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 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.
Department of Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea.
Dae-Gyun
DG
Ahn
Jin-Kyu
JK
Choi
Deborah R
DR
Taylor
Jong-Won
JW
Oh
D020413:3' Untranslated Regions; D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D016679:Genome, Viral; D008345:Manganese / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics
D020413:3' Untranslated Regions; D002413:Cations, Divalent; D003067:Coenzymes; D017931:DNA Primers; D012367:RNA, Viral; D011994:Recombinant Proteins; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase