Ma Yuanyuan, Shimakami Tetsuro, Luo Hong, Hayashi Naoyuki, Murakami Seishi
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
J Biol Chem. 2004 Jun 11;279(24):25474-82. doi: 10.1074/jbc.M401067200. Epub 2004 Mar 22.
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.
丙型肝炎病毒（HCV）NS5B是一种依赖RNA的RNA聚合酶（RdRP），是HCV RNA复制的核心催化酶。我们之前在JK-1分离株中鉴定出NS5B的五个新残基，它们在体外对RdRP活性不可或缺（Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728 - 737）。我们使用源自M1LE分离株的HCV复制子系统研究了这些残基在HCV RNA复制中的作用（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）。结果发现，M1LE中NS5B的这五个残基对HCV在体内的复制至关重要，并且对于体外RdRP活性以及纯化的细菌重组蛋白而言也是不可或缺的。我们还发现了JK-1和M1LE的嵌合复制子，其中仅源自JK-1的NS5B序列在Huh-7细胞中无法复制。通过HCV RNA复制子中NS5B M1LE和/或JK-1分离株的几种嵌合和取代形式，确定了导致该现象的残基。两个残基，即第220位和第288位氨基酸，至关重要，另外两个残基，即第213位和第231位氨基酸，对HCV的高效复制很重要。携带M1LE所有四个残基的突变型JK-1 NS5B在嵌合复制子中具有复制能力，并且与原始的M1LE复制子效率相同。通过比较NS5B各种嵌合和突变形式在体内的复制能力和体外的RdRP活性，发现HCV复制能力与RdRP活性密切相关。然而，在体外表现出较弱RdRP活性的对热和稀释敏感的NS5B被发现无复制能力，这表明HCV复制需要高于某个临界阈值的RdRP活性。
