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Journal of Virology, March 2004, p. 2738-2748, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2738-2748.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effect of Interaction between Hepatitis C Virus NS5A and NS5B on Hepatitis C Virus RNA Replication with the Hepatitis C Virus Replicon

Tetsuro Shimakami,1,2 Makoto Hijikata,3 Hong Luo,1,4 Yuan Yuan Ma,1 Shuichi Kaneko,2 Kunitada Shimotohno,3 and Seishi Murakami1*

Department of Molecular Oncology, Cancer Research Institute, Kanazawa University,1 Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Takara-Machi, Kanazawa, Ishikawa 920-0934,2 Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-Ku, Kyoto 606-8507, Japan,3 The Institute of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China4

Received 3 September 2003/ Accepted 10 November 2003

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.

* Corresponding author. Mailing address: Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-Machi, Kanazawa, 920-0934 Ishikawa, Japan. Phone: 81-76-265-2731. Fax: 81-76-234-4501. E-mail: semuraka{at}kenroku.kanazawa-u.ac.jp.

Journal of Virology, March 2004, p. 2738-2748, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2738-2748.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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