title: mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research.
abstract: Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m(7)GMP-nsP1 adduct. Subsequent transfer of m(7)GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m(7)G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m(7)GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research.
  
  
  OBJECTIVE
  Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.
authors.affiliation: CNRS, AFMB UMR 7257, Marseille, France Aix-Marseille Université, AFMB UMR 7257, Marseille, France.
authors.forename: Changqing
authors.initials: C
authors.lastname: Li
authors.affiliation: CNRS, AFMB UMR 7257, Marseille, France Aix-Marseille Université, AFMB UMR 7257, Marseille, France.
authors.forename: Jaime
authors.initials: J
authors.lastname: Guillén
authors.affiliation: CNRS, AFMB UMR 7257, Marseille, France Aix-Marseille Université, AFMB UMR 7257, Marseille, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Rabah
authors.affiliation: IBMM UMR 5247 CNRS-Université Montpellier-ENSCM, Montpellier, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Blanjoie
authors.affiliation: IBMM UMR 5247 CNRS-Université Montpellier-ENSCM, Montpellier, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Debart
authors.affiliation: IBMM UMR 5247 CNRS-Université Montpellier-ENSCM, Montpellier, France.
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Vasseur
authors.affiliation: CNRS, AFMB UMR 7257, Marseille, France Aix-Marseille Université, AFMB UMR 7257, Marseille, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: CNRS, AFMB UMR 7257, Marseille, France Aix-Marseille Université, AFMB UMR 7257, Marseille, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Decroly
authors.affiliation: CNRS, AFMB UMR 7257, Marseille, France Aix-Marseille Université, AFMB UMR 7257, Marseille, France bruno.coutard@afmb.univ-mrs.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Coutard
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D004666:Encephalitis Virus, Venezuelan Equine / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D012315:RNA Caps / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: D000998:Antiviral Agents; D012315:RNA Caps; D012333:RNA, Messenger