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

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.

Figure 1

Structure of the non-nucleoside compound DMB213.

Figure 2

Purification and activity of ZIKV recombinant NS5 polymerase domain WT (ZIKV NS5pol) and mutants. (a) Coomassie brilliant blue staining of purified NS5 polymerase domains after 10% SDS–PAGE. MW, molecular size standards in kDa. The positions of purified recombinant NS5 polymerase domains are indicated on the right. The mutant mGDD contains the active site substitutions GDD to GAA. (b) Activity of recombinant ZIKV NS5 polymerase domains as assessed using ZMG RNA template by filter-binding RdRp assay as described in the Materials and methods section. Values are the means of two independent experiments. Error bars represent standard deviations.

Figure 3

Docking of DMB213 into the active site of the Zika polymerase homology model. DMB213 docks in close proximity to the active site residues D536, D666 and D667. The active site is shown as surface coloured by standard CPK coloration. Blue represents surface-exposed nitrogen and red represents surface-exposed oxygen groups. The proximal location of Mg²⁺ was superimposed into the homology model from the dengue polymerase structure (2J7U) by structure overlay. A short double-stranded RNA was retrieved from the PDB structure 4WTA and overlaid on the homology model structure. Structure visualization was performed using PyMOL. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.