Chemicals

Ebselen (also known as PZ51, HY-13750; MedChemExpress (MCE)) was prepared at a concentration of 100 mM in DMSO (Sigma). Inositol (HY-B1411, MCE), L-690,330 (HY-101075, MCE), and LiCl (L9650, Sigma) were prepared at concentrations of 200 mM, 100 mM, and 3 M, respectively, in water. The solutions were stored at − 80 °C, except the LiCl solution, which was stored at room temperature (RT).

Cells

C6/36 cells (CRL-1660, ATCC) were cultivated in Leibovitz’s L-15 medium (L-15, Gibco) supplemented with 10% heat-inactivated FBS and incubated at 28 °C. Vero cells (Vero CCL-81, ATCC) were cultivated in the Minimum Essential Medium (MEM/EBSS; SH30024.02, HyClone) supplemented with 10% heat-inactivated FBS. Madin-Darby Canine Kidney (MDCK; CCL-34, ATCC) cells were cultivated in MEM (Gibco) supplemented with 10% heat-inactivated FBS. Huh-7 was cultivated in high glucose Dulbecco’s Modified Eagle Medium (DMEM, SH30285.02, HyClone) supplemented with 10% heat-inactivated FBS. Immortalized human hepatocyte cells (imHC) were cultivated in DMEM/F12 at a ratio of 1:1 (SH30023.02, HyClone) supplemented with 10% heat-inactivated fetal bovine serum (FBS). These cell lines were incubated at 37 °C with 5% CO₂.

Viruses

Dengue virus serotype 2 (DENV-2) strain 16,681, the Asian zika virus (ZIKV) strain (MU-DMSC-4/2017, accession number: MT377495)⁵⁶, and chikungunya virus (CHIKV) were propagated in C6/36 cells. Virus supernatants were collected at 5 days post-infection (dpi) for DENV-2, and at 7 dpi for ZIKV and CHIKV. Enterovirus 71 (EV71) subgenotype C4⁵⁷ was propagated in Vero cells. Influenza A virus (IAV) A/Nonthaburi/102/2009(H1N1) was propagated in MDCK cells. Virus supernatants of EV71 and IAV were collected at 3 dpi., then centrifuged to remove cell debris, aliquoted, and stored at − 80 °C.

Cell viability assay

The procedure for cell viability assay was described elsewhere³⁵. Cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well. The following day, cells were treated with twofold serial dilutions of ebselen or L690,330 in 2% FBS media for 48 h. Cells treated with media containing 0.5% DMSO served as the untreated control. Cell viability was assessed using MTT dye (Invitrogen) in duplicate. Percent cell viability was calculated relative to the untreated control. All assays were performed in duplicate.

Recombinant IMPA1 expression and purification

Human IMPA1 was cloned into the pET28a plasmid at the NdeI and PstI restriction sites. The recombinant protein was expressed in Escherichia coli BL21 (DE3) by culturing in 1 L of LB medium containing 50 µg/ml of kanamycin. The culture was incubated at 37 ℃ with 250 rpm shaking until the optical density at 600 nm reached 1.0. Protein expression was induced with 1 mM isopropyl β-D-thiogalactoside and the incubation continued at 20 ℃ with 200 rpm shanking for 20 h. The cells were harvested by centrifugation.

The cell pellets were resuspended in 20 mM sodium phosphate buffer (pH 7.4) containing 300 mM NaCl and 10 mM imidazole, then sonicated on ice for 5 min. The lysate was centrifuged at 12,500 rpm for 1 h to collect supernatant, which was then purified using immobilized metal affinity chromatography (TALON, Takara Bio, Japan). Unbound proteins were removed using 20 mM sodium phosphate buffer (pH 7.4), containing 300 mM NaCl and 20 mM imidazole. Elution of IMPA1 protein was carried out using 20 mM sodium phosphate buffer (pH 7.4) containing 300 mM NaCl and varying concentrations of imidazole (40, 100, 250, 400 mM). The eluted protein was dialyzed against 20 mM Tris-HCl pH 7.5 containing 10% glycerol at 4 ℃ for 8 h and stored at -20 °C until use.

In vitro enzymatic assays using recombinant IMPA1

The inhibitory effects of ebselen and lithium were assessed in a 20 µl reaction, following previously described protocols with minor modifications¹⁷. IMPA1 (5 ng) was incubated with various concentrations of ebselen (0‒10 µM) and lithium (0‒100 mM) for 30 min on ice. Subsequently, 1 mM of inositol monophosphate substrate and reaction mixture containing 50 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 3 mM MgCl_2, 150 mM KCl, 0.5 mg/mL bovine serum albumin (BSA) and 0.01% (v/v) Triton X-100 were added. The reaction was incubated at 37 ℃ for 1 h. Phosphate concentration was determined using a phosphate assay kit (Abcam, UK), following manufacturer’s protocols, and incubated for 30 min at RT in the dark. Absorbance at 595 nm was measured using a Sunrise absorbance reader (TECAN, Switzerland).

Cellular myo-inositol levels

Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. The following day, cells were treated with 25 µM ebselen, 30 mM LiCl, or 0.2% DMSO as the untreated control for 48 h. Cells were washed three times with phosphate-buffer saline (PBS) and collected by trypsinization. After cell counting, they were centrifuged, and the PBS was removed Cells were lysed with ice-cold inositol assay buffer. Lysates were then centrifuged, and the obtained supernatants were used for myo-inositol quantification using the myo-inositol assay kit (ab252896, Abcam), following the manufacturer’s protocol.

Evaluation of the antiviral activity of ebselen and L690330 against various RNA viruses

Vero or imHC cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well. The following day, imHC cells were infected with DENV-2 or ZIKV at a multiplicity of infection (MOI) of 1. Vero cells were infected with CHIKV, EV71 or IAV at MOI of 0.0025, 0.02 and 1, respectively. Following the virus adsorption step, cells were maintained in 2% FBS media containing twofold serial dilutions of ebselen or L690,330 for 48 h, or for 24 h in the case of CHIKV infection. Medium containing 0.5% DMSO was used as the untreated control. Subsequently, viral supernatants were harvested and virus titers were determined. Percent inhibition was calculated relative to the virus titer of the infected untreated control. Each experiment was performed in triplicate.

IMPA1 silencing using SiRNA

imHC cells were seeded in a 24-well plate at a density of 6.5 × 10⁴ cells/well. The following day, cells were transfected with 25 nM of siRNA targeting IMPA1 (si_IMPA1) from the ON-TARGETplus Human IMPA1 siRNA pool (Horizon Discovery, UK) using DharmaFECT™ Transfection Reagents (Horizon Discovery, UK), according to the manufacturer’s protocol. Irrelevant siRNA (si_irrelevant) from the ON-TARGETplus non-targeting pool (D-001810-10-05, Horizon Discovery, UK) was transfected as a non-targeting control. Two days post-transfection, cells were either mock-infected or infected with DENV-2 at a MOI of 2 for 2 h. After removing the viral inoculum, cells were maintained in media containing either 20 µM ebselen or no ebselen for 1 day under both si_irrelevant and si_IMPA1 conditions. Viral supernatants were then collected for virus titer quantification, and cells were harvested for western blot analysis to assess the expression levels of IMPA1, DENV-2 NS1, and GAPDH, which served as an internal loading control.

Reversion of Ebselen antiviral activity by inositol or PI

imHC cells were inoculated with DENV-2 or ZIKV at an MOI of 1. Alternatively, Vero cells were inoculated with EV71, IAV, or CHIKV at MOIs of 0.02, 1, or 0.0025, respectively. After 2 h of viral inoculation, the inoculum was removed and cells were maintained in media containing various concentrations of ebselen, inositol, mixtures of ebselen and inositol, PI, or mixtures of ebselen and PI. Virus supernatants were collected at 24 h post-infection (hpi) for CHIKV, and at 48 hpi for DENV-2, ZIKV, EV71, and IAV, and were subjected to viral titer quantification.

Viral quantification

DENV-2 and ZIKV quantification by focus forming assay

The procedure was described elsewhere³⁵. Briefly, Vero cells were seeded in 96-well plates at a density of 4 × 10⁴ cells/well. The following day, cells were infected with 10-fold serially diluted virus supernatants for 2 h. Thereafter, cells were overlaid with overlay medium (1.5% carboxymethylcellulose in 2% FBS-MEM, with penicillin/streptomycin) and incubated at 37 °C with 5% CO₂ for 3 days for DENV-2 or 2 days for ZIKV. After incubation, the overlay medium was removed, and the cells were washed several times with PBS. Cells were then fixed with 3.7% (v/v) formaldehyde in PBS for 10 min at RT. Following fixation, cells were permeabilized using 2% (v/v) Triton-X-100/PBS for 10 min at RT, then washed with with PBS. The cells were probed with anti-flavivirus envelope protein antibody (4G2) and a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG H + L (Invitrogen, 62–522) in 0.5% (v/v) Tween-20/PBS. Each antibody incubation was performed for 1 h at 37 °C, with three PBS washes between steps to remove excess antibody. Subsequently, the chromogenic substrate [100 mM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl₂, 0.335 mg/ml Nitroblue Tetrazolium (NDB0379, BioBasic), and 0.165 mg/ml BCIP-T (R0821, Thermo Scientific)] was added and incubated for 10 min at RT in the dark. Excess substrate was removed by washing with tap water. Foci were then counted, and virus titers were calculated as focus-forming units per ml (ffu/ml).

EV71 and CHIKV quantifications by plaque assay

Vero cells were seeded in 24-well plates at a density of 1.5 × 10⁵ cells/well. The following day, cells were infected with 10-fold serially diluted EV71 or CHIKV virus supernatants for 2 h. Then, the inoculum was removed, and cells were overlaid with 1 ml of overlay medium (1.2% microcrystalline cellulose (Avicel, RC-591) in 2% FBS-MEM) and incubated at 37 °C with 5% CO₂ for 3 days for EV71 or 2 days for CHIKV. The overlay medium was removed, and the cells were fixed with 10% (v/v) formaldehyde in PBS for 1 h. Cells were stained with 1% (w/v) crystal violet in 20% (v/v) ethanol for 5 minutes and washed with tap water to remove excess dye. The plaques were counted and the virus titers were calculated as plaque-forming units per ml (pfu/ml).

Plaque assay for IAV

MDCK cells were seeded in 24-well plates at a density of 1.5 × 10⁵ cells/well. The following day, cells were washed once with serum-free MEM containing 1 µg/ml of TPCK-treated trypsin (MEM + TPCK). Then, they were infected with 10-fold serial dilutions of viral samples in MEM + TPCK for 1.5 h. Subsequently, the inoculum was discarded, and cells were overlaid with 1 ml of 1.2% microcrystalline cellulose (Avicel, RC-591) in MEM + TPCK, and incubated at 37 °C with 5% CO₂ for 2 days. The cells were fixed and stained as described earlier. The number of plaques was counted, and virus titers were determined as plaque-forming units per ml (pfu/ml).

Immunofluorescence assays

imHC cells were seeded in 24-well plates at a density of 7 × 10⁴ cells/well with coverslips. The following day, cells were infected with ZIKV at MOI 1 for 2 h. After removal of the inoculum, cells were further cultivated in 2% FBS media containing ebselen or 0.5% DMSO as the untreated control. Then, cells were fixed with 4% (v/v) formaldehyde in PBS for 10 min at 4°C at 48 hpi. Subsequently, cells were permeabilized with 0.2% Triton-X-100 in PBS for 15 min at RT. Cells were probed with a 1:50 dilution of mouse anti-PI4P IgM (Z-P004, Echelon Biosciences) and a 1:1000 dilution of rabbit anti-ZIKV-NS1 IgG (GTX133306, Genetex) and incubated at 37 °C for 1 h. Then, cells were incubated with secondary antibody-conjugated fluorescence dyes and a 1:2000 dilution of Hoechst dye and incubated at 37 °C for 1 h. The images were taken using a confocal microscope.

Western blot analysis

After removing the media, cells were washed with PBS and lysed in RIPA buffer [50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.1% Triton X-100; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS); 1 mM sodium orthovanadate; and 1 mM NaF]. Cell lysates were prepared by centrifugation at 12,000 × g for 10 min at 4 °C to remove debris. Protein concentrations were determined using the Bradford assay. Lysates (15 µg) were mixed with sample loading dye and reducing reagent, then loaded onto NuPAGE 4–12% Bis-Tris protein gels (1.0 mm; NP0322BOX, Thermo Fisher Scientific) and subjected to electrophoresis. Separated proteins were transferred onto nitrocellulose membranes (PALL). Membranes were blocked with 3% (w/v) skim milk in 0.1% (v/v) Tween-20/TBS for 1 h at room temperature, then probed overnight at 4 °C with continuous rocking using a 1:2000 dilution of rabbit anti-IMPA1 (AB184165, Abcam), a 1:2000 dilution of DENV-2-NS1 (PA5-32207, Thermo Scientific), or a 1:10,000 dilution of mouse anti-GAPDH (sc-47724, Santa Cruz Biotechnology). After washing, membranes were incubated for 2 h at room temperature with a 1:2000 dilution of HRP-conjugated goat anti-rabbit IgG or a 1:5000 dilution of HRP-conjugated rabbit anti-mouse IgG. Protein bands were visualized using Clarity Western ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA) and imaged with ImageQuant LAS 4010 (GE Healthcare, Chicago, IL, USA). Band intensities were quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA) and normalized to GAPDH expression levels. The expression levels of IMPA1 and DENV-2 NS1 were presented as relative expression levels: IMPA1 was compared with untransfected cells, and DENV-2 NS1 was compared with cells not treated with ebselen.

Statistical analysis

An independent samples t-test was used to compare differences between two groups (untreated control vs. treated, or ebselen alone vs. ebselen combined with inositol or PI) using SPSS Statistics software (SPSS, Inc., Chicago, IL, USA); p ≤ 0.05 was considered statistically significant. The 50% cytotoxic concentration (CC₅₀) and half-maximal inhibitory concentration (IC₅₀) values were calculated from dose-response curves of antiviral treatment by non-linear regression analysis using GraphPad Prism 10 (GraphPad Software, Inc., CA).