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

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.