Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection

Abstract

Chikungunya virus (CHIKV) is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. There are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. We conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium ATPase, as a potent inhibitor of CHIKV infection. Treatment of human cells with digoxin or a related cardiac glycoside, ouabain, resulted in a dose-dependent decrease in infection by CHIKV. Inhibition by digoxin was cell type-specific, as digoxin treatment of either murine or mosquito cells did not diminish CHIKV infection. Digoxin displayed antiviral activity against other alphaviruses, including Ross River virus and Sindbis virus, as well as mammalian reovirus and vesicular stomatitis virus. The digoxin-mediated block to CHIKV and reovirus infection occurred at one or more postentry steps, as digoxin inhibition was not bypassed by fusion of CHIKV at the plasma membrane or infection with cell surface-penetrating reovirus entry intermediates. Selection of digoxin-resistant CHIKV variants identified multiple mutations in the nonstructural proteins required for replication complex formation and synthesis of viral RNA. These data suggest a role for the sodium-potassium ATPase in promoting postentry steps of CHIKV replication and provide rationale for modulation of this pathway as a broad-spectrum antiviral strategy.

Importance: Mitigation of disease induced by globally spreading, mosquito-borne arthritogenic alphaviruses requires the development of new antiviral strategies. High-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics and illuminate host pathways required for viral infection. Our study describes the potent inhibition of Chikungunya virus and related alphaviruses by the cardiac glycoside digoxin and demonstrates a function for the sodium-potassium ATPase in Chikungunya virus infection.

FIG 1

High-throughput screening to identify inhibitors of CHIKV infection. (A) U-2 OS cells were incubated with DMSO, 100 nM bafilomycin A1, or compounds from the NIH clinical collection at a concentration of 1 µM at 37°C for 1 h. Cells were adsorbed with SL15649 eGFP replicon particles at an MOI of ~5 IU/cell and incubated with compound at 37°C for 20 to 24 h. Cells were incubated with Hoechst dye to stain nuclei and imaged by automated, high-content fluorescence microscopy. (B) Robust Z scores were calculated for individual compounds. Shown are the average robust Z scores for compounds with robust Z scores of ≤−2 or ≥2 median absolute deviations from the median of each plate identified in three independent screening experiments. (C) Distribution of candidate compounds by known biological targets.

FIG 2

Digoxin potently inhibits CHIKV infection of human cells. (A) U-2 OS cells, (B) HSFs, (C) ST2 cells, or (D) C6/36 cells were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin for 1 h prior to adsorption with CHIKV strain SL15649 at an MOI of 5 PFU/cell. After 1 h of incubation, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the means. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, in comparison to DMSO, as determined by ANOVA followed by Tukey’s post hoc test.

FIG 3

CHIKV resistance to digoxin in murine cells correlates with decreased expression of the α3 subunit of the sodium-potassium ATPase. (A) ST2 cells or (B) C2C12 cells were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin for 1 h prior to adsorption with CHIKV strain SL15649 at an MOI of 5 PFU/cell. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as the percentages of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the means. (C) U-2 OS and ST2 cells were mock infected or infected with CHIKV 181/25 at an MOI of 5 PFU/cell. RNA was isolated and used for RT-PCR amplification of ATP1A1 (α1), ATP1A3 (α3), and GAPDH transcripts with human- or murine-specific primer sets (see Table S1 in the supplemental material). Reaction products were resolved by electrophoresis in 1% agarose gels (left). Band intensity was quantified by optical densitometry for four independent experiments (right). ***, P < 0.001, in comparison to DMSO, as determined by ANOVA followed by Tukey’s post hoc test.

FIG 4

Inhibition by digoxin occurs via the sodium-potassium ATPase. (A) U-2 OS cells were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin or the related cardiac glycoside, ouabain, for 1 h prior to adsorption with CHIKV SL15649 at an MOI of 5 PFU/cell. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were scored for infection by indirect immunofluorescence. Results are presented as percentage of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the means. (B) U-2 OS cells were incubated with DMSO or digoxin in standard medium or medium supplemented with increasing concentrations of NaCl (left) or KCl (right) for 1 h prior to adsorption with CHIKV 181/25 at an MOI of 5 PFU/cell. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or digoxin and the concentration of NaCl or KCl shown for 5 h. Cells were scored for infection by indirect immunofluorescence. Results are presented as percentages of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the means. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, in comparison to DMSO, as determined by ANOVA followed by Tukey’s post hoc test.

FIG 5

CHIKV inhibition by digoxin is not attributable to cytotoxicity. (A) U-2 OS cells were treated with DMSO, 10 µM STS, or increasing concentrations of digoxin for 6 h. Cell viability was quantified by PI staining. Results are expressed as percentages of viable cells normalized to DMSO-treated cells for individual experiments. Horizontal black lines indicate mean percentages of viability. (B) U-2 OS cells were treated with DMSO, 10 µM STS, or increasing concentrations of digoxin for 6 or 24 h. Cell viability was quantified by PrestoBlue fluorescence assay. Results are presented as percentages of viable cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the means. *, P < 0.05, and ***, P < 0.001, in comparison to DMSO, as determined by ANOVA followed by Tukey’s post hoc test.

FIG 6

Digoxin inhibits multiple alphaviruses, mammalian reovirus, and VSV. (A) U-2 OS cells were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin for 1 h prior to adsorption with CHIKV strains SL15649 and 181/25 at an MOI of 1 PFU/cell, RRV strain T48 at an MOI of 10 PFU/cell, or SINV strain TRSB at an MOI of 5 PFU/cell. After 1 h of incubation, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were stained with virus-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the mean. (B) HBMECs were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin for 1 h prior to adsorption with reovirus virions (left) or ISVPs (right) at an MOI of 1,500 particles/cell (~15 PFU/cell). After 1 h of incubation, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 20 h. Cells were scored for infection by indirect immunofluorescence. Results are presented as percentages of infected cells normalized to DMSO-treated cells for duplicate experiments. Error bars indicate standard errors of the means. Insets show schematics of reovirus virions and ISVPs. (C) U-2 OS cells were incubated with DMSO, 20 mM NH₄Cl, or digoxin at the concentrations shown for 1 h prior to adsorption with VSV-eGFP at an MOI of 10 PFU/cell. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were incubated with Hoechst stain to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate the standard errors of the means. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, in comparison to DMSO, as determined by ANOVA followed by Tukey’s post hoc test.

FIG 7

Digoxin inhibits CHIKV at postentry steps of the replication cycle. (A) U-2 OS cells were incubated with DMSO, 20 mM NH₄Cl, or 1 µM digoxin prior to (−60 to −15 min), during (0 to +45 min), or after (+60 to +240 min) adsorption with CHIKV 181/25 at an MOI of 5 PFU/cell for 1 h. Cells were incubated in the presence or absence of inhibitors for 5 h and scored for infection by indirect immunofluorescence. Results are presented as percentages of infected cells normalized to DMSO-treated cells for triplicate experiments. Error bars indicate standard errors of the means. (B) U-2 OS cells were incubated with DMSO, 10 µM 5-NT, or digoxin at the concentrations shown for 1 h prior to adsorption with CHIKV strain 181/25 (left) or SINV strain TRSB (right) at an MOI of 100 PFU/cell at 4°C for 1 h. Unbound virus was removed, and cells were treated at 37°C for 5 min with either acidic medium (pH 5.5 [black bars]) to trigger viral fusion at the plasma membrane or neutral medium (pH 7.4 [white bars]) as a control. Cells were incubated at 37°C for 18 h with medium containing DMSO or inhibitor and NH₄Cl to block subsequent rounds of infection. Cells were scored for infection by indirect immunofluorescence. Results are presented as percentages of infected cells for triplicate experiments. Error bars indicate standard errors of the means. *, P < 0.05, and ***, P < 0.001, in comparison to DMSO, as determined by ANOVA followed by Tukey’s post hoc test.

FIG 8

Passage of CHIKV in the presence of digoxin enriches for drug-resistant viruses. U-2 OS cells were incubated with DMSO, 10 µM 5-NT, or increasing concentrations of digoxin for 1 h prior to adsorption with CHIKV stocks that had been passaged 14 times in the presence of either DMSO (SL15649_DMSO) or digoxin (SL15649_Digoxin) at an MOI of 5 PFU/cell. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or inhibitor for 5 h. Cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells. Error bars indicate standard errors of the means. (B) U-2 OS cells were incubated with DMSO or 500 nM digoxin for 1 h prior to adsorption at an MOI of 5 PFU/cell with virus clones that were plaque purified from either the SL15649_DMSO or SL15649_Digoxin stock. After 1 h, virus was removed, and cells were incubated with medium containing DMSO or digoxin for 5 h. Cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percentages of infected cells normalized to DMSO-treated cells for duplicate experiments. Error bars indicate standard errors of the means. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, in comparison to DMSO-treated or DMSO-passaged virus-infected cells as determined by ANOVA followed by Tukey’s post hoc test.