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

TL;DR

Monensin and salinomycin, ionophore antibiotics, show broad antiviral effects against enteroviruses by disrupting acidic conditions in cells.

Contribution

The study reveals that monensin and salinomycin inhibit enterovirus infection by neutralizing endolysosomal pH during viral entry.

Findings

Monensin and salinomycin reduce EV71 infection in a dose-dependent manner with IC50 values of 0.25 μM and 1.49 μM, respectively.

Both agents inhibit EV71 at the entry stage, independent of viral binding and internalization.

They neutralize acidic endolysosomal pH and block viral maturation, showing broad-spectrum antiviral activity against multiple enterovirus serotypes.

Abstract

Enteroviruses, which belong to the Picornaviridae family, are implicated in a variety of illnesses that range from mild to severe, with some infections potentially being life-threatening. Among these, Enterovirus 71 (EV71) is recognized as one of the most virulent members of the enterovirus genus. Currently, there are no effective treatments available for EV infections. Ionophore antibiotics are small, hydrophobic, and lipophilic molecules approved for use in veterinary medicine as anti-coccidial feed additives. Notably, ionophore antibiotics such as monensin (MON) and salinomycin (SAL) have shown antiviral activity against specific virus groups, although the modes of actions are not yet well understood. This study investigates the antiviral effects and mechanisms of MON and SAL against enteroviruses. Our findings reveal a dose-dependent reduction in EV71 infection, with 50% inhibitory concentrations of 0.25 μM for MON and 1.49 μM for SAL. Mechanistic investigations demonstrated that both agents primarily inhibit EV71 infection at the entry stage, independent of viral binding and internalization. Furthermore, these agents effectively neutralized low pH levels within endolysosomes, which was associated with a decrease in antiviral efficacy when acidic conditions were maintained in the medium. Additionally, both agents showed the ability to block viral maturation, which requires an acidic environment. The antiviral effects of MON and SAL were also observed against various serotypes of enteroviruses. In summary, MON and SAL exhibit antiviral efficacy by neutralizing endolysosomal pH during the viral entry process, suggesting a potential broad-spectrum antiviral strategy that could be applicable to other viruses with similar replication pathways.

Figures

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.

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 a

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

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

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 (NH_4_Cl) at 20 mM used as a control. After incubation for an additional 3 hours, cells were harvested with their proteins ext

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 me

Full text

Introduction

Enteroviruses (EVs) are classified within the Picornaviridae family and are characterized as non-enveloped, positive-sense RNA viruses. The Enterovirus genus includes several human pathogens that commonly circulate, such as rhinoviruses, coxsackieviruses (types A and B), echoviruses, and numbered enteroviruses. While the majority of EV infections are mild or asymptomatic [1], they can also lead to serious and potentially life-threatening conditions, including myocarditis, pancreatitis, meningitis, encephalitis, and acute paralysis. Among these viruses, EV71 (or EV-A71) is a notable cause of hand, foot, and mouth disease, particularly in young children and infants. A considerable proportion of individuals infected with EV71 may suffer from severe neurological complications, which can, in some instances, result in fatalities, especially within the Asia-Pacific region [2, 3]. Currently, vaccines are available to address poliovirus and EV71 infections [4, 5]; however, the development of vaccines for all EVs presents considerable challenges due to the extensive diversity of serotypes. Furthermore, there are no approved antiviral treatments for EV infections at present, underscoring the urgent need for broad-spectrum antiviral medications to effectively manage the diverse and potentially severe impacts of these viruses.

The life cycle of EVs begins with their specific binding to one or more cell surface receptors, thereby initiating the process of receptor-mediated endocytosis. This essential step varies according to the serotype of the virus and the particular host cell involved. Following the binding process, alterations in pH within the endosome facilitate trafficking, ultimately resulting in the release of the viral genome into the cytoplasm. Upon entering the cytosol, the positive-stranded viral genome serves as messenger RNA (mRNA) and undergoes translation to produce a polyprotein [6]. This translation process employs an internal ribosome entry site located in the 5' untranslated region of the viral genome, enabling efficient, cap-independent translation [7]. The ensuing polyprotein is subsequently cleaved by virus-encoded proteases, specifically 2 A and 3 C, generating essential structural proteins known as VP1-4, in addition to critical non-structural proteins, including proteases and polymerases. The replication of the viral genome is facilitated by the enzyme RNA-dependent RNA polymerase (3D) [8]. The newly synthesized positive-sense progeny viral RNAs are packaged into capsids, resulting in the formation of new virions. These virions are then released from the host cells through either lytic or non-lytic mechanisms [8].

Ionophore antibiotics are small molecules that exhibit low solubility in water (hydrophobicity) and a high affinity for lipid environments (lipophilicity). They selectively bind to metal cations, facilitating their transport across lipid bilayer membranes. Among the various types of ionophores, polyether ionophores are versatile carboxyl ionophores with potential antimicrobial properties [9]. Several members of this group have been approved by the U.S. Food and Drug Administration (FDA) for use in veterinary medicine as anti-coccidial feed additives. To date, only monensin (MON) and salinomycin (SAL) from Streptomyces species, and narasin and maduramicin from Actinomadura species, have been reported to possess antiviral activity [10]. Research has shown that MON and SAL exhibit antiviral activities against a range of viruses, including human cytomegalovirus (HCMV) [11], Herpes simplex virus-1 (HSV-1) [11–13], human immunodeficiency virus-1 (HIV-1) [14], influenza virus [15, 16], human coronavirus OC43 [17, 18] and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [17, 19, 20]. In contrast, narasin and maduramicin have only been shown to be active against SARS-CoV-2 [21]. Collectively, studies indicate that these compounds can interfere with several steps in the viral replication cycle. However, the precise antiviral mechanisms of the polyether ionophores are not currently understood.

In this study, we investigated the antiviral properties of two representative polyether ionophores, MON and SAL, focusing on their ability to inhibit EV infections. Our findings demonstrated that both MON and SAL have potent, broad-spectrum antiviral effects. Importantly, our data support the idea that MON and SAL displayed the antiviral efficacy by deacidifying the endolysosomes and the acidic cellular milieu, thereby targeting viral entry and viral maturation, respectively.

Materials and methods

Cells and viruses

The human rhabdomyosarcoma (RD) (ATCC, CCL-136) cells [22] were cultured in a minimum essential medium (MEM) (Gibco-BRL Inc.) containing 10% fetal bovine serum (FBS) and 100 μg/mL penicillin/streptomycin. The viral stocks analyzed in this study comprised EV71 (strain BrCr), coxsackievirus A16 (CVA16), coxsackievirus B3 (CVB3), and Echovirus 9 (Echo 9) [23]. These viruses were propagated in human rhabdomyosarcoma (RD) cells, which were maintained in a modified culture medium consisting of minimal essential medium (MEM) supplemented with 2% fetal bovine serum (FBS), henceforth referred to as MEM-2. To measure the viral titer, we prepared cell-associated viruses from cell lysates that were collected after three cycles of freezing and thawing, followed by centrifugation at 15,300×g for 10 minutes (min). Infectious titers were determined using a 50% tissue culture infectious dose (TCID₅₀) assay conducted on RD cells. This was achieved by analyzing clarified supernatants that contained cell-associated virus particles, according to the method described by Reed and Muench [24].

Chemicals and compounds

MON (CAS 22373-78-0), SAL (CAS 53003-10-4), and suramin (CAS 129-46-4) were purchased from Cayman Inc. and chloroquine (CAS 50–63-5) was obtained from Merck Inc.. All the compounds were dissolved in dimethyl sulfoxide (DMSO), with the final DMSO concentration in the culture medium staying below 0.05%, a threshold found to be non-cytotoxic across all cell lines evaluated. All compound-free controls were prepared with a concentration of 0.05% DMSO.

Cell viability assay

The cytotoxic effects of the test compounds were evaluated using a cell proliferation assay, as referenced in [25]. To achieve concentrations of 1, 3, 10, 30, and 100 μM, a series of dilutions of MON and SAL in culture medium was prepared. Next, 100 µL of each dilution was added to the corresponding wells of a 96-well plate containing RD cells, and the plate was incubated for 12 hours. Control wells were included: one containing only cells (untreated control) and another with only the medium (blank control). After the incubation period, 20 µL of CellTiter 96 AQueous One Solution (Promega) was added to each well, followed by an additional incubation of 3 hours. Absorbance was then measured at 490 nm using a microplate reader (SunriseTM, TECAN). The data were expressed as a percentage of the untreated control, with the blank control values subtracted from each test compound's readings. The 50% cytotoxicity concentration (CC_50_) was determined using GraphPad Prism 9 software.

Western blot

The Western blot procedure was adapted from a validated method [26]. To detect the EV71 VP1 and VP2 antigens, we used a mouse anti-EV71 antibody (Ab, 1:1000; MAB979, Chemicon) for VP1 and a rabbit anti-VP2 Ab (1:3000; GTX132340, GeneTex) for VP2 as the primary Abs. For the secondary Abs, we employed a horseradish peroxidase (HRP)-conjugated goat anti-mouse Ab (1:1000; sc-2060, Santa Cruz) for VP1 and an HRP-conjugated goat anti-rabbit Ab (1:3000; sc-2004, Santa Cruz) for VP2. Alpha tubulin served as a loading control and was detected using a rabbit anti-α tubulin primary Ab (1:1000; GTX112141, GeneTex) and an HRP-conjugated goat anti-rabbit secondary Ab (1:3000; sc-2004S, Santa Cruz,). Protein detection was achieved using an enhanced chemiluminescence Western blot kit (Amersham).

Time of addition assay

We followed a protocol reported in a previous study with some modifications [27]. The confluent monolayers of RD cells in 6-well plates were pretreated with MON, SAL, or DMSO for 1 hour (time −2–−1 hour). After washing with phosphate-buffered saline (PBS), the cells were inoculated with EV71 stocks at a multiplicity of infection (MOI) of 0.5 at 37 ℃ for 1 hour (time −1–0 hour). After viral adsorption, the cells were washed with PBS and cultured in MEM-2. MON, SAL, or DMSO was added at different time intervals to assess their effects on distinct stages of viral infection: (i) Full-time treatment (−2 to 8 hours): Cells were treated with the indicated compounds during the pretreatment phase, virus adsorption period, and continuously for 8 hours post-infection. (ii) Entry-phase treatment (−2 to 1 hour): Compounds were applied during the pretreatment and virus adsorption phases and maintained for 1 hour post-infection, after which the medium was replaced with compound-free MEM-2. (iii) Post-entry treatment (2 to 8 hours): No compounds were added before or during the virus adsorption phase. Cell treatments began 2 hours after infection and continued until 8 hours post-infection. At the end of each condition, cells were harvested for Western blotting analysis.

The binding and internalization assay

The assays for viral binding and entry were conducted according to previously reported procedures [27]. RD cells were pretreated with 5 or 10 μM of MON or SAL at 37 °C for 2 hours. To assess viral binding, cells were transferred to ice and incubated with binding buffer (PBS supplemented with 1% BSA and 0.1% sodium azide) for 10 min, followed by infection with EV71 at an MOI of 20 for 1 hour on ice. Unattached virions were removed by washing with cold PBS, and surface-bound virus was stripped using cold 0.25% trypsin. For the internalization assay, cells were warmed to 37 °C after virus adsorption to allow entry for 1 hour in the presence of MON or SAL. Subsequently, residual surface-bound virus was removed with 0.25% trypsin at 37 °C for 3 min. Total RNA was then extracted, and the levels of EV71 RNA were quantified using RT-qPCR.

Acidic organelle staining

This assay was adapted from a previously reported method [28, 29]. RD cells were pre-incubated with MON or SAL at 37 °C for 2 hours. Subsequently, the cells were treated with LysoTracker DND-99 at 150 nM (catalog number L7528, Thermo Fisher Inc.), maintaining the same concentration of the compounds for an additional hour, also at 37°C. Following the staining procedure, the cells were subjected to three washes with PBS and subsequently fixed in 500 μL of 4% paraformaldehyde. Cells grown on glass coverslips were mounted with glycerol mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI). The coverslips were sealed and the samples were examined with a fluorescence microscope (DM6000B, Leica Microsystems) using an excitation wavelength of 545 ± 30 nm and an emission filter of 610 ± 75 nm. Fluorescence intensities were quantified using ImageJ software (NIH, USA).

The low-pH exposure assay

This experiment was conducted according to a previously established method [27]. Cells were pretreated with culture medium containing 5 μM of either MON or SAL at 37 °C for 1 hour. Following the pretreatment, EV71 was introduced at an MOI of 0.5, and virus adsorption was performed on ice while maintaining the presence of 5 μM MON or SAL. Subsequent to the binding of the virus, the inoculum was discarded, and any unbound virus was removed through washing with ice-cold PBS. The cells were then incubated at 37 °C for 1 hour in fresh medium containing either MON or SAL. Subsequently, the supernatant was discarded, and the media adjusted to varying pH levels (7.4, 6.5, 5.5, or 5.0) containing the compounds were applied for 10 min. Finally, the media were removed, and cells were cultured in pH 7.4 medium supplemented with 5 μM MON or SAL at 37 °C for 8 hours.

RNA extraction and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)

RNA preparation and RT-qPCR were carried out based on a previously published protocol [25]. Total RNA, including both cellular and viral RNA, was extracted using TRIzol reagent (Invitrogen). Reverse transcription was conducted with AMV Reverse Transcriptase XL (Takara), and quantitative PCR was carried out using the FastStart Universal SYBR Green Master Kit (Roche Applied Science), following the manufacturer's instructions. Primer sets targeting the VP1 region of the EV71 (BrCr strain) genome and human β-actin (used as an internal reference) were obtained from a prior study [30].

Immunofluorescence assay

Immunofluorescence staining was performed as previously described [25]. RD cells were cultured in 24-well plates and treated with 4% paraformaldehyde for fixation, followed by permeabilization using 0.2% Triton X-100. To detect various enterovirus serotypes, the following primary Abs were employed: mouse anti-EV71 Ab (1:1000; MAB979, Merck Inc., Kenilworth, NJ, USA) for EV71 and CVA16 detection; mouse anti-coxsackievirus B group Ab (1:1000; MAB9410, Merck) for CVB3; and mouse anti-echovirus group Ab (1:1000; MAB9670, Merck) for Echo9. A fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary Ab (1:100; Jackson ImmunoResearch) was used as a secondary Ab. Nuclear staining was performed using DAPI at a concentration of 0.1 μg/mL (Merck Inc., Kenilworth, NJ, USA). Fluorescence microscopy was performed using a Leica microscope (DM6000B), equipped with FITC and UV filter sets. Quantification of EV antigen-positive and DAPI-stained nuclei was conducted by counting cells in at least five randomly selected fields for each experiment, utilizing MetaMorph software (Molecular Devices, Nashville, TN, USA).

Results

Chemical structures of MON and SAL and their antiviral potency

The chemical structures of polyether ionophore antibiotics, such as MON and SAL (Fig. 1A and 1B), consist of carboxylic acids, multiple cyclic ethers, and various oxygen-containing groups. We evaluated the anti-EV71 effectiveness of MON and SAL using Western blot analysis at specific concentrations in RD cells. Treatment with either compound resulted in a dose-dependent decrease in viral protein levels (Fig. 1C and 1D). To assess cytotoxicity, we conducted a cell viability assay to determine the CC_50_ of the test compounds. The results revealed that the CC_50_ values were 34.96 μM for MON (Fig. 2A) and 41.92 μM for SAL (Fig. 2B). It was noted that the treatments with concentrations of 10 μM or lower resulted in undetectable cytotoxicity, indicating that the antiviral effects at 10 μM or lower are independent of compounds’ cytotoxicity. Moreover, we measured viral titers in response to MON and SAL treatments using the TCID_50_ assay, showing 50% inhibitory concentration (IC₅₀) values of 0.25 μM for MON and 1.49 μM for SAL (Fig. 2C and 2D). The selectivity index (SI), calculated as CC_50_ divided by IC_50_ (SI = CC_50_/IC_50_), for MON and SAL was obtained, with the values at 139.84 and 28.13, respectively (Fig. 2C and 2D).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 tubulinFig. 2Cytotoxicity 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. IC_50_ and CC_50_ values were calculated using GraphPad Prism9 software. The SI value was calculated by value of CC_50_ divided by that of IC_50_. Data represented the means of triplicated experiments and the standard error of the mean (SEM). Compound-free wells contained DMSO

MON and SAL targeted the viral entry, independent of viral binding and internalization

To identify which stage of the EV71 life cycle is influenced by MON and SAL, a time-of-addition assay was performed over an 8-hour infection period (Fig. 3A). RD cells were treated with MON or SAL at concentrations of 3 μM and 10 μM at various time points: (a) 1 hour before virus adsorption, throughout the 1-hour adsorption period, and continuing for the entire 8-hour infection (whole treatment); (b) from 1 hour before to 2 hours after virus adsorption, after which the compounds were removed (entry phase); or (c) starting 2 hours post-adsorption and continuing for the remainder of the 8-hour infection (post-entry). Western blot analysis of viral protein levels indicated that both MON and SAL, at a concentration of 3 μM, as well as SAL at 10 μM, primarily exerted their antiviral effects during the entry stage of the viral lifecycle. However, treatment with 10 μM of MON demonstrated strong inhibition at both the entry and post-entry stages. This enhanced inhibition may be attributed to its excessively high concentration, which is approximately 40 times greater than its IC_50_ value (Fig. 3B and 3C). To understand how MON and SAL affect various stages of EV71 entry, we investigated their effects on virus binding and internalization. In our binding assay, cells were incubated with the virus at ice temperature for 1 hour, both with and without the compounds. This condition allows the virus to attach to the cells but prevents internalization. We then initiated internalization by shifting the temperature to 37 °C for another hour, which facilitates the uptake of virus that was bound to the cell surface. Any virions that had not been internalized were removed using trypsin treatment. Suramin, a known inhibitor of EV71 binding and internalization, was used as a control at effective concentrations for the assays [23]. We quantified viral RNA levels using RT-qPCR for each condition. Treatment with suramin reduced viral RNA levels in a dose-dependent manner during both binding and internalization. In contrast, there were no significant differences in viral RNA levels between the MON- or SAL-treated groups and the untreated group, indicating that neither MON nor SAL interferes with viral binding or internalization (Fig. 3D and 3E).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 bound to the cell surface. For the internalization assay, after the initial virus adsorption on ice, the cells were incubated at 37 °C for an additional hour to enable viral internalization. The cells were then washed with PBS and treated with trypsin again to eliminate any remaining virus on the cell surface. Suramin (SUR) at concentrations of 40 μM and 80 μM was used as a control in both the binding and internalization assays. Finally, the viral RNA levels from both assays were quantified using RT-qPCR. RNA level was normalized with human β-actin as an internal control. The results are shown as relative levels compared to a compound-free control containing 0.05% DMSO. These data were obtained from three independent experiments. ns= not significance

MON and SAL neutralized the pH-dependent virus entry

MON and SAL are monovalent cation ionophores that disrupt intracellular ion gradients by exchanging Na⁺ or K⁺ for H⁺ across biological membranes. This process results in the dissipation of the proton gradient in acidic organelles [31, 32], such as lysosomes, which is essential for viral entry. Therefore, we investigated whether treatment with MON or SAL resulted in an elevation of pH in acidic cellular compartments in RD cells. These compartments were labeled with LysoTracker dye, which emits red fluorescence exclusively in acidic environments, such as endolysosomes. Chloroquine (CQ), a known inhibitor of endosomal acidification [33], was used as a control. The results showed that compared to the untreated control group, there were significant decreases in fluorescence intensity after treatment with MON or SAL, in a dose-dependent manner (Fig. 4). Since endolysosomal acidification is crucial for virus entry, this suggests that MON and SAL could obstruct EV71 entry by alkalizing the endolysosome.Fig. 4MON 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)

To test this, RD cells were exposed to acidic pH media following viral attachment. The cells were pretreated with MON or SAL before being inoculated with EV71 stock while kept on ice. After 1 hour of incubation, the viral inoculum was removed, and unbound particles were washed away. Viral internalization was initiated by incubating the cells at 37 °C with the respective compounds for an additional hour. Subsequently, the cells were exposed for 10 min to compound-containing media adjusted to different pH levels (7.4, 6.5, 5.5, or 5.0), followed by an 8-hour recovery period in pH 7.4 medium still containing the compound. Cell lysates were then collected for Western blot analysis to quantify viral protein levels at 8 hours post-infection (Fig. 5A). We observed pronounced increases in viral protein expression when cells were exposed to an acidic medium at pH 5, in conjunction with treatment using MON or SAL. In contrast, only minor increases in viral protein expression were noted at pH 6.5 (Fig. 5B). This suggests that early endosomes, which have a pH of 6.0 to 6.5, are not the primary site of viral uncoating. These findings align with our earlier observation that treatment with MON and SAL raised the pH of acidic compartments (Fig. 4). Together, these results support the conclusion that these ionophores inhibit EV71 entry by targeting a low pH-dependent step, likely involving endolysosomal acidification.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

Blockade of the viral maturation by MON and SAL

During the late stages of poliovirus infection, the cleavage of the viral structural protein VP0 into VP2 and VP4 is critical for the maturation of the virus and the subsequent production of progeny virions. This cleavage occurs following the assembly of the genome-containing virion in acidic cellular vesicles. It is important to note that if the acidification of cellular vesicles is inhibited, this essential cleavage process is hindered [34]. We next investigated if MON or SAL block viral maturation in the acidic environment. MON or SAL at the indicated concentrations were used to treat RD cells at 9 hours post-infection for 3 hours, and the viral proteins VP0 and its cleavage product VP2 were analyzed using Western blot analysis (Fig. 6). Ammonium chloride (NH_4_Cl), a weak base that is taken up by intracellular vesicles, was used as a control to assess acidity inhibition. Our results showed that the generation of VP2 was progressively reduced with increasing concentrations of MON or SAL. This suggests that MON and SAL likely block acid-dependent viral maturation.Fig. 6MON 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 (NH_4_Cl) 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

Antiviral spectrum

We further evaluated the antiviral activity of MON and SAL against a range of enterovirus serotypes, including CVA16, CVB3, Echo 9, and EV71, using an immunofluorescence assay (Fig. 7). Both compounds demonstrated antiviral activity across these EV serotypes, albeit to varying extents. For MON, EC_50_s ranged from 0.189 μM to 0.403 μM, with SIs ranging from 86.75 to 184.97. In contrast, SAL showed EC_50_s ranging from 1.823 μM to 2.463 μM and SIs between 17.02 and 23.00. The data indicate that both compounds exhibit broad-spectrum antiviral activity across all tested serotypes, with MON demonstrating a wider therapeutic window compared to SAL. These findings suggest that cellular processes such as acidic endolysosomal activity and acidic cellular environments are critical common pathways that EVs exploit.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). IC_50_ values were calculated using GraphPad Prism9 software. The SI value was calculated by value of CC_50_ divided by that of IC_50_

Discussion

EVs from various serotypes present significant global health challenges, especially among immunocompromised adults and pediatric populations. Among these, EV71 is particularly noted for causing severe neurological complications in infants and young children across the Asia-Pacific region. At present, there are no approved antiviral agents available for the treatment of EV infections. Since EV-related diseases are caused by multiple serotypes, there is an urgent need for broad-spectrum antiviral strategies. In this study, we evaluated the antiviral activity of the polyether ionophores MON and SAL against multiple EV serotypes. Both compounds demonstrated dose-dependent inhibition of EV71 infection and exhibited high SI values, with MON achieving an SI of 139.84 and SAL reaching 28.13 (Fig. 1 and 2). High SI values were consistently observed in antiviral activities against all tested serotypes, with MON exhibiting lower IC_50_ values and higher SI values compared to SAL (Fig. 7). These findings indicate that their antiviral activities were specific, effective, and independent of cytotoxic effects. A mechanistic study has revealed that MON and SAL primarily exert their antiviral effects at the entry stage of the virus (Fig. 3B and 3 C), without significantly influencing virus binding or internalization (Fig. 3D and 3E). This effect is, at least in part, due to the compounds’ ability to neutralize the low pH levels present in endolysosomal vesicles (Fig. 4). Notably, the restoration of viral infection was observed in cells pretreated with MON or SAL when the medium was supplemented with acidic conditions (Fig. 5). Furthermore, the antiviral activity is associated with the disruption of the acidic environment necessary for the cleavage of VP0, which subsequently produces VP2 and VP4 later in the infection process (9 hours post-infection, Fig. 6). All antiviral measurements in this study were conducted using RD cell line. However, both MON and SAL have shown antiviral activity against certain other EVs in different cell types, such as poliovirus in HeLa cells [35] and enterovirus G in Marc-145 cells [36]. Although the specific mechanisms behind their antiviral actions were not investigated in these studies, this suggests that the observed antiviral potency in our study is not dependent on the type of cell used. Collectively, these findings elucidate a mechanism through which MON and SAL neutralize pH levels within the endolysosomal pathway and acidic vesicles, thereby playing a crucial role in both viral entry and the maturation of EVs.

MON and SAL have been known for their ability to demonstrate multiple antiviral mechanisms against a range of viruses. Studies indicate that they can effectively suppress infections caused by SARS-CoV-2, coronavirus OC43, HSV-1, HIV-1, and influenza through the inhibition of intracellular protein transport and glycoprotein processing of viral envelope proteins [12–17]. However, the antiviral mechanism is likely independent of EVs, as they are non-enveloped viruses. Moreover, SAL disrupts endosomal acidification by inactivating the proton transport function of the M2 protein of influenza virus [15]. MON exhibits antiviral activity against HSV-1 and HCMV by targeting viral replication; however, the precise mechanisms involved remain to be fully elucidated [11]. Furthermore, SAL is shown to impede both the entry and replication of SARS-CoV-2 [19], although the specific mechanisms behind this action are largely still unknown.

Polyether ionophores, such as MON and SAL, are recognized for their distinctive structural and physicochemical properties, which enable the selective binding of metal cations, mainly sodium and potassium, and their efficient transport across cell membranes. Within the cellular environment, these cations are released from their binding with the ionophore, thereby disrupting the established concentration gradient of cations. This influx of cations induces the efflux of protons, leading to the alkalinization of acidic vesicles [37, 38]. It is proposed that this process may elucidate the mechanisms by which MON and SAL contribute to the pH-elevation of the endolysosomal system (Fig. 4) and acidic vesicles (Fig. 6), key sites for viral entry and maturation, respectively. To our knowledge, this is the only report that indicates MON and SAL display anti-EV effect targeting both viral entry and viral maturation by causing deacidification.

MON is known for its diverse chemotherapeutic potential against various infectious agents. The repurposing of MON as an antiviral agent for human use may be an effective strategy to ameliorate outbreaks of emerging viruses that currently lack vaccines and approved treatments. In parallel, SAL has been utilized in clinical trials for the treatment of different types of cancer by targeting cancer stem cells. SAL demonstrated partial tumor regression without significant side effects [39]. Only two studies have investigated the antiviral activity of MON and SAL in mice. Oral administration of MON slightly reduced the levels of RNA and the titer of the Zika virus on day one; however, this effect was not sustained [40]. Similarly, oral SAL treatment did not improve survival rates in mice infected with influenza [15]. Using suitable nanocarriers for the delivery of MON and SAL could enhance their therapeutic efficacy by improving solubility, stability, and biocompatibility, while also reducing non-specific toxicity [41]. In a landscape where therapeutic options are limited, harnessing the alkalizing effects and host-targeting properties of both MON and SAL could provide a promising approach to treating EV infections. Further preclinical studies are crucial to thoroughly assess the therapeutic potential of MON and SAL on antiviral activity for future clinical applications.