title: Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2'-modified nucleotide analogues.
abstract: In targeting the essential viral RNA-dependent RNA-polymerase (RdRp), nucleotide analogues play a major role in antiviral therapies. In the Flaviviridae family, the hepatitis C virus (HCV) can be eradicated from chronically infected patients using a combination of drugs which generally include the 2'-modified uridine analogue Sofosbuvir, delivered as nucleotide prodrug. Dengue and Zika viruses are emerging flaviviruses whose RdRp is closely related to that of HCV, yet no nucleoside drug has been clinically approved for these acute infections. We have purified dengue and Zika virus full-length NS5, the viral RdRps, and used them to assemble a stable binary complex made of NS5 and virus-specific RNA primer/templates. The complex was used to assess the selectivity of NS5 towards nucleotide analogues bearing modifications at the 2'-position. We show that dengue and Zika virus RdRps exhibit the same discrimination pattern: 2'-O-Me > 2'-C-Me-2'-F > 2'-C-Me nucleoside analogues, unlike HCV RdRp for which the presence of the 2'-F is beneficial rendering the discrimination pattern 2'-O-Me > 2'-C-Me ≥ 2'-C-Me-2'-F. Both 2'-C-Me and 2'-C-Me-2'-F analogues act as non-obligate RNA chain terminators. The dengue and Zika NS5 nucleotide selectivity towards 2'-modified NTPs mirrors potency of the corresponding analogues in infected cell cultures.
authors.affiliation: Aix-Marseille Université, AFMB (Laboratoire d'Architecture et Fonction de Macromolécules Biologiques) UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France.
authors.forename: Supanee
authors.initials: S
authors.lastname: Potisopon
authors.affiliation: Aix-Marseille Université, AFMB (Laboratoire d'Architecture et Fonction de Macromolécules Biologiques) UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France.
authors.forename: François
authors.initials: F
authors.lastname: Ferron
authors.affiliation: Aix-Marseille Université, AFMB (Laboratoire d'Architecture et Fonction de Macromolécules Biologiques) UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Fattorini
authors.affiliation: Aix-Marseille Université, AFMB (Laboratoire d'Architecture et Fonction de Macromolécules Biologiques) UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France. Electronic address: barbara.selisko@afmb.univ-mrs.fr.
authors.forename: Barbara
authors.initials: B
authors.lastname: Selisko
authors.affiliation: Aix-Marseille Université, AFMB (Laboratoire d'Architecture et Fonction de Macromolécules Biologiques) UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, 13288 Marseille, France. Electronic address: bruno.canard@afmb.univ-mrs.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism; D003716:Dengue Virus / Q000201:enzymology*; D006801:Humans; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000069474:Sofosbuvir / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000071244:Zika Virus / Q000201:enzymology*
chemical_list: D000998:Antiviral Agents; C582223:NS5 protein, dengue virus; D009711:Nucleotides; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D000069474:Sofosbuvir
keywords: 2′-modified NTP analogue; Dengue virus; Elongation complex; NS5; RNA-dependent RNA polymerase; Zika virus