Development of a luminescence-based method for measuring West Nile Virus MTase activity and its application to screen for antivirals

Abstract

West Nile virus (WNV) is a flavivirus responsible for causing febrile illness and severe neurological diseases, with an increasing impact on human health around the world. However, there is still no adequate therapeutic treatment available to struggle WNV infections. Therefore, there is an urgent need to develop new techniques to accelerate the discovery of drugs against this pathogen. The main protein implicated in the replication of WNV is the non-structural protein 5 (NS5). This multifunctional protein contains methyltransferase (MTase) activity involved in the capping formation at the 5'-end of RNA and the methylation of internal viral RNA residues, both functions being essential for viral processes, such as RNA translation and escape from the innate immune response. We have developed a straightforward luminescence-based assay to monitor the MTase activity of the WNV NS5 protein with potential for high-throughput screening. We have validated this method as a sensitive and suitable assay for the identification of WNV MTase inhibitors assessing the inhibitory effect of the broad MTase inhibitor sinefungin, a natural nucleoside analog of the universal methyl donor S-adenosyl methionine (SAM). The screening of a small series of purine derivatives identified an adenosine derivative as a dose-dependent inhibitor of the MTase activity. The antiviral efficacy of this compound was further confirmed in WNV infections, displaying a measurable antiviral effect. This result supports the utility of this novel method for the screening of inhibitors against WNV MTase activity, which can be of special relevance to the discovery and development of therapeutics against WNV.

Keywords: NS5; WNV; antiviral; flavivirus; methyltransferase.

Graphical abstract

Fig. 1

Luminescence-based MTase assay using WNV NS5. A) Schematic of the steps involved in the luminescence-based methyltransferase assay developed in this work using the The MTase-Glo™ kit. The standard MTase activity assay stage is indicated. B) SDS-PAGE analysis of WNV NS5 WT, WNV NS5-GAA, and WNV NS5–4A recombinant proteins expressed in E. coli BL21(DE3)-pRIL and purified by affinity chromatography. “M”, molecular marker. The molecular weight (in kDa) of each band is indicated. C) Relative activity of different recombinant WNV NS5 after 60 min of reaction in the presence (+) or absence (−) of the indicated reagents. The concentration of each reagent is the same as used in a standard MTase activity assay. 100 % activity (in black) corresponds to the standard MTase activity assay performed with WNV NS5 WT as established in Materials and Methods. D) Time course MTase activity of WNV NS5 in a standard MTase activity assay in the presence (circles) or in the absence (triangles) of WNV NS5 WT as described in Materials and Methods. Black circle corresponds to the activity displayed in a “standard MTase activity assay”. Averages and ±SD of three independent experiments are represented.

Fig. 2

Optimization of conditions for the WNV NS5 methylations. Relative activity of WNV NS5 varying the pH (A), temperature (B), concentrations of NaCl (C), concentration of WNV NS WT (D) and methyl acceptor RNA (E), while keeping the other three parameters constant at the standard MTase activity assay levels. For each parameter, activities are related to the optimal level, expressed as 100 % (black circles or bar). Averages and ±SD of three independent experiments are represented. The R squared value of the linear function that relates the amount of WNV NS WT to the activity is given in D.

Fig. 3

Inhibitory effects of different inhibitors against the WNV NS5 WT Mtase or RdRp activities. A) Chemical structures of sinefungin and compound 1. B) MTase activity dose-response inhibition curves and IC₅₀ values of the WNV NS5 WT exerted by sinefungin. C) MTase activity dose-response inhibition curves and IC₅₀ values of the WNV NS5 WT exerted by compound 1. D) RdRp activity dose-response inhibition curve and IC₅₀ value of the WNV NS5 WT exerted by compound 1. IC₅₀ calculation for each compound is indicated within the corresponding graph. Averages and ±SD of at least three independent experiments are represented.

Fig. 4

Antiviral activity of compound 1 against WNV. A) Effect of compound 1 on WNV multiplication. Vero cells were infected at a MOI of 1 PFU/cell, treated with 10, 50 µM of compound 1 or the same amount of vehicle (DMSO) as a control and the virus yield released to the culture medium at 24 h post-infection was determined by plaque assay. B) Effect of compound 1 on cellular ATP levels. The cytotoxicity of compound 1 was analyzed in uninfected cells treated in parallel as in (A) by quantification of the amount of cellular ATP. C) Evaluation of the cytotoxicity of compound 1 by MTT assay. Uninfected cells treated were in parallel as in (A) and cell viability was determined analyzed by MTT assay. Averages and ±SD of four to six biological replicates are represented. *** for P<0.0007 for ANOVA and Dunnet's multiple comparison test in (A).