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The juxtamembrane sequence of the Hepatitis C virus polymerase can affect RNA synthesis and inhibition by allosteric polymerase inhibitors

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Abstract

The Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), nonstructural protein 5B (NS5B), is anchored in the membrane through a C-terminal helix. A sequence of ca. 12 residues that connects the catalytically competent portion of the RdRp and the C-terminal helix, the juxtamembrane sequence (JMS), has a poorly defined role in RdRp function in a large part since it is translated from a cis-acting RNA element (CRE) that is essential for HCV replication. Using a HCV replicon that transposed a second copy of CRE to the 3′ UTR of the HCV replicon, we demonstrate that amino acid substitutions in the JMS were detrimental for HCV replicon replication. Substitutions in the JMS also resulted in a defect in de novo-initiated RNAs synthesis in vitro and in a cell-based reporter assay. A nonnucleoside inhibitor of the NS5B that binds to the catalytic pocket was less potent in inhibiting NS5B in the presence of JMS mutations. The JMS mutants exhibit reduced stability in thermodenaturation assays, suggesting that the JMS helps confer a more stable conformation to NS5B that could impact RNA synthesis.

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Abbreviations

HCV:

Hepatitis C virus

NS5B:

Nonstructural protein 5B

RdRp:

RNA-dependent RNA polymerase

JMS:

Juxtamembrane sequence

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Acknowledgments

We thank R. Bartenschlager for the HCV PI-ins3.1-3 plasmid. We thank Laura B. Kao for editing of the manuscript.

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Author notes

  1. C. T. Ranjith-Kumar

    Present address: Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, Gurgaon, 122016, Haryana, India

Authors and Affiliations

  1. Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, 47405, USA

    Y. Wen, X. Lin, B. Fan & C. C. Kao

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  1. Y. Wen
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  2. X. Lin
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Correspondence to C. C. Kao.

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Edited by Hartmut Hengel.

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Wen, Y., Lin, X., Fan, B. et al. The juxtamembrane sequence of the Hepatitis C virus polymerase can affect RNA synthesis and inhibition by allosteric polymerase inhibitors. Virus Genes 51, 1–11 (2015). https://doi.org/10.1007/s11262-015-1199-4

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  • DOI: https://doi.org/10.1007/s11262-015-1199-4

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