Monensin and salinomycin exhibit broad-spectrum antiviral activity against enteroviruses by disrupting lysosomal acidification

Figures

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  Fig. 1. Dose-dependent decreases in EV71 protein levels by MON and SAL. Chemical structures of MON (A) and SAL (B), two polyether ionophores used in this study. RD cells were pretreated for 1 hour with MON (C) or SAL (D) at concentrations of 0.1, 0.3, 1, 3, and 10 μM, and then infected with EV71 at an MOI of 0.5 for 1 hour. Following PBS wash, the cells were maintained in media containing the compounds for an additional 8 hours. Cell lysates were prepared and analyzed by Western blot using anti-EV71 VP1 Ab and anti-α tubulin Ab as an internal control. The band intensities were quantified using ImageJ. The percentages shown below each lane represent the intensity of viral VP1 protein relative to that of α tubulin

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  Fig. 2. Cytotoxicity and antiviral activity of MON and SAL. Compound cytotoxicity was determined by treating RD cells with MON (A) or SAL (B) at 1, 3, 10, 30, and 100 μM for 12 hours. Cell viability was measured using a cell proliferation assay and expressed as the percentage of compound-free (DMSO-treated) cells. RD cells were pretreated with MON or SAL at the concentrations indicated for 1 hour, followed by infection with EV71 at an MOI of 0.5 for 8 hours, with test compounds maintained throughout the infection. Titers of cell-associated EV71 from cells treated with MON (C) or SAL (D) were measured by the TCID₅₀ assay. IC50 and CC50 values were calculated using GraphPad Prism9 software. The SI value was calculated by value of CC50 divided by that of IC50. Data represented the means of triplicated experiments and the standard error of the mean (SEM). Compound-free wells contained DMSO

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  Fig. 3. Timing of EV71 inhibition by MON and SAL. A time-of-addition assay was conducted, as illustrated in the schematic representation (A). MON or SAL was added to RD cells at specified times relative to viral infection, with an MOI of 0.5. The initiation of viral adsorption is indicated at −1 hour and its completion at 0 hour. The solid and dashed lines represent the periods with and without compound treatment, respectively. The treatment windows are categorized as follows: Whole (W): −2 to 8 hours; Entry (En): −2 to 1 hours; Post-Entry (P-En): 2 to 8 hours. Western blot analysis was performed to assess viral VP1 expression in RD cells treated with either 3 or 10 μM of MON (B) or SAL (C) at the indicated times. For the binding (D) and internalization assays (E), RD cells were pretreated with MON or SAL at 5 μM or 10 μM at 37 °C for 2 hours. Following this, the cells were placed on ice and treated with binding buffer for 10 min. Next, 20 MOI of EV71 was added, and the mixture was incubated on ice for 1 hour to allow the virus to adsorb onto the cells. For the binding assay, unbound virus was washed off with cold-PBS, and the cells were treated with trypsin to remove any virus that was…

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  Fig. 4. MON and SAL demonstrated alkalizing effects in cellular vesicles. RD cells were treated with MON or SAL at concentrations of 1, 2, 5, or 10 μM for 2 hours, followed by staining with 150 nM LysoTracker Red (LTR) for 1 hour. Two control groups were included: one without LysoTracker treatment (LTR−) and another that received only LysoTracker treatment (LTR+) without the treatment of either test compound. Treatment of chloroquine at 10 μM was used as a control. Fluorescence microscopy was employed to capture representative images of the LTR signal (n=5) under each condition (A). The corresponding quantification of mean fluorescence intensity was conducted using ImageJ software and is presented as a bar graph (B)

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  Fig. 5. The exposure of RD cells to low pH conditions reduced the antiviral activity of MON and SAL. The low-pH exposure assay is illustrated in the schematic representation below (A). In this assay, RD cells were pretreated with MON or SAL at 5 μM for 1 hour. After pretreatment, the cells were inoculated with the EV71 virus at an MOI of 0.5 while still on ice for another hour, in the presence of MON or SAL at 5 μM. The supernatant, which contained unbound virus, was then removed, and the cells were incubated with medium containing the corresponding compounds at 37 °C for 1 hour. Next, the cells were exposed to media with pH values of 7.4, 6.5, 5.5, and 5.0, each containing MON or SAL at 5 μM for 10 min. Following this exposure, the media was replaced with pH 7.4 medium that contained the corresponding compounds. After 8-hour of incubation with the different pH media, the levels of viral VP1 protein and tubulin, which served as an internal control, were measured using Western blot analysis, with representative blots shown (B). The percentages displayed beneath each lane represent the intensity of viral VP1 in relation to the intensity of tubulin

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  Fig. 6. MON and SAL suppressed EV71 at the virus maturation stage. (A) Schematic representation of the experimental procedure for assessing the inhibition of EV71 maturation. RD cells were infected with EV71 at an MOI of 0.5 at −1 hour. At 0 hour post-infection, cells were washed with PBS and replaced with maintenance medium. At 9 hours post-infection, MON or SAL (B) at the concentrations indicated were added to the infected RD cell cultures, with a weak base ammonium chloride (NH4Cl) at 20 mM used as a control. After incubation for an additional 3 hours, cells were harvested with their proteins extracted and subjected to Western blot analysis using anti-VP2 and anti-α tubulin Abs. The percentages shown below each lane represent the intensity of viral VP2 relative to that of VP0, compared with that of α tubulin

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  Fig. 7. Antiviral activity of MON and SAL against various EV serotypes. RD cells were pretreated with MON or SAL for 1 hour. Subsequently, cells were infected with EV71, CVB3, CVA16, and Echo 9 stocks at an MOI of 0.5 for 12 hours, with the corresponding concentration of MON (A-D) and SAL (E-H) maintained throughout the infection. The IFA was conducted using the aforementioned protocol. The percentage of infection from EV71-infected cells treated with medium containing 0.05% DMSO and that from mock-infected cells were arbitrarily set as 0% and 100% inhibition rates, respectively. The graphs present mean values along with standard deviations (n = 3). IC50 values were calculated using GraphPad Prism9 software. The SI value was calculated by value of CC50 divided by that of IC50