title: The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain.
abstract: Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.
authors.affiliation: Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors.forename: Supanee
authors.initials: S
authors.lastname: Potisopon
authors.affiliation: Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Priet
authors.affiliation: Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors.forename: Axelle
authors.initials: A
authors.lastname: Collet
authors.affiliation: Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France CNRS, AFMB UMR 7257, 13288 Marseille, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Decroly
authors.affiliation: Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France CNRS, AFMB UMR 7257, 13288 Marseille, France Barbara.Selisko@afmb.univ-mrs.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.affiliation: Aix-Marseille Université, AFMB UMR 7257, 13288 Marseille, France CNRS, AFMB UMR 7257, 13288 Marseille, France Barbara.Selisko@afmb.univ-mrs.fr.
authors.forename: Barbara
authors.initials: B
authors.lastname: Selisko
mesh_terms: D017434:Protein Structure, Tertiary; D012313:RNA / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D061805:Transcription Elongation, Genetic; D061785:Transcription Initiation, Genetic; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C582223:NS5 protein, dengue virus; C034477:RNA primers; D017361:Viral Nonstructural Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase