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Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates

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Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The RdRp activity was strictly dependent on Mn2+. The nsp12 preferentially copied homopolymeric pyrimidine RNA templates in the absence of an added oligonucleotide primer. It was also able to initiate de novo RNA synthesis from the 3’-ends of both the plus- and minus-strand genome of SARS-CoV, using the 3’-terminal 36- and 37-nt RNA, respectively. The in vitro RdRp assay system established with a full-length nsp12 will be useful for understanding the mechanisms of coronavirus replication and for the development of anti-SARS-CoV agents.

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Abbreviations

SARS-CoV:

Severe acute respiratory syndrome coronavirus

RdRp:

RNA-dependent RNA polymerase

Nsp12:

Nonstructural protein 12

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Acknowledgments

This work was supported by the Seoul R&BD Program (Grant 10580), Technology Innovation Program (MKE 10035159) funded by the Ministry of Knowledge Economy (MKE, Korea), and in part by grants from the National Research Foundation of Korea (NRF-2011-0001236, 2011-0027650, and 2010-0029116).

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Authors and Affiliations

  1. Department of Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120-749, Korea

    Dae-Gyun Ahn, Jin-Kyu Choi & Jong-Won Oh

  2. Translational Research Center for Protein Function Control, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120-749, Korea

    Jong-Won Oh

  3. Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, 20892, USA

    Deborah R. Taylor

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  1. Dae-Gyun Ahn
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  2. Jin-Kyu Choi
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Correspondence to Jong-Won Oh.

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Ahn, DG., Choi, JK., Taylor, D.R. et al. Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates. Arch Virol 157, 2095–2104 (2012). https://doi.org/10.1007/s00705-012-1404-x

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