Antimicrob Agents Chemother. 2017 Feb 23;61(3). pii: e01967-16.

Analysis of Ribonucleotide 5‘-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.

Lu G1, Bluemling GR1,2, Collop P1, Hager M1, Kuiper D1, Gurale BP1, Painter GR1,2,3, De La Rosa A1,2, Kolykhalov AA4,2.

1Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA.
2Drug Innovation Ventures at Emory (DRIVE), Atlanta, Georgia, USA.
3Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA.
4Emory Institute for Drug Development (EIDD), Emory University, Atlanta, Georgia, USA a.kolykhalov@emory.edu.

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-Barr√© 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 an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-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 of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

KEYWORDS: RdRp; ZIKV; Zika virus; nonradioactive assay; polymerase assay; polymerase inhibitor

PMID: 27993851