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INTRODUCTION
The genus Enterovirus, within the family Picornaviridae, comprises 15 species (Enterovirus A–L and Rhinovirus A–C), encompassing more than 200 serotypes. Among these, group B coxsackieviruses (CVBs) include six distinct serotypes, designated CVB1 through CVB6. CVBs are characterized as small (approximately 30 nm), non-enveloped, single-stranded positive-sense RNA viruses possessing a genome of approximately 7.4 kb in length. CVBs exhibit high infectivity and are predominantly transmitted via fecal-oral and respiratory routes, infecting individuals across all age groups. Following infection, CVBs initially replicate within the gastrointestinal tract and subsequently disseminate systemically, potentially leading to widespread infection. Clinical manifestations associated with CVB infection range from mild illnesses, such as fever, hand-foot-and-mouth disease (HFMD), and upper respiratory tract infections (URTIs), to severe complications, including viral encephalitis, aseptic meningitis, myelitis, myocarditis, pancreatitis, and occasionally fatal outcomes. Notably, CVBs exhibit a marked tropism for pancreatic acinar cells, leading to pancreatitis with variable severity, ranging from mild inflammation to severe pancreatic injury (1, 2, 3).
Autophagy is an evolutionarily conserved biological process that maintains cellular homeostasis by degrading damaged organelles and proteins, playing a critical role in immune responses including pathogen elimination. Autophagy initiates with the sequestration of cytoplasmic components within a double-membrane structure known as the phagophore, which subsequently matures into an autophagosome. The autophagosome then fuses with a lysosome to form an autolysosome, wherein the enclosed contents are degraded by lysosomal enzymes. Autophagy is induced under various physiological conditions, such as nutrient starvation, cellular differentiation, development, and stress responses. As a defense mechanism against viral infections, autophagy can target cytoplasmic viral particles or viral components for degradation by delivering them to lysosomes. However, certain viruses have been reported to suppress or evade autophagy, thereby neutralizing this host defense mechanism. Furthermore, other viruses have evolved strategies to manipulate or exploit the autophagic machinery, creating favorable environments for viral replication and enabling evasion of host immune responses (4, 5, 6, 7, 8, 9,10).
Cycloheximide, a known inhibitor of protein synthesis, has been reported to suppress protein degradation pathways, including autophagy. Furthermore, cycloheximide elevates intracellular amino acid levels, thereby activating mTORC1 signaling, which serves as a central negative regulator of autophagy (11). Given that cycloheximide may influence autophagy through either the blockade of de novo protein synthesis or the modulation of mTORC1 signaling, the present study aimed to elucidate the antiviral effects of cycloheximide on CVB3 infection by assessing its capacity to inhibit autophagy via mTORC1 activation.
MATERIALS AND METHODS
Cell culture, viruses, and reagents
The coxsackievirus B1 (CVB1), coxsackievirus B2 (CVB2), coxsackievirus B3 (CVB3), coxsackievirus B4 (CVB4), coxsackievirus B5 (CVB5) and coxsackievirus B6 (CVB6) were obtained from the division of vaccine research of the Korea Center Disease Control and prevention, and was propagated at 37 °C in Vero cells (ATCC, Manassas, VA, USA), which are kidney epithelial cells that originated from an African green monkey. Vero cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. MEM, FBS, trypsin-EDTA, and antibiotic-antimycotic solution were purchased from Gibco BRL (Invitrogen Life Technologies, Karlsruhe, Germany). Tissue culture plates were purchased from Falcon (BD Biosciences, San Jose, CA, USA). Cycloheximide and rupintrivir were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Sulforhodamine B (SRB) assays
Assays of antiviral activity and cytotoxicity were evaluated by the sulforhodamine (SRB) method using the cytopathic effect (CPE) induced by viral infection as recently reported (12). A day prior to infection, Vero cells were seeded at a density of 3 × 104 cells per well in a 96-well culture plate. The following day, the culture medium was removed, and cells were washed with 1× phosphate-buffered saline (PBS) (Corning Incorporated, New York, USA). Viral infectivity was evaluated by monitoring the virus-induced cytopathic effect (CPE), which was assessed by measuring cell viability. After inoculating cells with 1 × 10³ PFU of CVB3, the number of intact cells remaining attached to the plate was quantified. The antiviral drug candidates were added to each well at the indicated time points and the cytopathic effect was monitored at 48 h post-infection. The absorbance of SRB in each well was read at 540 nm using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) and a reference absorbance of 620 nm. The antiviral activity of each test compound in CVB3-infected cells was calculated as a percentage of the corresponding maximum survival of non-infected cells after normalization with untreated infected control cells.
Quantitative RT-PCR (RT-qPCR)
Total RNA was extracted from Vero cells using a QIAamp® viral RNA mini kit (Qiagen, California, USA). Taqman real-time PCR and reverse transcription PCR were carried out using AgPath-ID™ One-Step RT-PCR Reagents (Applied Biosystems, California, USA) and a Bio-Rad CFX96 thermal cycler (Bio-Rad, California, USA). The CVB 5′ noncoding region (NCR) of the gene was detected using qRT-PCR. The following CVB 5’NCR primers were used: forward primer 5′-GCGATTGTCACCATWAGCAGYCA-3,’ reverse primer 5′-GGCCCCTGAATGCGGCTAATCC-3,’ and probe primer 5′-CCGACTACTTTGGGWGTCCGTGT-3’. The following GAPDH primers were used: forward primer 5′-GGTCTCCTCTGACTTCAACA-3′, reverse primer 5′-AGCCAAATTCGTTGTCATAC-3′, and probe primer 5′-CCCTCAACGACCACTTTGTCAAG-3′. The cycling conditions were as follows: heating at 45 °C for 10 min for reverse transcription, reverse transcription inactivation and initial denaturation at 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s and at 62 °C for 45 s. The results were analyzed using the real-time system AB 7900HT software (Thermo Fisher Scientific, Waltham, MA, USA) and all values were normalized to GAPDH levels. A Bio-Rad CFX96 thermal cycler was used at the Core Facility for Innovative Cancer Drug Discovery (CFICDD) at Kangwon National University.
Western blot
Total cellular proteins were extracted using 200 μL of PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea) and subjected to sonication. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions. For Western blot analysis, 20–30 μg of total protein was mixed with 2× SDS sample buffer, boiled at 95 °C for 5 minutes, and then separated by electrophoresis on a 10% SDS-PAGE gel, followed by transfer to a membrane. Protein levels were evaluated in the lysate of CVB-infected veroa cells with primary antibodies including: LC3B: 2775S, mTORC1: 2972S, P-mTORC1: 2971S, S6: 2217S (Cell Signaling Technologies, Danvers, MA, USA), anti-rabbit Cytoskeletal Actin Antibody: A300-491A (Bethyl Laboratories, Montgomery, TX, USA) and secondary antibodies including: goat anti-mouse IgG F(ab')2, polyclonal antibody (HRP conjugate) (Enzo Life Sciences., Farmingdale, NY, USA) and anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technologies). The elevated chemi-luminescence substrate used was femtoLUCENT™ PLUS HRP Kit (G-biosciences, St. Luise, MO, USA). Images were obtained with ImegeQuantTM LAS 4000 mini system (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) and analyzed using Image J software (NIH, Bethesda, MD, USA).
RESULTS
Cycloheximide Inhibits CVB3-Induced Cytopathic Effects and Viral Replication in Vero Cells
To evaluate the antiviral activity of cycloheximide against coxsackievirus B3 (CVB3), cell viability was assessed using the sulforhodamine B (SRB) assay in Vero cells. Cycloheximide was tested at concentrations of 2, 0.4, 0.08, and 0.016 μM. Treatment with cycloheximide effectively suppressed CVB3-induced cytopathic effects by more than 80% at concentrations up to 0.4 μM, demonstrating antiviral activity comparable to that of the positive control, rupintrivir (Fig. 1A). Cytotoxicity evaluation in uninfected Vero cells indicated that cycloheximide did not exhibit cytotoxicity at concentrations up to 2 μM (Fig. 1B). To further investigate whether cycloheximide inhibits CVB3 replication, real-time PCR analysis was performed. Vero cells were infected with CVB3 and treated with cycloheximide at concentrations of 10, 2, 0.4, and 0.08 μM. After 24 h, infection was terminated, and viral RNA was extracted from the cells. RT-qPCR results showed that CVB3 RNA was undetectable even at the lowest tested concentration of 0.08 μM (Fig. 1C), indicating effective inhibition of viral replication. Subsequently, we assessed viral protein expression using western blot analysis. Vero cells infected with CVB3 were treated with cycloheximide at concentrations of 2, 0.4, and 0.08 μM. After 24 h, viral infection was terminated, and the expression of the viral capsid protein VP3 was analyzed. Cycloheximide treatment completely inhibited VP3 expressions at concentrations as low as 0.08 μM (Fig. 1D).
Fig. 1
Antiviral activity of cycloheximide against CVB3 in vitro. Vero cells were infected with 1 × 10³ PFU of CVB3 and treated with cycloheximide. (A, B) Cell viability and cytotoxicity following cycloheximide treatment were assessed using the sulforhodamine B (SRB) assay. (C) Relative expression of the CVB3 5′ noncoding region (NCR) gene in CVB3-infected Vero cells was quantified by real-time PCR. (D) Western blot analysis of VP3 protein expression in CVB3-infected cells treated with vehicle or cycloheximide at concentrations of 0.08, 0.4, and 2 µM for 24 h. Band intensities were quantified, and the ratio of VP3 to β-actin was calculated. Rupintrivir (2 µM) was used as a positive control.
Cycloheximide Suppresses CVB3-Induced Autophagy via Activation of mTORC1 Signaling
Some RNA viruses are known to induce autophagosome formation to facilitate viral genome replication (13). Cycloheximide has been reported to regulate autophagy by activating mTORC1, a master negative regulator of autophagy (11). Based on this, we investigated whether cycloheximide modulates autophagic activity during CVB3 infection. Vero cells were exposed to CVB3, and autophagy was evaluated 8 h and 24 h after infection. CVB3 infection led to a slight increase in LC3 expression, indicating autophagy activation. However, treatment with cycloheximide at concentrations of 10, 2, and 0.4 μM resulted in a dose-dependent reduction in LC3 levels, suggesting suppression of autophagy (Fig. 2). To elucidate the mechanism by which cycloheximide suppresses autophagy, we investigated the activation status of mTORC1, a key negative regulator of the autophagy pathway. Vero cells infected with CVB3 were treated with 10 μM cycloheximide, and the phosphorylation level of mTORC1 was assessed 8 h post-infection as an indicator of mTORC1 activation. The results demonstrated that 10 μM cycloheximide increased the phosphorylation of mTORC1 in CVB3-infected Vero cells, suggesting enhanced mTORC1 signaling (Fig. 3). These findings indicate that cycloheximide suppresses CVB3-induced autophagy by activating the mTORC1 signaling pathway, thereby contributing to its antiviral activity.
Fig. 2
Analysis of LC3 expression following cycloheximide treatment during CVB3 infection. Representative immunoblot results showing the expression of LC3-II and β-actin in Vero cells that were either infected or uninfected with CVB3 and treated with cycloheximide. Vero cells were infected with CVB3 and subsequently treated with cycloheximide at concentrations of 0.4, 2, and 10 μM. Autophagy levels were evaluated at 8 h (A) and 24 h (B) post-infection. All samples were derived from the same experiment, and the gels/blots were processed in parallel. The ratio of LC3-II to β-actin was calculated based on densitometric analysis of the immunoblot bands.
Fig. 3
Analysis of mTORC1 and phospho-mTORC1 expression following cycloheximide treatment in CVB3-infected Vero cells. Vero cells were infected with CVB3, and 10 μM cycloheximide was administered 8 h post-infection. mTORC1 and phospho-mTORC1 (p-mTORC1) levels were assessed by immunoblotting. All samples were derived from the same experiment, and the gels/blots were processed in parallel. The bar graphs show the densitometric quantification of mTORC1 and p-mTORC1 levels normalized to β-actin, as well as the ratio of p-mTORC1 to total mTORC1.
Broad-Spectrum Antiviral Activity of Cycloheximide Against Coxsackievirus B Serotypes
To evaluate whether cycloheximide exerts antiviral effects against other members of the coxsackievirus B (CVB) group, Vero cells were infected with coxsackievirus B1 (CVB1), B2 (CVB2), B4 (CVB4), B5 (CVB5), or B6 (CVB6). Cycloheximide was then administered at concentrations of 2, 0.4, 0.08, and 0.016 μM, and cell viability was assessed 48 h post-infection. Similar to the results observed with CVB3, treatment with cycloheximide maintained over 90% cell viability at concentrations up to 0.4 μM for all tested CVB serotypes. These results indicate potent antiviral activity across the CVB group, comparable to that of the positive control, rupintrivir (Fig. 4A). To further validate the antiviral efficacy of cycloheximide, we performed quantitative reverse transcription PCR (RT-qPCR). Vero cells were infected with various CVB serotypes and treated with cycloheximide at concentrations of 10, 2, 0.4, and 0.08 μM. After 24 h, viral RNA was extracted from the cells, and viral replication levels were analyzed by RT-qPCR. Cycloheximide treatment completely inhibited viral RNA replication for all tested CVB serotypes at concentrations as low as 0.08 μM (Fig. 4B). These findings demonstrate that cycloheximide possesses broad-spectrum antiviral activity against multiple members of the CVB group by effectively inhibiting viral replication.
Fig. 4
In vitro antiviral activity of cycloheximide against CVB1, CVB2, CVB4, CVB5, and CVB6. Vero cells were infected with 1 × 10³ PFU of CVB1, CVB2, CVB4, CVB5, or CVB6 and treated with cycloheximide at concentrations of 0.016, 0.08, 0.4, or 2 µM. (A) Antiviral activity was assessed by the sulforhodamine B (SRB) assay following cycloheximide treatment. (B) The relative expression levels of the 5′ noncoding region (NCR) gene of each CVB serotype were quantified by real-time PCR. For this analysis, Vero cells were infected with each virus and treated with cycloheximide at 0.08, 0.4, or 2 µM, and viral gene expression was evaluated 24 h post-infection.
DISCUSSION
Viruses induce cellular stress and stimulate autophagy, particularly RNA viruses, which exploit autophagy to form double-membrane vesicles (DMVs) for replication and immune evasion. Poliovirus promotes DMV formation through the viral proteins 2BC and 3A by stimulating LC3 lipidation (14). Flaviviruses such as Zika virus (ZIKV) activate autophagy by inhibiting the AKT-mTOR signaling pathway (15). Influenza A virus (IAV) uses its M2 protein to relocalize LC3, promoting autophagosome formation, particularly through the AKT-TSC2-mTOR pathway in H5N1 strains (16, 17). Human parainfluenza virus type 3 (HPIV3) accumulates autophagosomes by inhibiting lysosome fusion. Coronaviruses (SARS-CoV, MHV) similarly induce DMV formation (18). Picornaviruses, including coxsackievirus B3 (CVB3) and foot-and-mouth disease virus (FMDV), also utilize autophagy; CVB3 increases replication when autophagosome maturation is inhibited (19), and FMDV induces ATG5-dependent autophagosome formation, accumulating viral VP1 protein (20, 21, 22). Hepatitis C virus (HCV) promotes autophagosome formation but blocks lysosomal fusion, creating a favorable environment for viral replication, with its NS5B protein interacting with ATG5 early in infection (23). Influenza virus accumulates autophagosomes via M2 to facilitate viral assembly and release (16, 17). In conclusion, diverse RNA viruses actively manipulate host autophagy to create replication compartments, evade immune responses, and effectively enhance viral replication (14).
Cycloheximide is known to inhibit starvation-induced autophagy by activating mTORC1 through an increase in intracellular amino acid pools (11). In our study, cycloheximide exhibited antiviral activity against CVB3, suppressed autophagy in CVB3-infected Vero cells, and activated mTORC1 signaling. Furthermore, cycloheximide also demonstrated antiviral activity against other CVB groups in vitro. Collectively, our findings suggest that cycloheximide’s inhibition of autophagy effectively suppresses CVB group replication, highlighting its potential as a therapeutic approach for CVB infections. These results further support the concept that viruses exploit autophagy to enhance their replication.
However, cycloheximide is a potent inhibitor of global protein synthesis, which raises concerns about its inherent cytotoxicity and low selectivity. At a concentration of 2 µM or less, which is the condition of this experiment, did not reveal significant cytotoxic effects in Vero cells, higher concentrations may severely impair essential cellular functions by broadly suppressing protein synthesis. Consequently, the clinical applicability of cycloheximide is severely limited due to its potential toxicity and lack of selectivity. Future research should therefore prioritize developing novel antiviral agents that harness the beneficial autophagy-inhibitory effects observed with cycloheximide, but with enhanced selectivity and significantly reduced toxicity.
In conclusion, our findings reinforce the concept that RNA viruses exploit host autophagy mechanisms to enhance replication. Targeting autophagy pathways offers a promising strategy for antiviral therapeutic development. Further investigations into selective autophagy modulators with improved safety profiles are warranted.
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