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

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.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…In addition to the RdRp domain, nsp12 also contains an N-terminal domain that is essential for RdRp activity (122) and probably interacts with nsp5, nsp8, and nsp9 (123). In vitro, full-length nsp12 drives RNA synthesis in a primer-dependent manner on both homo- and heteropolymeric RNA templates (124, 125). However, the in vitro nsp12 RdRp activity is weak and nonprocessive, in contrast with the efficient replication of the RNA genome in vivo.…”

Atlas of coronavirus replicase structure

“…Cheng and coworkers suggest that the N-terminal domain is required for polymerase activity possibly via involvement in template-primer binding (Cheng et al, 2005). Snijder and coworkers were able to confirm that the full-length nsp12 has robust, primer-dependent RNA polymerase activity (te Velthuis et al, 2010), a finding generally confirmed by the later study of Ahn et al (2012). There has been some success in the use of inhibitors of viral polymerases as therapeutics.…”

SARS-CoV ORF1b-encoded nonstructural proteins 12–16: Replicative enzymes as antiviral targets

“…SARS-CoV nsp12 fused to a GST-tag exhibits weak polymerase activity in vitro using poly (rA)/oligo dT 12-18 as a template (Cheng et al, 2005). Neither metal requirements nor the ability to initiate RNA synthesis with or without an RNA primer are clearly established, as reported by several authors which used full-length histidine-tagged nsp12 (Ahn et al, 2012;Cheng et al, 2005;te Velthuis et al, 2010). In any case, the in vitro nsp12 polymerase activity was weak, contrasting with the replication of $30 kb-long coronavirus RNA genomes.…”