Zika Virus Replicons for Drug Discovery

Abstract

The current epidemic of Zika virus (ZIKV) has underscored the urgency to establish experimental systems for studying viral replication and pathogenesis, and countermeasure development. Here we report two ZIKV replicon systems: a luciferase replicon that can differentiate between viral translation and RNA synthesis; and a stable luciferase replicon carrying cell line that can be used to screen and characterize inhibitors of viral replication. The transient replicon was used to evaluate the effect of an NS5 polymerase mutation on viral RNA synthesis and to analyze a known ZIKV inhibitor. The replicon cell line was developed into a 96-well format for antiviral testing. Compare with virus infection-based assay, the replicon cell line allows antiviral screening without using infectious virus. Collectively, the replicon systems have provided critical tools for both basic and translational research.

Keywords: Drug discovery; Flavivirus; Replicon; Zika.

Fig. 1

Characterization of ZIKV luciferase replicon. (a) Diagram for ZIKV replicon construction. C₃₈ and E₃₀ represent DNA sequences encoding the first 38 amino acids of C protein and the last 30 amino acids of E protein, respectively. Rluc2A represents the gene cassette expressing Renilla luciferase (Rluc) and foot-and-mouth disease virus 2A protease (Rluc2A). HDVr, hepatitis delta virus ribozyme sequence. (b) Transient replicon assay. Equal amount of wild-type (WT) and NS5ΔGDD mutant RNAs (10 μg) were electroporated into Huh7 cells. Cellular Rluc signals were measured at indicated time points. The means and standard deviations from three independent experiments are shown. (c) Antiviral activity of NITD-008. Huh7 cells were electroporated with 10 μg of WT Rluc replicon. The transfected cells were treated immediately with NITD-008 (1 or 5 μM) or 0.9% DMSO as a control. The relative Rluc activities harvested at 24 and 32 h p.t. are indicated in percentages of the Rluc activities derived from the DMSO control cells (set as 100%). N.S., not significant; *, significant (p < 0.05); **, highly significant (p < 0.01).

Fig. 2

A Huh7 cell line stably expressing luciferase and Neo ZIKV replicon (Huh7 Rep-Neo cell). (a) Schematic diagrams of the full-length cDNA clone of ZIKV (top) and the cDNA clone of ZIKV Rep-Neo (bottom). In the ZIKV Rep-Neo construct, a fragment containing internal ribosome entry site (IRES) and neomycin resistance gene (Neo) was inserted downstream of the first 28 nucleotides of 3′UTR. The nucleotide lengths of complete 5′UTR and 3′UTR are indicated. (b) Development of Huh7 Rep-Neo cell line. The flowchart outlines the major steps to generate the stable replicon carrying cell line. (c) Transient luciferase assay. Huh7 cells were electroporated with equal amounts (10 μg) of WT Rep-Neo RNA or an NS5 polymerase GDD active site mutant replicon (Rep NS5 ∆ GDD) RNA. At 24 and 48 h p.t., luciferase signals were measured from lysates of about 20,000 transfected cells or naïve Huh7 cells. The average and standard deviations from three independent measurements are presented. (d) Detection of viral dsRNA. Rep-Neo cells (P6) were analyzed for dsRNA by IFA using mAb J2 and goat-anti-mouse IgG conjugated with Alexa Fluor®488 as primary and secondary antibodies, respectively. Nucleus was stained by DAPI. (e) Detection of viral NS4B protein. The expression of NS4B protein was detected in Rep-Neo cells (P6) using mAb 44-4-7 and goat-anti-mouse IgG conjugated with Alexa Fluor®488 as primary and secondary antibodies, respectively. (f) Luciferase assay of P6 Rep-Neo cells. Luciferase activities were measured from the lysates of 20,000 P6 Rep-Neo cells or naive Huh7 cells. (g) Antiviral testing using Rep-Neo cells. Rep-Neo cells (P6) were seeded into a 96-well plate. The cells were incubated with NITD-008 at indicated concentrations and measured for luciferase activities at 48 h post-treatment. See experimental details in Materials and Methods.