mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

• DOI: 10.1128/jvi.00599-15
• Publication Date: 2015-06-04T11:25:31Z
• Authors: Changqing Li; Jaime Guillén; Nadia Rabah; Alexandre Blanjoie; Françoise Debart; Jean-Jacques Vasseur; Bruno Canard; Etienne Decroly; Bruno Coutard
• Keywords: Aurintricarboxylic acid; RNA ligase

ABSTRACT Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing transfer of methyl group from S -adenosylmethionine (AdoMet) N7 position GTP molecule followed by formation an m 7 GMP-nsP1 adduct. Subsequent GMP onto 5′ end has not been demonstrated in vitro yet. Here we report biochemical characterization Venezuelan equine encephalitis virus (VEEV) We have developed enzymatic assays uncoupling different reactions steps catalyzed The MTase GT reaction were using nonhydrolyzable (GIDP) substrate original Western blot assay anti-m 3 G/m G-cap monoclonal antibody, respectively. is stimulated -adenosyl- l -homocysteine (Ado-Hcy), product preceding reaction, metallic ions. covalent linking between involves phosphamide bond nucleotide histidine residue. Final guanylyltransfer RNA was observed for first time with alphavirus 5′-diphosphate oligonucleotide whose sequence corresponds genome. Alanine scanning mutagenesis residues H37, H45, D63, E118, Y285, D354, R365, N369, N375 revealed their respective roles MT reactions. Finally, inhibitory effects sinefungin, aurintricarboxylic acid (ATA), ribavirin triphosphate on investigated, providing possible avenues antiviral research. IMPORTANCE Emergence or reemergence alphaviruses represents serious health concern, elucidation replication mechanisms prerequisite development specific inhibitors targeting enzymes. In particular, able, through sequence, add cap required protection against cellular nucleases initiation proteins translation. this study, diphosphate synthetic mimicking time. performed 1 (nsP1). Reference compounds target inhibited functions, highlighting value development.