title: Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.
abstract: Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.
authors.affiliation: From the CNRS and Aix-Marseille Universités, IMR Laboratory (UPR 3243), Institut de Microbiologie de la Méditérannée, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Adrien
authors.initials: A
authors.lastname: Lugari
authors.affiliation: From the CNRS and Aix-Marseille Universités, IMR Laboratory (UPR 3243), Institut de Microbiologie de la Méditérannée, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors.forename: Stephane
authors.initials: S
authors.lastname: Betzi
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Decroly
authors.affiliation: INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Bonnaud
authors.affiliation: INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Hermant
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Guillemot
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Debarnot
authors.affiliation: INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Borg
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Mickaël
authors.initials: M
authors.lastname: Bouvet
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: From the CNRS and Aix-Marseille Universités, IMR Laboratory (UPR 3243), Institut de Microbiologie de la Méditérannée, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Electronic address: morelli@ifr88.cnrs-mrs.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Morelli
authors.affiliation: INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France. Electronic address: patrick.lecine@inserm.fr.
authors.forename: Patrick
authors.initials: P
authors.lastname: Lécine
mesh_terms: D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D020125:Mutation, Missense; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C032117:cap I RNA (nucleoside-2'-)methyltransferase