In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positivestranded RNA genome of coronaviruses encodes a replication/ transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARSCoV nsp7 and nsp8 activate and confer processivity to the RNAsynthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.replicative complex reconstitution | processivity factor A virus-encoded RNA-dependent RNA polymerase (RdRp) is the central enzyme in the replicative cycle of RNA viruses (1). In the case of mammalian positive-strand RNA (+RNA) viruses, the enzyme is generated by the translation of the incoming viral genome, which yields a polyprotein precursor from which the RdRp-containing subunit is released by proteolytic cleavage. Subsequently, the RdRp is integrated into a membraneassociated viral enzyme complex that drives the production of negative-strand RNA (−RNA), new genome molecules, and in many virus groups also subgenomic (sg) messenger RNAs (mRNAs) (2-4). Compared with the replication of either viral or cellular DNA sequences, RNA virus genomes are copied with relatively low fidelity, because the products of replication are believed to be neither proofread nor edited (5). This property is a major factor in the evolution, adaptation, and epidemiology of RNA viruses.Among +RNA viruses, coronaviruses (CoVs) (order Nidovirales) stand out for having the largest single-stranded RNA genomes known to date (6,7). Research into the molecular and structural biology of CoVs was boosted significantly by the emergence, in 2003, of a previously undiscovered CoV that caused the severe acute respiratory syndrome (SARS) epidemic ...

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.