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Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) 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. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/β domain and a long-α-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 oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His(6) tag. Remarkably, the same protein harboring 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 of 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 noncanonical RNA polymerase capable of synthesizing RNA of up to template length.

Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity