3ub0

Publication Abstract from PubMed

Non-structural proteins 7 and 8 of SARS coronavirus have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His(6)-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. Here we present the 2.6-A crystal structure of the Feline Coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the alpha-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an alpha/beta domain and a long-alpha-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size-exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribomucleotides of up to 6 nucleotides in length when carrying an N-terminal hexahistidine tag. Remarkably, the same protein harbouring the sequence GPLG instead of the His(6)-tag at its N-terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template-length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of Human Coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain-length of six. These results indicate that in case of FCoV as well as HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a non-canonical RNA polymerase capable of synthesizing RNA of up to template-length.

Non-structural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer that Exhibits Primer-independent RNA Polymerase Activity.,Xiao Y, Ma Q, Restle T, Shang W, Svergun DI, Ponnusamy R, Sczakiel G, Hilgenfeld R J Virol. 2012 Feb 8. PMID:22318142^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.