The viral capping enzyme nsP1: a novel target for the inhibition of chikungunya virus infection

Abstract

The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.

Figure 1. Chemical structures of MADTP-314, MADTP-346, MADTP-372 and MADTP-393.

Figure 2. Mechanism of action of the MADTP series.

(a) Huh 7.5.1 cells treated with different MADTP compounds were infected with CHIKV pseudoparticles. Arbidol and chloroquine were used as positive controls. The entry of CHIKVpp was determined by measuring the luciferase activity. The average values ± SD of three independent experiments are shown. (b) Cells were transfected with a replication-deficient CHIKV RNA encoding an nsP3-Rluc fusion protein, in the presence or absence of MADTP-314. Subsequently, luciferase activity was determined at 2.5, 5 and 7.5 h post-transfection. (c) CHIKV-infected Vero E6 cells (MOI 5), treated with 50, 100 and 250 μM MADTP-314, were metabolically labeled with ³H-uridine from 6–7 h p.i. Total RNA was isolated and separated by denaturing agarose gel electrophoresis, followed by fluorographic detection of ³H-labeled RNA. (d) In vitro RNA synthesizing activity of isolated CHIKV RTCs in the presence or absence of 250 μM MADTP-314, quantified by measuring the incorporation of ³²P-CTP. RNA II is a 7.5 kb CHIKV RNA that corresponds to the 5′-proximal 7.5 kb of the CHIKV genome up to the subgenomic promoter region.

Figure 3. Characterization of the CHIKV LS3-nsP1-P34S mutant.

(a) Plaque morphology of CHIKV LS3 and mutant CHIKV LS3-nsP1-P34S at 3 days post infection. The plaque assay was performed on Vero E6 cells as described in ref. . (b) Growth curve of CHIKV LS3 (black) and mutant CHIKV LS3-nsP1-P34S (grey) on Vero E6 cells (MOI 0.05). Virus titers were determined by plaque assay on Vero E6 cells at 3 days p.i. Data shown are average values of two independent experiments. (c) The expression of nsP1 in Vero E6 cells infected with WT CHIKV LS3 or the nsP1-P34S mutant virus at various time points post infection (MOI 0.05) analyzed by Western blot.

Figure 4. Sensitivity of WT and nsP1-P34S mutant virus to MADTP-314.

Effect of various concentrations of MADTP-314 on the expression of nsP1 and the structural protein E2 of WT CHIKV LS3 and the P34S-LS3 mutant CHIKV in infected Vero E6 cells (MOI 0.1) that were analyzed by Western blotting at 28 h p.i. Percentages of untreated control are presented below the blots. The anti-E2 antibody was a gift of Dr. G. Pijlman, the anti-nsP1 antibody was published before in ref. .

Figure 5. Effect of MADTP compounds on the methyltransferase and the guanylyltransferase activity of VEEV nsP1.

(a) Effect of different concentrations of MADTP-372, MADTP-314 and MADTP-346 on the in vitro guanylylation of VEEV nsP1. The m⁷GMP-nsP1 complex was detected by Western blot with an anti-methyl^3/7Gp antibody. (b) Dose-response curves of MADTP-314 (circles), MADTP-346 (triangles), and MADTP-372 (squares) on the methyltransferase activity of VEEV nsP1. The nsP1-MTase product (3H-methyl) GIDP was measured by a scintillation counter. The average values ± SD of two independent experiments are shown. (c) Relative nsP1 guanylylation activity (% WT) of the D34S-nsP1 mutant in the absence of compounds. (d) Relative nsP1 guanylylation activity (% untreated control) of WT and D34S VEEV nsP1 when treated with different concentrations of MADTP-372. The average values ± SD of two independent experiments are shown.