title: Analysis of Ribonucleotide 5'-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.
abstract: Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed anin vitrononradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry outin vitroRNA-dependent RNA synthesis in this assay. Our results show that Mn2+is required for enzymatic activity, while Mg2+is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility ofin vitropolymerase assays in the identification of potential ZIKV inhibitors.
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.
authors.forename: Gaofei
authors.initials: G
authors.lastname: Lu
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.|Drug Innovation Ventures at Emory (DRIVE), Atlanta, Georgia, USA.
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Bluemling
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Collop
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Hager
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.
authors.forename: Damien
authors.initials: D
authors.lastname: Kuiper
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.
authors.forename: Bharat P
authors.initials: BP
authors.lastname: Gurale
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.|Drug Innovation Ventures at Emory (DRIVE), Atlanta, Georgia, USA.|Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: George R
authors.initials: GR
authors.lastname: Painter
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.|Drug Innovation Ventures at Emory (DRIVE), Atlanta, Georgia, USA.
authors.forename: Abel
authors.initials: A
authors.lastname: De La Rosa
authors.affiliation: Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA a.kolykhalov@emory.edu.|Drug Innovation Ventures at Emory (DRIVE), Atlanta, Georgia, USA.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Kolykhalov
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001681:Biological Assay; D002413:Cations, Divalent; D017931:DNA Primers / Q000138:chemical synthesis / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism; D000072417:Protein Domains; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism / Q000494:pharmacology*; D013194:Staining and Labeling / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*; D000071244:Zika Virus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D002413:Cations, Divalent; D017931:DNA Primers; D004277:DNA, Single-Stranded; D011122:Polyphosphates; D012367:RNA, Viral; D012265:Ribonucleotides; D017361:Viral Nonstructural Proteins; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase; C005692:triphosphoric acid
keywords: RdRp; ZIKV; Zika virus; nonradioactive assay; polymerase assay; polymerase inhibitor