7 cells and selection with G418, cell colonies can be generated that carry persistently replicating HCV replicons [24, 25]. JK5BBg/II For 5BAgeI Rev 5AAatII For JK5BBg/II Rev 5B SEF5 Eco81I For NR5B For NR5Bt Rev JK5B For JK5Bt Rev
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
