PMID:22039154

Citation	te Velthuis, AJ, van den Worm, SH and Snijder, EJ (2012) The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic Acids Res. 40:1737-47
Abstract	Uniquely among RNA viruses, replication of the ~30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.
Links	PubMed PMC3287201 Online version:10.1093/nar/gkr893
Keywords	Amino Acid Motifs; Amino Acid Sequence; Cations, Divalent/chemistry; DNA-Directed RNA Polymerases/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Multimerization; Protons; RNA/metabolism; RNA Replicase/chemistry; RNA Replicase/genetics; RNA Replicase/metabolism; RNA, Double-Stranded/metabolism; SARS Virus/enzymology; Sequence Homology, Amino Acid; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CVHSA:R1AB	enables	GO:0003725: double-stranded RNA binding	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
CVHSA:R1AB	involved_in	GO:0039690: positive stranded viral RNA replication	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete

References

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