Apoptosis is a key immune defense mechanism against viral infections that prevents viruses from establishing themselves in host cells (Barber, 2001; Galluzzi et al., 2012). However, several studies have reported that various viruses exploit apoptosis to evade host immunity, thereby exacerbating viral infections (Belov et al., 2003; Buenz and Howe, 2006; Galluzzi et al., 2008). Human rhinovirus (HRV) manipulates the apoptotic pathway to facilitate replication and proliferation. By inhibiting the apoptosis pathway during the early stages of HRV infection, the virus enables increased replication within host cells, which in turn worsens the inflammatory response caused by the virus (Croft et al., 2017; Lötzerich et al., 2018). Such inflammatory responses induced by HRV infection are associated with an increased risk of progression to asthma or chronic obstructive pulmonary disease (COPD) in affected individuals (Johnston et al., 1995). Therefore, developing antiviral agents that target mechanisms that suppress apoptosis can be an important therapeutic strategy for inhibiting the progression of HRV infection and alleviating viral infection-associated inflammatory responses.

HRVs are responsible for >50% of upper respiratory tract infections, commonly referred to as the common cold, which typically resolves within 5-7 d. Symptoms include nasal congestion, sneezing, coughing, and sore throat, although approximately 12-32% of children <4 years of age with HRV infections are asymptomatic (Jacobs et al., 2013). The presence of >100 HRV serotypes has hindered the development of approved vaccines and antiviral agents, leaving palliative care as the only treatment option (Blaas and Fuchs, 2016). Although HRV infections are generally mild and self-limiting in otherwise healthy individuals, they can be life-threatening when superimposed on chronic conditions, such as COPD, asthma, or cystic fibrosis (CF). This highlights the urgent need for effective antiviral therapies (Blaas and Fuchs, 2016).

Natural extracts have long been used in traditional medicine, and numerous compounds with potential antiviral activities have recently been identified (Zakaryan et al., 2017; Ninfali et al., 2020). Serratula coronata, a plant species of the thistle tribe of the daisy family, is native to Eurasia and synthesizes a wide range of ecdysteroids and flavonoids, making it a valuable source of herbal remedies (Báthori et al., 2004). Flavonoid compounds such as quercetin-3-methyl ether (Q3ME) have anti-inflammatory (Wei et al., 2001; Mohamed et al., 2014), neuroprotective (Al-Dabbas et al., 2006), antioxidant (Fendrick et al., 2003; Mohamed et al., 2014), and apoptosis-inducing properties (Zeng et al., 2024). However, its antiviral activity against HRV1B has not been reported.

In the present study, we demonstrated that extracts of S. coronata and the flavonoid Q3ME, isolated from S. coronata, inhibited HRV1B in vitro in a concentration-dependent manner. In addition, in vivo experiments showed that these compounds reduced HRV1B levels in the lungs of infected mice. Notably, Q3ME increased apoptosis during the early stages of viral infection, suggesting that early activation of apoptosis may confer antiviral activity against HRV1B.

Plant material

S. coronata was obtained from the Plant Extract Bank of Korea Research Institute of Bioscience and Biotechnology. The antiviral efficacy of the methanol extract against HRV1B was evaluated using the Sulforhodamine B (SRB) assay. This extract demonstrated approximately 80% inhibition of HRV1B at a concentration of 10 μg/mL, as shown in Supplementary Fig. 1. The aerial parts of S. coronata were collected and analyzed by Professors Yong Soo Kwon and Hee Jung Yang at the College of Pharmacy, Kangwon National University, to identify antiviral bioactive constituents.

Extraction and isolation

The dried powdered aerial parts of S. coronata (200 g) were extracted with methanol (MeOH; 3 L×4) at 25°C. The combined extracts were concentrated to yield a residue (16.6 g), which was suspended in 200 mL distilled water and successively partitioned into hexane (500 mL×3), chloroform (500 mL×3), and butanol (BuOH; 500 mL×5). Each fraction was concentrated to yield hexane (2.2 g), chloroform (1.8 g), BuOH (4.1 g), and aqueous (8.1 g) fractions, which were tested for SRB-based cytotoxicity and antiviral activity. The BuOH fraction exhibited high antiviral activity (Supplementary Fig. 2). The BuOH residue was fractionated using ODS column chromatography (YMC Triet C18, 30×350 mm), eluted with water and a gradually increasing proportion of methanol (75:25→0:100). Eight fractions were collected: SCI-1 (600.8 mg), SCI-2 (351.2 mg), SCI-3 (334.7 mg), SCI-4 (1268 mg), SCI-5 (188.1 mg), SCI-6 (311.9 mg), SCI-7 (196.5 mg), and SCI-w (99 mg). Among the eight fractions of S. coronata, SCI-6 exhibited the most potent antiviral activity. The active compound was isolated using SCI-6 guided by the SRB bioassay. Fraction SCI-6 was repeatedly purified using a Shiseido CAPCELL PACK C18 AQ column (20×250 mm) and eluted with an acetonitrile-water gradient to yield active compound 1 (81.3 mg) (Supplementary Fig. 3). The structure of SCI-6C was determined using spectroscopic analyses, including electron ionization mass spectrometry (EI-MS), proton nuclear magnetic resonance (¹H-NMR), and carbon-13 nuclear magnetic resonance (¹³C-NMR). Nuclear resonance signals from ¹H-NMR and ¹³C-NMR were used to identify the structure of the active compound, quercetin 3-methyl ether (Supplementary Fig. 4).

Q3ME (3) HRESI-MS m/z: 315.0518 [M-H]^-, (calcd for C₁₆H₁₁O₇); ¹H-NMR (400MHz, dimethyl sulfoxide [DMSO]-d₆, δ); 7.54 (¹H, d, J=2.0, H-2’), 7.44 (¹H, dd, J=8.4, 2.0, H-6’), 6.90 (¹H, d, J=8.4, H-5’), 6.41 (¹H, d, J=1.2, H-8), 6.19 (¹H, d, J=1.2, H-6), 3.78 (3H, s, O-methyl); ¹³C-NMR (100MHz, DMSO-d₆, δ) 177.9 (C-4), 164.1 (C-7), 161.3 (C-5), 156.3 (C-9), 155.6 (C-2), 148.7 (C-4’), 145.2 (C-3’), 137.7 (C-3), 120.8 (C-1’), 120.6 (C-6’), 115.8 (C-2’) 115.4 (C-5’), 104.2 (C-10), 98.5 (C-6), 93.6 (C-8), 59.7 (O-methyl) (Báthori et al., 2004).

Viruses, cells, and reagents

HRV1B was provided by the American Type Culture Collection (ATCC; Manassas, VA, USA) and were propagated in human epithelioid carcinoma cervix (HeLa) cells at 32°C. HeLa cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 0.01% antibiotic-antimycotic. Antibiotics, antimycotics, FBS, and MEM were supplied by Gibco BRL (Grand Island, NY, USA). Tissue culture plates were purchased from Falcon (BD Biosciences, NJ, USA). SRB was purchased from Sigma-Aldrich (St. Louis, MO, USA). All the other chemicals were of reagent grade. Stock solutions (10 mg/mL) of the antiviral compounds were prepared in DMSO and subsequently diluted in the appropriate culture media. The final DMSO concentration reached a maximum of 0.1% maximum, which did not affect the cell culture. Therefore, 0.1% DMSO was also added to all no-drug control samples.

Antiviral activity assay

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 (Song et al., 2015). The day before infection, 3×10⁴ cells/well were seeded into a 96-well a culture plate with HeLa cells. The following day, the culture medium was aspirated, and the cells were washed with 1×phosphate-buffered saline (PBS). To assess cytopathic effect (CPE) inhibition, 90 μL of diluted HRV1B (1.8×10³ pfu) suspension, 30 mM MgCl₂, and 1% fetal bovine serum (FBS), was treated with 10 μL of the candidate compound. Test compounds were prepared using a 5-fold serial dilution method. Antiviral activity was evaluated at four concentrations, ranging 0.4-50 μg/mL. Three wells were used as virus controls (virus-infected, non-drug-treated cells) and three wells were used as cell controls (non-infected, non-drug-treated cells). The 96-well culture plates were incubated at 33°C in 5% CO₂ for 2-3 days until 70-80% of CPE. After infection, the medium was aspirated, and the cells were washed with 1×PBS. Next, the cells were fixed with ice-cold 70% acetone (100 μL/well) for 30 min in –20°C freezer and stained with 0.4% SRB in 1% acetic acid. The SRB-stained cells were solubilized with 10 mM unbuffered Tris base solution, and the absorbance was measured at 562 nm by using a SpectraMax^® i3 microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance of 620 nm. The SpectraMax^® i3 microplate reader were measured at the Core-Facility for Innovative Cancer Drug Discovery (CFICDD) at Kangwon National University. The results were then calculated as percentages of the controls, and the percentage protection achieved by the test compound in the virus-infected cells was calculated using the following formula : {(OD_t)_virus(OD_c)_virus}÷{(OD_c)_mock-(OD_c)_virus}×100 (expressed in %), where (OD_t)_virus is the optical density measured with a given CFS in virus-infected cells, (OD_c)_virus is the optical density measured for the control untreated virus-infected cells, and (OD_c)_mock is the optical density. In addition, the morphology of the cells was observed under a microscope at 32×10 magnification (Axiovert 10; Zeiss, Wetzlar, Germany), and images were recorded.

Replicon assay

The pDP1Luc/VP3 mutant clone, which contains the firefly luciferase gene instead of the P1 capsid-coding region of the HRV14 genome, was provided by Stanley M. Lemon (University of North Carolina, Chapel Hill, USA). The HRV14 replicon plasmid linearized by Mlu I was used for in vitro RNA transcription with the RiboMAX™ Large Scale RNA Production System (Promega, Madison, WI, USA). We transfected 3×10⁵ HeLa cells/well in a six-well plate with 0.4 μg HRV14 replicon RNA using Lipofectamine^® 2000 (Promega). The transfected cells were seeded into 96-well plates (2×10⁴ cells/well) and simultaneously treated with various doses of Q3ME and rupintrivir. After 12 h, the cells were analyzed for firefly luciferase activity using the ONE-Glo Luciferase Assay System (Promega). Cell viability was measured using the CellTiter-Glo^® Luminescent Cell Viability Assay (Promega).

Time-of-addition

A time-of-addition assay was designed to determine the mechanism of action of antiviral compounds (Basu et al., 2011; Daelemans et al., 2011). Q3ME (10 μg/mL), pleconaril (10 μM), and rupintrivir (2 μg/mL) were administered to cells either 1 h prior to HRV1B infection, at the time of infection (0 h), or 1, 2, 4, 6, and 8 h post-infection. For the pre-infection treatment, each compound was applied for 1 h and subsequently washed out before HRV1B infection. After 10 h, real-time polymerase chain reaction (PCR) analysis was performed using the THUNDERBIRD^® SYBR^® quantitative (q)PCR mix (Toyobo, Osaka, Japan), and CFX96 optics module real-time PCR system (Bio-Rad, Hercules, CA, USA) (Bae et al., 2021).

Time-course

HeLa cells infected with HRV1B were harvested at 4. 6, 8, 10, and 12 h post-infection, after which, Q3ME 10 μg/mL, 10 μM pleconaril and 2 μg/mL rupintrivir was added. Total RNA was extracted at the indicated post-infection time points, and real-time PCR analysis was performed using the THUNDERBIRD^® SYBR^® qPCR mix (Toyobo), and CFX96 optics module real-time PCR system (Bio-Rad).

Microarray data analysis

Cell lysates were prepared and sent to MACROGEN (Seoul, Korea) for transcriptome analysis. The microarray data were generated following the standard protocol of the Agilent SurePrint G3 Human GE 8X60K, V3 Microarrays (Agilent^®, CA, USA). Briefly, RNA labeling and hybridization were performed using 100 ng of total RNA from each sample. A total of 600 ng of Cy3-labeled cRNA from each sample was purified using the RNeasy Mini Kit (Qiagen), fragmented, and hybridized to the Agilent SurePrint G3 Human GE 8X60K, V3 Microarrays (Agilent^®). The hybridized arrays were promptly scanned using an Agilent Microarray Scanner D (Agilent^®), and raw expression data were extracted using the Agilent Feature Extraction Software (v11.0.1.1). From the raw data text file, probes flagged as ‘A’ in any sample were filtered out. The selected gProcessedSignal values were log-transformed and normalized using the quantile method. A heatmap was created along with a dendrogram derived from the hierarchical clustering analysis using the ‘hclust’ function in R Foundation for Statistical Computing, Vienna, Austria.

Western blot

Total protein lysates from cells were prepared using sonication with PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea). Protein levels were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes. The membranes were subsequently incubated with primary antibodies for 24 h at 4°C. Primary antibodies: c-JUN, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), CHOP (Cell Signaling, Denver, MA, USA). Thereafter, secondary Abs, goat anti-mouse IgG F(ab′)2, polyclonal Abs (HRP conjugated) (Enzo Life Sciences, Farmingdale, NY, USA) was added for 2 h at 20°C. Proteins were detected using West Femto Maximum Sensitivity Substrate (Abbkine, Atlanta, GA, USA) and visualized using an ImageQuant LAS 4000 Mini System (Cytiva, Marlborough, MA, USA). Chemiluminescence intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA) (Song et al., 2014).

Real-time PCR

Total RNA was isolated using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcription was performed using an RNase inhibitor, oligo (dT) 15 primers, a dNTP mixture, and Moloney murine leukemia virus reverse transcriptase (Promega) in 5×buffer. qPCR analysis was performed, according to the established protocol as previously described (Yi et al., 2023), using the THUNDERBIRD^® SYBR^® qPCR mix (Toyobo), and CFX96 optics module real-time PCR system (Bio-Rad). The following primers were used: HRV 5’-NCR sense, 5’-TCC TCC GGC CCC TGA ATG-3’ and HRV 5’-NCR-antisense, 5’-GAA ACA CGG ACA CCC AAA G-3’; and human β-actin-sense, 5’-CCA TCA TGA AGT GTG ACG TGG-3’, and human β-actin-antisense, 5’-GTC CGC CTA GAA GCA TTT GCG-3.’ The PCR conditions were set as follows: 95°C for 3 min for 1 cycle and 95°C, 30 s; 60°C, 30 s; and 72°C, 30 s (35 cycles) (Song et al., 2018).

Mice and virus infection

Female BALB/c mice aged 4 weeks were purchased from the SPL Laboratory Animal Company (Koatech Bio, Pyeongtaek, Korea) and maintained for 7 d for stabilization. They were anesthetized with intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg) and intranasally infected thrice with 1.8×10⁷ pfu/mouse of HRV1B with 10 μL/PBS at intervals of 10 min. The mice were maintained in the experimental facility at Kangwon National University and the experiments were approved by the Institutional Animal Care and Use Committee of Kangwon National University (KW-140811-2).

Cytokine and Chemokine assay

Cytokine levels in the supernatants of HRV1B infected mouse lung lysates were evaluated using the enzyme-linked immunosorbent assay (ELISA). ELISA kits for TNF-α, IL-1β, IL-6, and CCL2 were purchased from e-Bioscience (eBioscience, San Diego, CA, USA), and ELISA kit for CXCL1 was purchased from R&D Systems (Minneapolis, MN, USA). Lungs were obtained from mice infected with HRV1B, and similar amount of lung tissues were homogenized in CK28 (Bertin Technology, Orsay, France) using 2.8-mm ceramic beads with 600 μL PBS and using a Minilys homogenizer (Bertin Technology) at 6000 rpm for 15 s, twice at –20°C. The clear supernatants were collected following centrifugation at 10,000×g for 10 min at 4°C. The levels of cytokines and chemokines in the lung supernatants were evaluated according to the manufacturer’s instructions (Seo et al., 2011). The absorbance was read at 450 nm using a SPECTRA MAX 340 (Molecular Devices) (Song et al., 2018).

Antiviral activity of Q3ME isolated from S. coronata against HRV1B

We evaluated the antiviral activity of Q3ME, a compound purified from the SCI fraction separated from the BuOH fraction, against HRV1B. The antiviral activity of Q3ME was assessed in HeLa cells using an SRB assay. The results of the SRB assay demonstrated that Q3ME inhibited the cytopathic effect (CPE) caused by HRV1B by >80% at a concentration of 10 µg/mL (Fig. 1A). However, no cytotoxicity was observed at the same concentration (Supplementary Fig. 5). Additionally, the antiviral activity experiment using the SRB assay showed that Q3ME exhibited an IC50 value of 4.45 µg/mL against HRV1B. Cell morphology analysis revealed that cells infected with HRV1B but not treated with the drug developed severe CPE. In contrast, cells treated with 10 µg/mL of Q3ME after HRV1B infection exhibited normal morphology. This morphology was similar to that observed in cells treated with 2 µg/mL of rupintrivir, which served as a positive control group (Fig. 1B). To validate the antiviral activity of Q3ME, HeLa cells were infected with HRV1B and then treated with Q3ME at various concentrations. 24 h post-treatment, the inhibition of HRV1B replication by Q3ME was confirmed using real-time PCR. The results indicated that Q3ME effectively inhibited HRV1B replication at concentrations above 0.4 µg/mL (Fig. 1C). To further investigate the inhibitory efficacy of Q3ME against HRV1B replication, we transfected HeLa cells with in vitro transcribed HRV14-replicon RNAs. This was followed by simultaneous treatment with Q3ME at various concentrations (50, 10, 2, 0.4, 0.08, 0.016, and 0.0032 μg/mL) and 2 μg/mL of rupintrivir for a period of 12 h. After the treatment period, the activity of HRV14-replicon RNAs was evaluated by measuring luciferase activity. The results indicated that Q3ME demonstrated potent inhibitory effects up to a concentration of 0.4 μg/mL, and significant activity was still observed at a concentration as low as 0.016 μg/mL (Fig. 1D). In the cytotoxicity analysis, including the HRV14-replicon experiment, the toxicity was evaluated using CellTiter-Glo reagent at concentrations of 50, 10, 2, 0.4, 0.08, 0.016, and 0.0032 μg/mL. The results demonstrate that Q3ME exerted minimal impact on the viability of HeLa cells, even at the highest concentration of 50 μg/mL (Fig. 1E).

Figure 1. Antiviral activity of Q3ME against HRV1B in vitro. (A) HeLa cells were infected with HRV1B (1.8×10³ pfu) and treated with Q3ME. Cell viability and cytotoxicity following Q3ME treatment were measured using the SRB assay. (B) Captured images confirming CPE inhibition activity of Q3ME. (C) Relative HRV1B 5’NCR gene expression in HRV1B-infected HeLa cells was determined using RT-qPCR. (D) HeLa cells were transfected with in vitro-transcribed HRV14 replicon RNA, promptly treated with the indicated concentrations of Q3ME for 12 h, and assessed for firefly luciferase activity. (E) HeLa cells without transfection of HRV14 replicon were analyzed for cell viability using CellTiter-Glo® reagent. The results are shown as mean ± standard error of the mean (SEM). *p<0.05, **p<0.01, and ***p<0.001 for comparison with the HRV1B-infected vehicle-treated group (Veh) based on one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. For the validation assessment, (A) was obtained through three independent experiments and (C, D, E) were obtained through two independent experiments.

Analysis of the antiviral action mechanism of Q3ME against HRV1B through time course and addition time experiments

Based on the findings from the HRV14 replicon study, it can be inferred that the antiviral action of Q3ME does not stem from inhibition of the entry step of HRV or the virus particle itself. This suggests that Q3ME targets other phases of the viral infection cycle, such as uncoating, protease-mediated polyprotein processing, replication, and translation. Therefore, to analyze the mode of antiviral action of Q3ME against HRV1B, time-course and time-of-addition experiments were conducted. For the time course experiment, HeLa cells infected with HRV1B were treated with 10 μg/mL of Q3ME, and reverse transcription (RT)-qPCR analyses were performed at 4, 6, 8, 10, and 12 h post-treatment. Rupintrivir, an HRV 3C protease inhibitor, and pleconaril, a viral capsid binding inhibitor, were included as controls (Patick et al., 2005). Expression of the HRV1B 5’ NCR gene was detected after 6 h; however, Q3ME delayed the detection of viral RNA expression until 12 h post-infection. Q3ME inhibited viral RNA expression more robustly for up to 12 h, similar to rupintrivir, an inhibitor of the HRV 3C protease (Fig. 2A). Based on these results, we propose that the mechanism of action of Q3ME is similar to that of rupintrivir. Based on the results of these time-course experiments, we conducted additional time-of-addition experiments to identify specific stages affected by Q3ME. In this experiment, 10 μg/mL of Q3ME was added to the culture medium at various time points: –1, 0, 1, 2, 4, 6, and 8 h post-infection. We then analyzed the expression of the HRV1B 5’ NCR gene at 10 h post-infection. Treatment of HeLa cells with Q3ME 1 h before HRV1B infection did not inhibit the viral infection. However, administration of Q3ME for up to 4 h after infection effectively suppressed viral RNA expression (Fig. 2B). These findings indicated that Q3ME targets an early stage of the viral replication and translation cycle.

Figure 2. Microarray analysis comparing HRV1B-infected and HRV1B-infected cells treated with Q3ME. RNA was obtained 12 h after infection. (A) Heat map of microarray analysis comparing HRV1B-infected and HRV1B-infected cells treated with Q3ME. Gene ontology (GO) analysis of 317 differentially expressed genes (DEGs). (B) Heat maps of microarray analysis of apoptosis-related genes.

Q3ME modulates apoptosis-related gene expression to exert antiviral effects during early HRV1B infection

Through time-course and time-of-addition analysis, we confirmed that Q3ME exhibited antiviral activity during the early stages of HRV1B infection. To elucidate the mechanism through which Q3ME exerts its antiviral effects during the initial phase of viral infection, we conducted a microarray analysis. This analysis identified 160 upregulated and 157 downregulated genes, highlighting significant changes in the patterns of differentially expressed genes (DEGs; Fig. 3A). The resulting heatmap comparing the virus-infected and Q3ME-treated groups demonstrated that gene expression was significantly modulated when Q3ME was administered, compared to the virus-only group. Gene Ontology (GO) analysis revealed that Q3ME treatment was highly associated with apoptosis (Supplementary GO analysis). It has been reported that HRV facilitates viral invasion by suppressing apoptosis during the early stages of infection (Croft et al., 2017; Lötzerich et al., 2018). To investigate whether Q3ME exerts its antiviral activity by modulating apoptosis during early infection, we performed a heatmap analysis focusing on apoptosis-related genes. Following Q3ME treatment, the expression of anti-apoptotic genes was downregulated, whereas that of pro-apoptotic genes was upregulated, indicating that Q3ME activates the early apoptotic pathway (Fig. 3B). We also compared the mock control and Q3ME-treated mock control groups. The analysis revealed that apoptosis-related genes were also upregulated in the Q3ME-treated mock control group, indicating that Q3ME modulates apoptosis even in the absence of viral infection (Supplementary Fig. 6).

Figure 3. Q3ME inhibited HRV1B replication at an early stage of viral infection. HeLa cells were infected with HRV1B at 50% tissue culture infective dose (TCID50) and harvested at the indicated time points (4, 6, 8, 10, and 12 h) after treatment with 10 µg/mL Q3ME, 10 μM pleconaril, and 2 μg/mL rupintrivir. HeLa cells were treated with 10 µg/mL Q3ME, 10 μM pleconaril, and 2 μg/mL rupintrivir at the time of or after infection with HRV1B at the indicated time points. Viral mRNA was analyzed 8 h post-infection. Pleconaril and rupintrivir were used as positive controls. Total RNA was isolated and viral RNA was analyzed using RT-qPCR. The results are shown as mean ± SEM. *p<0.05; ***p<0.001, ^††p<0.01, ^†††p<0.001, ^##p<0.01, and ^###p<0.001 for comparison with the HRV1B-infected Veh group based on one-way ANOVA with Bonferroni’s multiple comparison test. For the validation assessment, (A, B) were obtained through three independent experiments.

Q3ME enhances apoptosis to counteract HRV1B-induced immune evasion during early infection

Microarray analysis revealed that Q3ME activated apoptosis-related genes during the early stages of infection. To validate this finding, we analyzed the expression of key pro-apoptotic genes such as DDIT3 and c-Jun using qPCR. The results demonstrated a significant increase in the expression of both genes at higher Q3ME concentrations. Consistent with the qPCR findings, western blotting analysis revealed a concentration-dependent increase in DDIT3 and c-Jun protein levels (Fig. 4A, 4B).

Figure 4. RT-qPCR and western blot analysis confirmed gene expressions and protein levels in the apoptosis-upregulating group. (A) RT-qPCR analysis confirmed DDIT3 and c-Jun gene expression levels among the apoptosis-upregulating genes. (B) Western blotting of DDIT3 and c-Jun protein levels among the apoptosis-upregulating genes. (C) RT-qPCR analysis confirmed the levels of caspase 8, BH3 protein group, and mitochondrial protein group, which promote apoptosis). RNA was obtained 12 h after infection. The results are shown as mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001 for comparison with the HRV1B-infected Veh group based on one-way ANOVA with Bonferroni’s multiple comparison test. For the validation assessment, (A, C) were obtained through two independent experiments.

HRV has been reported to suppress apoptosis during the early stages of infection by disrupting the RIPK1-TRIF-FADD complex, thereby inhibiting the activation of death receptors (Lötzerich et al., 2018). To determine whether Q3ME-mediated apoptosis activation could counteract this HRV immune evasion mechanism, we analyzed downstream signals using qPCR. The results showed an increase in the expression of caspase-8 and BH3-only proteins with increasing concentrations of Q3ME, suggesting that Q3ME enhanced apoptosis and thereby counteracted RV-induced apoptosis suppression. Furthermore, the expression levels of AIF, EndoG, and Omi/HtrA2 were analyzed using qPCR, and a significant increase in these genes was observed following Q3ME treatment (Fig. 4C). These findings suggest that Q3ME induces programmed cell death in host cells, thereby enhancing the host immune response to viral invasion.

In conclusion, Q3ME activated apoptosis during the early stages of viral infection. By reversing the suppression of apoptosis, a mechanism HRV1B uses to evades host immunity, Q3ME may enhance the host’s immune response.

Antiviral activity of Q3ME against HRV1B was evaluated and verified in vivo

We used a mouse model to assess the antiviral efficacy of S. coronata extract and Q3ME against HRV1B in vivo. Mice were administered S. coronata extract (50 mg/kg) and Q3ME (10 mg/kg) intraperitoneally at 1 h before and 4 h after HRV1B infection, respectively (Fig. 5A). Eight hours post-infection, the mice were sacrificed and lung tissues were harvested. The collected lung tissues were homogenized to obtain supernatants, which were then subjected to real-time PCR targeting viral mRNA. This allowed us to quantify the amplification levels of negative-strand and positive-strand RNA, which are critical indicators of viral replication stages. The results demonstrated that Vehicle-treated HRV1B-infected mice showed elevated levels of both positive- and negative-stranded viral RNAs. In contrast, mice treated with S. coronata extract and Q3ME showed significant suppression of both viral RNA strands (Fig. 5B). These findings indicate that S. coronata extract and Q3ME effectively inhibited HRV1B replication. In addition, viruses isolated from the lung tissues of Q3ME-treated mice were used to infect cells and cell viability was assessed. Cells infected with viruses from untreated mice showed a viability reduction to <10%. However, cells infected with viruses from Q3ME-treated mice maintained >60% viability. This suggests that Q3ME significantly attenuated the infectivity of HRV1B in vivo. Finally, to assess changes in inflammatory cytokine production due to HRV1B infection, we measured the levels of TNF-α, IL-1β, IL-6, and CCL2 in mouse lung tissues. HRV1B-infected mice exhibit a significant increase in the expression of inflammatory cytokines. In contrast, Q3ME-treated mice exhibited a marked decrease in cytokine levels (Fig. 5C). Collectively, these results confirmed that S. coronata extract and Q3ME possess potent antiviral activity against HRV1B in vivo. They mitigate HRV1B-induced pathogenesis by inhibiting viral replication and reducing the production of inflammatory cytokines. These findings suggested that Q3ME is a promising candidate for the development of therapeutic agents against HRV1B infection.

Figure 5. Antiviral activity of Q3ME against HRV1B in vivo. (A) BALB/c mice were infected intraperitoneally with a 1.8×10⁷ pfu/mouse of HRV1B. Q3ME treatment was administered intraperitoneally at 10 mg/kg twice at 1 h before and 4 h after HRV1B infection; the mice were sacrificed 8 h post-infection. (B) HRV1B RNA levels in the lungs of mice treated with Q3ME. RNA was isolated and viral RNA was analyzed using RT-qPCR. (C) Cell viability, confirmed through HRV1B obtained from the lungs of infected mice, was evaluated using the SRB assay, and the results were determined based on absorbance at 562 nm. Cytokine and chemokine levels in the lung tissues of mice were confirmed using the ELISA. The results are shown as mean ± SEM. **p<0.01 and ***p<0.001 for comparison with the HRV1B-infected Veh group based on one-way ANOVA with Bonferroni’s multiple comparison test. For the validation assessment, (B, C) were obtained through three independent experiments.

HRV is globally recognized as a major cause of common cold and respiratory illnesses. In addition to upper respiratory tract infections, HRV can exacerbate chronic respiratory diseases such as asthma, COPD, and CF (Jacobs et al., 2013; Moore et al., 2013). HRV, an RNA virus belonging to the Picornaviridae family, presents significant public health challenges due to its diverse infection profiles and rapid transmission. HRV is classified into three species groups, A, B, and C, each of which is further subdivided into serotypes based on the capsid gene sequences: 78 serotypes in group A, 30 in group B, and 51 in group C. The presence of >100 HRV serotypes makes vaccine development highly complex and challenging. Moreover, the lack of approved vaccines or antiviral agents for the prevention or treatment of HRV infections underscores the urgent need for novel therapeutic strategies to manage HRV-related diseases (Jacobs et al., 2013).

Apoptosis, also known as programmed cell death, is a process in which cells systematically activate intrinsic death pathways in response to various stimuli, leading to self-elimination (Kerr et al., 1972). Apoptosis plays a significant role in viral infections by acting as a key immune defense mechanism in the interactions between viruses and the host (Barber, 2001; Galluzzi et al., 2012). Cell death can be induced by various stimuli, and the type of stimulus determines the mode of cell death. Upon the activation of death signals, a cascade of signaling proteins is initiated (Gavrieli et al., 1992; Ankarcrona et al., 1995; Tidball et al., 1995; Sakahira et al., 1998). Upon activation of cytoplasmic helicase receptors that recognize 5′-triphosphate RNA or long double-stranded RNA such as RIG-I or MDA-5 these receptors interact with the mitochondria-bound adapter protein IPS-1 through homotypic caspase activation and recruitment domains (CARDs). Upon activation, IPS-1 recruits IKKε and TBK1 to phosphorylate IRF-3 (Kumar et al., 2006). Phosphorylated IRF-3 then translocates to the nucleus to induce the expression of IFN-β or binds with Bax to form pores in the mitochondrial membrane, thereby leading to apoptosis (Chattopadhyay and Sen, 2014). IPS-1-dependent activation of IRF-3 leads to the activation of pro-apoptotic Bcl-2 family proteins (Besch et al., 2009) and BH3 domain proteins (Chattopadhyay et al., 2010), which in turn activate caspase-9, thereby promoting apoptosis (Okazaki et al., 2013).

Several studies have reported that viruses exploit apoptosis to evade host immune defenses, thereby exacerbating viral infections (Belov et al., 2003; Buenz and Howe, 2006; Galluzzi et al., 2008). Picornaviruses are known to inhibit this apoptotic pathway by cleaving RNA sensors such as RIG-I through various viral proteases. Studies have reported that poliovirus, echovirus type 1, encephalomyocarditis virus, and minor-group HRVs suppress apoptosis by cleaving RIG-I between 8 and 14 h post-infection (Barral et al., 2009). In addition, several viruses have been found to cleave MAVS and TRIF, thereby disrupting virus-induced signaling pathways. Specifically, during Coxsackievirus B3 infection, MAVS is cleaved 5 h post-infection and TRIF is cleaved 3 h post-infection (Mukherjee et al., 2011). Furthermore, the 3ABC proteases of HRV1a and hepatitis A virus are known to cleave IPS-1 (MAVS) at approximately 15 h post-infection, contributing to the evasion of host immune responses (Drahos and Racaniello, 2009). Another study revealed that the 3C protease (3C^pro) of HRV regulates programmed cell death. The cells infected with HRV-A16 for 15 h expressed 3C^pro, which inhibited apoptosis. Activated 3C^pro reduces programmed cell death and enhances viral spread (Lötzerich et al., 2018).

Flavonoids are secondary plant metabolites responsible for the coloration and fragrance of flowers and possess antimicrobial, antiviral, antioxidant, anti-allergic, and anti-inflammatory properties (Batra and Sharma, 2013; Panche et al., 2016; Abotaleb et al., 2018). Furthermore, during carcinogenesis, flavonoids interfere with various signaling pathways, thereby limiting proliferation, angiogenesis, and metastasis, or increasing apoptosis (Ravishankar et al., 2013). Quercetin, the most abundant flavonoid, is widely available in tomatoes, apples, berries, grapes, onions, tea leaves, Brassica vegetables, capers, shallots, nut bark, flowers, and seeds (Kelly, 2011). The mitochondria-mediated apoptotic effects of quercetin have been demonstrated in HL-60 cells through the regulation of Cox-2, caspase-3, Bax, Bad, Bcl-2, cytochrome c, and PARP (Niu et al., 2011). In BC1, BC3, and BCBL lymphoma cells, quercetin induced apoptosis by downregulating the PI3K/AKT/mTOR and STAT3 signaling pathways (Granato et al., 2017; Varghese et al., 2018). Kaempferol is a non-toxic dietary flavonoid found in various foods, including tea, kale, beans, onions, tomatoes, strawberries, broccoli, cabbage, apples, and grapes (Calderón-Montaño et al., 2011; Chen and Chen, 2013). Kaempferol has been reported to induce intrinsic apoptosis in A2780/CP70, A2780wt, and OVCAR-3 cell lines through the mitochondrial pathway, which involves an increase in caspase-3, caspase-7, p53, Bax, and Bad and a decrease in Bcl-xL (Luo et al., 2011). In addition, in HeLa cell lines, kaempferol has been shown to elevate the Bax/Bcl-2 ratio, which is associated with the regulation of mitochondrial function and apoptosis (Kashafi et al., 2017).

In the present study, S. coronata extract and Q3ME exhibited potent antiviral activity against HRV1B both in vitro and in vivo. Time-of-addition and time-course experiments indicated that Q3ME operated through a mechanism similar to that of rupintrivir, which is known for its antiviral activity against HRV1B. This suggests that Q3ME protects the cells from the early stages of viral infection. To elucidate the underlying mechanism, we conducted a microarray analysis. The results revealed significant changes in the gene expression patterns upon Q3ME treatment, which were highly associated with apoptosis. Examination of apoptosis-related genes indicated that Q3ME treatment increased apoptotic processes. These findings confirm that Q3ME exerts antiviral effects by activating apoptosis during the early phase of viral infection. Our study demonstrated that Q3ME has a significant antiviral effect against HRV1B infection (Fig. 6), suggesting its potential as an antiviral therapeutic agent against HRV infections.

Figure 6. Proposed antiviral mechanism of Q3ME. Q3ME has an antiviral mechanism that induces programmed cell death, leading to apoptosis in HRV1B-infected cells. The figure was Created in BioRender (https://BioRender.com/l53x620).

This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2022-NR074811). This research was further supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (RS-2023-KH134750, HI23C0195). We would like to thank Editage (www.editage.co.kr) for English language editing.

The authors declare no conflict of interests.