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J. Biol. Chem., Vol. 279, Issue 24, 25474-25482, June 11, 2004

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

Yuanyuan Ma, Tetsuro Shimakami, Hong Luo, Naoyuki Hayashi, and Seishi Murakami¶

From the Department of Molecular Oncology, Cancer Research Institute and the Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa University, 13-1 Takara-Machi, Kanazawa, Ishikawa 920-0934, Japan

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.

Received for publication, January 30, 2004 , and in revised form, March 2, 2004.

^* This work was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety, by research grants-in-aid for scientific research (B) and development, and by a grant-in-aid for scientific research on priority areas (C) in oncogenesis from the Ministry of Education, Sports, Culture, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-76-265-2731; Fax: 81-76-234-4501; E-mail: semuraka{at}kenroku.kanazawa-u.ac.jp.

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