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J. Biol. Chem., Vol. 278, Issue 50, 50301-50308, December 12, 2003

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Hepatitis C Virus Non-structural Proteins in the Probable Membranous Compartment Function in Viral Genome Replication^*

Yusuke Miyanari, Makoto Hijikata, Masashi Yamaji, Masahiro Hosaka, Hitoshi Takahashi, and Kunitada Shimotohno

From the Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan

The molecular mechanism of hepatitis C virus(HCV) RNA replication is still unknown. Recently, a cell culture system in which the HCV subgenomic replicon is efficiently replicated and maintained for a long period in Huh-7 cells has been established. Taking advantage of this replicon system, we detected the activity to synthesize the subgenomic RNA in the digitonin-permeabilized replicon cells. To elucidate how and where this viral RNA replicates in the cells, we monitored the activity for HCV RNA synthesis in the permeabilized replicon cells under several conditions. We obtained results suggesting that HCV replication complexes functioning to synthesize the replicon RNA are protected from access of nuclease and proteinase by possible cellular lipid membranes. We also found that a large part of the replicon RNA, including newly synthesized RNA, was present in such a membranous structure but a large part of each NS protein was not. A small part of each NS protein that was resistant to the proteinase action was shown to contribute sufficiently to the synthesis of HCV subgenomic RNA in the permeabilized replicon cells. These results suggested that a major subcellular site of HCV genome replication is probably compartmentalized by lipid membranes and that only a part of each NS protein forms the active replication complex in the replicon cells.

Received for publication, May 30, 2003 , and in revised form, August 20, 2003.

^* This work was supported by grants-in-aid for cancer research and for the second-term comprehensive 10-year strategy for cancer control from the Ministry of Health, Labor, and Welfare, through grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, grants-in-aid of research for the future from the Japanese Society for the Promotion of Science, and by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan. 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: 53 Kawahara-cho Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: 81-75-751-4046; Fax: 81-75-751-3998; E-mail: mhijikat{at}virus.kyoto-u.ac.jp.

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