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

Abstract

f 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 SARSCoV 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 ␣-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an ␣/␤ domain and a long-␣-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical crosslinking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARSCoV 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.

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

“…It has been hypothesized that coronavirus RNA synthesis involves structurally and functionally separable RNA synthesising complexes [33]. The nsp7+nsp8 complex from three different coronaviruses have recently been shown to have primer-independent RNA polymerase activity [33,34] in contrast to the nsp12 RdRP which is primer dependent [35]. Currently the details regarding coronavirus RNA synthesis are very limited and do not explain how the polymerases distinguish between templates or prime synthesis.…”

Biogenesis and Dynamics of the Coronavirus Replicative Structures

“…Nsp8 has been shown to associate together with nsp7 into a hexadecameric complex, consisting of eight copies of each protein, thereby forming a channel that can harbor RNA and may serve as a processivity factor for nsp12 [23]. Nsp8 also interacts with nsp7 in a 1:2 ratio [24] and is able to synthesize much longer transcripts [24,25]. …”

RNA structure analysis of alphacoronavirus terminal genome regions

“…Also, there is evidence that a heteromultimeric complex formed by nsp7 and nsp8 interacts with (and serves as a processivity factor for) the RNA-dependent RNA polymerase (RdRp, nsp12) (Zhai et al, 2005). Additional interactions between individual subunits of the RTC have been suggested on the basis of two-hybrid screening data (Pan et al, 2008;von Brunn et al, 2007) and there is evidence that a substantial number of coronavirus nsps form homo-and/or heterooligomeric complexes (Anand et al, 2002(Anand et al, , 2003Bouvet et al, 2014;Chen et al, 2011;Ricagno et al, 2006;Su et al, 2006;Xiao et al, 2012;Zhai et al, 2005).…”