title: Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.
abstract: The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.
authors: From the Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France, CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors: Mickaël
authors: M
authors: Bouvet
authors: Cancer Research Center of Marseille (CRCM), CNRS UMR7258, INSERM U1068, Institut Paoli-Calmettes, Aix-Marseille Université, F-13009 Marseille, France.
authors: Adrien
authors: A
authors: Lugari
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, P. O. Box 9600, 2300RC Leiden, The Netherlands.
authors: Clara C
authors: CC
authors: Posthuma
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, P. O. Box 9600, 2300RC Leiden, The Netherlands.
authors: Jessika C
authors: JC
authors: Zevenhoven
authors: Cancer Research Center of Marseille (CRCM), CNRS UMR7258, INSERM U1068, Institut Paoli-Calmettes, Aix-Marseille Université, F-13009 Marseille, France.
authors: Stéphanie
authors: S
authors: Bernard
authors: Cancer Research Center of Marseille (CRCM), CNRS UMR7258, INSERM U1068, Institut Paoli-Calmettes, Aix-Marseille Université, F-13009 Marseille, France.
authors: Stéphane
authors: S
authors: Betzi
authors: From the Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France, CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors: Isabelle
authors: I
authors: Imbert
authors: From the Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France, CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors: Bruno
authors: B
authors: Canard
authors: From the Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France, CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors: Jean-Claude
authors: JC
authors: Guillemot
authors: CIRI, INSERM U1111, CNRS UMR5308, Université Lyon 1, ENS de Lyon, 69007 Lyon, France, and.
authors: Patrick
authors: P
authors: Lécine
authors: Institute of Virology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.
authors: Susanne
authors: S
authors: Pfefferle
authors: Institute of Virology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.
authors: Christian
authors: C
authors: Drosten
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, P. O. Box 9600, 2300RC Leiden, The Netherlands.
authors: Eric J
authors: EJ
authors: Snijder
authors: From the Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France, CNRS, AFMB UMR 7257, 13288 Marseille, France, etienne.decroly@afmb.univ-mrs.fr.
authors: Etienne
authors: E
authors: Decroly
authors: Cancer Research Center of Marseille (CRCM), CNRS UMR7258, INSERM U1068, Institut Paoli-Calmettes, Aix-Marseille Université, F-13009 Marseille, France, xavier.morelli@inserm.fr.
authors: Xavier
authors: X
authors: Morelli
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D018352:Coronavirus Infections / Q000201:enzymology* / Q000473:pathology; D018360:Crystallography, X-Ray; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D060066:Protein Interaction Maps / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; C558878:Nsp10 protein, SARS virus; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C558879:Nsp16 protein, SARS virus; C023894:methylcobalamin-coenzyme M methyltransferase; C525596:nsp14 protein, SARS coronavirus; D005095:Exoribonucleases
keywords: Genetics; Protein-Protein Interaction; RNA Methyltransferase; RNA Virus; Viral Replication; Viral Transcription