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Updated: Mar 9, 2026
High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication
Published on: October 30, 2021
Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors
Hong-Tao Xu1, Said A Hassounah1, Susan P Colby-Germinario1
1Jewish General Hospital, McGill University AIDS Centre, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.
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.
Insights
Researchers developed a Zika virus RNA-dependent RNA polymerase (RdRp) assay to find new antiviral drugs. The assay identified two inhibitors, sofosbuvir and DMB213, showing promise against Zika virus replication.
Area of Science:
- Virology
- Drug Discovery
- Molecular Biology
Background:
- Zika virus (ZIKV) is a re-emerging pathogen with no available antiviral treatments.
- Viral RNA-dependent RNA polymerase (RdRp) is crucial for Flaviviridae family virus replication and a key target for antiviral development.
Purpose of the Study:
- To establish a functional assay for screening Zika virus RdRp inhibitors.
- To identify and characterize small molecules with anti-ZIKV RdRp activity.
Main Methods:
- Purified recombinant ZIKV NS5 polymerase was used to establish an RdRp activity assay.
- Inhibition was assessed using IC50 and EC50 values in enzymatic and cell-based assays, respectively.
- Drug resistance mutations and enzyme kinetics were analyzed.
Main Results:
- Sofosbuvir triphosphate and DMB213 inhibited ZIKV RdRp with IC50 values of 7.3 μM and 5.2 μM, respectively.
- Cell-based assays confirmed anti-ZIKV activity for both compounds (EC50s of 8.3 μM and 4.6 μM).
- A specific RdRp mutation conferred resistance to sofosbuvir but not DMB213, which acts competitively with natural substrates.
Conclusions:
- The developed ZIKV RdRp assay is effective for screening antiviral compounds.
- DMB213 shows potential as a non-nucleoside inhibitor for ZIKV, with a distinct resistance profile.
- This assay can facilitate the discovery of novel agents targeting ZIKV replication.
