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Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain

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Abstract

Hepatitis C virus (HCV) JFH1 efficiently replicates and produces infectious virus particles in cultured cells. We compared polymerase activity between JFH1 and 1b strains in vitro. The RNA polymerase activity of 1b was 6.4% of that of JFH1. In order to study the mechanism and identify domains responsible for the high polymerase activity of JFH1, we converted the amino acids of 1b RdRp to those of JFH1, and compared their Km, Vmax and template binding activity. Four amino acid mutations in the thumb domain of 1b RdRp, S377R, A450S, E455N and Y561F increased 1b polymerase activity, and their activity was 23.1, 45.8, 28.9, and 36.1% of JFH1, respectively. Vmax and RNA binding activity of JFH1, 1bwt and 1bA450S was JFH1 > 1bA450S > 1b, which indicated both high processivity and slightly higher template binding activity contributed to the high polymerase activity of JFH1.

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Abbreviations

HCV:

Hepatitis C virus

RdRp:

RNA-dependent RNA polymerase

IRES:

Internal ribosome entry site

UTR:

Untranslated region

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Acknowledgments

This work was supported by grants from Chinese Academy of Sciences (O514P51131 and O812P1A131) and Chinese National Key Project (2008ZX10002-014).

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Authors and Affiliations

  1. Unit of Viral Genome Regulation, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 225 South Chongqing Road, 200025, Shanghai, People’s Republic of China

    Leiyun Weng, Jingling Zhou & Tetsuya Toyoda

  2. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, 200031, Shanghai, China

    Jiamu Du & Jianping Ding

  3. Department of Virology II, National Institute of Health, 1-23-1 Toyama, Shinjuku, Tokyo, 132-8640, Japan

    Takaji Wakita

  4. Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Biology, 3-18-22 Honkomagome, Bunkyo-Ku, Tokyo, 113-8613, Japan

    Michinori Kohara

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  1. Leiyun Weng
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  2. Jiamu Du
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  3. Jingling Zhou
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  7. Tetsuya Toyoda
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Correspondence to Tetsuya Toyoda.

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Weng, L., Du, J., Zhou, J. et al. Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain. Arch Virol 154, 765–773 (2009). https://doi.org/10.1007/s00705-009-0366-0

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  • DOI: https://doi.org/10.1007/s00705-009-0366-0

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