Abstract
Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.
Highlights
Zika virus (ZIKV) is an emerging human pathogen of the Flaviviridae family, a group of single-stranded RNA enveloped viruses
We show that recombinant ZIKV RNA-dependent RNA polymerase (RdRp) can synthesize RNA de novo, which can be quantified in real-time using a fluorescent dye (SYTO 9), which detects the presence of double-stranded RNA generated after the copy of single-stranded homopolymeric RNA
We demonstrate the potential value of this protocol to screen antiviral compounds by using known broad-spectrum antivirals, which reproduce their inhibitory activities in this assay. The versatility of this method was further established with RdRps from two distinct viruses: foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV)
Summary
Zika virus (ZIKV) is an emerging human pathogen of the Flaviviridae family, a group of single-stranded (ss) RNA enveloped viruses. Members of this family include the human pathogens dengue virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, Japanese encephalitis virus and hepatitis C virus (HCV)[1]. We demonstrate the potential value of this protocol to screen antiviral compounds by using known broad-spectrum antivirals, which reproduce their inhibitory activities in this assay The versatility of this method was further established with RdRps from two distinct viruses: foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV). The implications of these results for high-throughput approaches are discussed
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