Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors

J Antimicrob Chemother. 2017 Mar 1;72(3):727-734. doi: 10.1093/jac/dkw514.

Abstract

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available.

Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase.

Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 μM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 μM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates.

Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

MeSH terms

  • Antiviral Agents / pharmacology*
  • Drug Discovery / methods
  • Enzyme Inhibitors / pharmacology*
  • HIV Integrase / metabolism
  • HIV Reverse Transcriptase / antagonists & inhibitors
  • RNA, Viral / metabolism
  • RNA-Dependent RNA Polymerase / antagonists & inhibitors*
  • RNA-Dependent RNA Polymerase / chemistry
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Recombinant Proteins / metabolism
  • Sofosbuvir / pharmacology
  • Virus Replication / drug effects
  • Zika Virus / drug effects
  • Zika Virus / enzymology*
  • Zika Virus / physiology

Substances

  • Antiviral Agents
  • Enzyme Inhibitors
  • RNA, Viral
  • Recombinant Proteins
  • HIV Integrase
  • reverse transcriptase, Human immunodeficiency virus 1
  • RNA-Dependent RNA Polymerase
  • HIV Reverse Transcriptase
  • Sofosbuvir
  • p31 integrase protein, Human immunodeficiency virus 1