title: One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.
abstract: 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 positive-stranded 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 SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing 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.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, 13288 Marseille, France;
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Subissi
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2300RC, Leiden, The Netherlands; and.
authors.forename: Clara C
authors.initials: CC
authors.lastname: Posthuma
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, 13288 Marseille, France;
authors.forename: Axelle
authors.initials: A
authors.lastname: Collet
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2300RC, Leiden, The Netherlands; and.
authors.forename: Jessika C
authors.initials: JC
authors.lastname: Zevenhoven-Dobbe
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2300RC, Leiden, The Netherlands; and Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119899, Russia.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Gorbalenya
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, 13288 Marseille, France;
authors.forename: Etienne
authors.initials: E
authors.lastname: Decroly
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2300RC, Leiden, The Netherlands; and.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, 13288 Marseille, France; isabelle.imbert@afmb.univ-mrs.fr bruno.canard@afmb.univ-mrs.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, Aix-Marseille Université, 13288 Marseille, France; isabelle.imbert@afmb.univ-mrs.fr bruno.canard@afmb.univ-mrs.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Imbert
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005095:Exoribonucleases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D015203:Reproducibility of Results; D059386:Reverse Genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D045169:Severe Acute Respiratory Syndrome / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D046912:Multiprotein Complexes; D050505:Mutant Proteins; C034477:RNA primers; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D005095:Exoribonucleases
keywords: processivity factor; replicative complex reconstitution