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

Arch Virol. 2012 Nov;157(11):2095-104. doi: 10.1007/s00705-012-1404-x. Epub 2012 Jul 13.

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 Mn(2+). 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Cations, Divalent / metabolism
  • Coenzymes / metabolism
  • DNA Primers / genetics
  • Genome, Viral
  • Manganese / metabolism
  • RNA, Viral / metabolism*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • SARS Virus / enzymology*
  • SARS Virus / genetics

Substances

  • 3' Untranslated Regions
  • Cations, Divalent
  • Coenzymes
  • DNA Primers
  • RNA, Viral
  • Recombinant Proteins
  • Manganese
  • RNA-Dependent RNA Polymerase