Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses

Abstract

No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2'-C-methylcytidine (2'-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2'-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2'-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2'-CMC, AHFV variants with reduced susceptibility to 2'-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2'-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2'-CMC.

FIG 1

2′-Modified nucleoside analogues inhibit Alkhurma hemorrhagic fever virus (AHFV). (A) Inhibition of AHFV-induced cell death. 2′-Modified nucleoside analogues were dissolved in DMSO and added to A549 cells to a final concentration of 50 μM and 0.5% DMSO. Cells were infected with AHFV at a multiplicity of infection (MOI) of 0.5 and incubated for 3 days, and cell viability was determined. (B) Effect of 2′-modified nucleosides on cell viability. A549 cells were treated as described for panel A but were mock infected. Cell viability was measured 3 days after the addition of compound. Cells were treated with 10 μM dasatanib as a positive control to inhibit cell viability. Mean values from four replicate wells are shown, and error bars indicate the standard errors of the means. A representative of three independent experiments is shown. Abbreviations: DMSO, dimethyl sulfoxide; 2′-CMA, 2′-C-methyladenine; 2′-CMC, 2′-C-methylcytidine; 2′-CMG, 2′-C-methylguanine; 2′-CMU, 2′-C-methyluridine; 2′-OMA, 2′-O-methyladenine; 2′-OMC, 2′-O-methylcytidine; 2′-OMG, 2′-O-methylguanine; 2′-OMU, 2′-O-methyluridine.

FIG 2

Effect of compounds on AHFV titers. A549 cells were treated with 2′-CMC (A), 6-azaU (B), or IFN-α2a (C) and infected with AHFV at an MOI of 0.5. Twenty-four h postinfection, the cultures were harvested, frozen, and thawed three times, and the viral titers in the resulting supernatants were determined. Mean titers from three biological replicates are depicted, and error bars indicate standard errors of the means.

FIG 3

Antiviral effect of 6-azaU is inhibited by uridine or cytidine but not guanosine. A549 cells were treated with 4 μM 6-azaU and diluent (black bar) or with various concentrations of uridine (white columns), cytidine (shaded columns), or guanosine (crosshatched columns). The cells were then infected with AHFV at an MOI of 0.5 and then incubated for 3 days before cell viability was determined. Mean values from eight replicate wells are shown, and error bars indicate the standard deviations.

FIG 4

Reporter replicon system for AHFV. (A) Schematic of the subgenomic, luciferase reporter, AHFV replicon. The T7 promoter is followed by the 5′ untranslated region of AHFV and then the N-terminal 27 amino acids of the AHFV C protein (C*) fused to firefly luciferase (FF-luc), the EMCV IRES, and the signal sequence from the AHFV E1 protein (E1ss) and the nonstructural genes NS1, 2A, 2B, 3, 4A, 4B, and 5. The location of the NS5 polymerase active site is indicated (GDD). (B) Replicon luciferase signals over time. Wild-type or polymerase-inactivated (pol−) replicons were electroporated into Huh7 cells, and luciferase activity was determined at various times posttransfection. To account for differences in transfection efficiency, luciferase signals were normalized to those obtained at 4 h posttransfection. Mean values from four replicate wells are shown, and error bars indicate the standard deviations. (C) Immunofluorescence of Huh7 cells electroporated with pol⁻ or wild-type replicons, fixed and stained at 48 h posttransfection. Cell nuclei are blue; green staining indicates AHFV proteins.

FIG 5

Amino acid substitutions associated with AHFV growth in the presence of 2′-CMC assessed using the reporter replicon. (A) Susceptibility of mutant AHFV replicons to 2′-CMC. Wild-type or mutant replicon RNA was electroporated into Huh7 cells, 2′-CMC was added 4 h posttransfection, and the cultures were incubated for 48 h before harvesting for luciferase assay. Luciferase signals were normalized to that at 4 h posttransfection and are shown as percentages of the normalized signal obtained for that replicon incubated without 2′-CMC. Mean values from three experiments, performed with RNA transcribed from two independently generated templates, is shown with error bars representing the standard errors of the means. (B) Replication capacity of mutant replicons. Mutant replicons were electroporated into Huh7 cells, and cells were incubated for 48 h before harvesting for luciferase assay. Mean values from three experiments, performed with RNA transcribed from two independently generated templates, is shown with error bars representing the standard errors of the means.

FIG 6

Modeling of amino acid substitutions associated with AHFV resistance to 2′-CMC. (A) Active site of the HCV NS5B polymerase (Protein Data Bank accession number 1C2P). The S282T change is associated with resistance to 2′-CMC. (B) AHFV RdRp sequence modeled on the WNV NS5 RdRp structure (Protein Data Bank accession number 2HFZ). The residues of the GDD motif are shown in blue, and the S604 and C666 residues associated with resistance to 2′-CMC are shown in red. (C) AHFV RdRp sequence modeled on the WNV NS5 RdRp structure, showing the predicted location of the M644 residue relative to the GDD motif (blue) and the S603 and C666 residues (red).