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 ...

The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses".