ST cells and HEK293A cells were purchased from China Institute for Veterinary Drug and cultured in Dulbecco’s Minimal Essential Medium (DMEM; Hyclone) with 10% fetal bovine serum (FBS; Sigma). All cells were maintained in 37°C 5% CO₂ incubator. The PSV was isolated from PSV-infected piglets in Hunan Province, China, and kept in our laboratory. Z-VAD-FMK, ZIETD-FMK, and Z-LEHD-FMK obtained from Beyotime. The PSV VP1 protein-specific monoclonal antibody was prepared by our laboratory. The GAPDH, Bax, Bcl-2, and Cyt c antibodies were purchased from ABclonal. Antibodies against caspase-3 (D3R6Y), caspase-8 (1C12) and caspase-9 (Human Specific) were obtained from Cell Signaling Technologies. The antibody against PARP1 was purchased from Beyotime. The anti-Tomm 20 antibody were purchased from Abcam. The antibody against Flag was purchased from Sigma. HRP-goat anti-mouse lgG, HRP-goat anti-rabbit lgG, Alexa Fluor 549-goat anti-mouse lgG, and Alexa Fluor 488-goat anti-mouse lgG antibodies were obtained from Proteintech.

All fragments corresponding to PSV-encoded proteins were amplified from the PSV cDNA and inserted into pCAGGS-3×Flag. The mutation fragments of 2A gene were obtained by homologous recombination and also inserted into the pCAGGS-3×Flag. These recombinant plasmids were identified by DNA sequencing. Primers designed based on the genomic sequence of PSV ( Table 1 ).

The apoptotic cells were analyzed by the TUNEL Detection Kit (Beyotime). Briefly, ST or HEKHEK293A cells were cultured in 24-well plates. After PSV infected or pCAGGS-Flag-2A transfected, the cells were fixed with 4% paraformaldehyde and permeabilized using 0.2% Triton X-100 in 0.1% sodium citrate. Then, the cells were overlaid with TUNEL reaction mixture according to the manufacturer’ manual. Next, the cells were stained using VP1-specific mAb and Alexa Fluor 549-goat anti-mouse antibody. Finally, the cells were stained with DAPI and detected by the confocal microscope (Zeiss).

The cell apoptosis was determined using an FITC Annexin V Apoptosis Detection Kit (Solarbio) following the manufacturer’ instructions. Briefly, the cells were collected and washed twice with cold PBS by centrifugation at 300 × g for 5 min, and then stained with Annexin V and propidium iodide (PI). The percentage of apoptotic cells was examined by flow cytometry. At least 10⁵ events were recorded for each data.

To analyze the mitochondrial membrane potential by the MitoProbe Assay Kit (Solarbio), HEK293A cells were transfected with pCAGGS-Flag-2A using different doses for 24 h. According to the manufacturer’ manual, the cells were incubated with JC-1 dye (2 nM) at 37°C for 30 min, and then cells were examined by flow cytometry using emission at wavelengths of 529 nm (green) and 590 nm (red). At least 10⁴ events were recorded for each data.

The cells were seeded into six-well plates and collected with lysis buffer (Beyotime) containing protease and phosphatase inhibitors. The samples were then separated on a SDS–PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in the Tris-buffered saline (TBS) containing Tween 20 for 2 h and then incubated with the primary antibodies at 4°C overnight. After three times washing with TBST, the membranes were incubated by HRP-conjugated secondary antibodies, and thereafter target proteins were visualized using an ECL system (Tanon). The gray density was performed using the ImageJ software.

The cells were mock-infected or infected with PSV. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 in PBS. Then, the cells were blocked with 1% BSA for 30 min. After washing three times in PBS, the cells were labeled with mouse anti-VP1 mAb at 4°C overnight. After another washing with PBS, the cells were incubated with Alexa Fluor 488-goat anti-mouse lgG for 1 h at RT, followed by staining with DAPI. Fluorescence images were captured using the confocal microscope (Zeiss).

To isolate the cytosolic and mitochondrial fractions, the Mitochondria/Cytosol Fractionation Kit (Beyotime) was used. According to its manufacturer’s manual. HEK293A cells were transfected with pCAGGS-Flag-2A using different doses. After 24 h, the cells were harvested though centrifugation at 600 × g for 10 min at 4°C. After washing in the cold PBS, the centrifuged cells were resuspended in lysis buffer containing protease inhibitors. Subsequently, the cell suspensions were homogenized with the glass homogenizers. After 10-30 times of homogenization, the complete homogenates were centrifuged at 600 × g for 10 min at 4°C, and then transferred the supernatants to new tubes for another centrifugation at 12,000 × g for 10 min at 4°C. The supernatants were harvested as the cytosolic fraction, while the pellet was collected as mitochondrial fraction using mitochondrial lysis buffer.

Data were shown as the mean ± standard deviation (SD). Results were performed by one-way ANOVA. Significance was analyzed using GraphPad Prism software. Differences were considered statistically significant when the p-value (*) was less than 0.05 and highly significant when the p-value (**) was less than 0.01; ns indicated not significant.

To investigate whether PSV could trigger cell apoptosis, PSV-infected cells were observed by optical microscopy after staining with DAPI. The CPEs were visible in PSV-infected ST and HEK293A cells at 24 hpi and there were a marked cell shrinkage, nuclear diameter reduction and chromatin condensation in most of the infected cells ( Figure 1A ). Then, the intracellular fragmented DNA in PSV-infected cells were detected by the TUNEL assay. The results showed that the TUNEL positive signals were only exanimated in PSV-infected cells (MOI = 1, 24 hpi) ( Figure 1D ). To detect the precise apoptotic rate, the ST and HEK293A cells were infected with rising doses of PSV and stained with Annexin V/PI. The percentages of the viable, apoptotic, and necrotic cells were quantitatively determined by flow cytometry. PSV infection produced readily apparent apoptosis (Annexin V positive) at 24 hpi, compared with the mock-infected cells. The percentage of apoptotic cells increased with the dose of viral-infection and reached 30.73% and 18.15% in the ST and HEK293A cells, respectively ( Figures 1B, C ). These data demonstrated that PSV infection could induce cell apoptosis.

Caspase is a cysteine protease, which plays a basic role in cell apoptosis response to different stimuli (17, 18). The mitochondrial and death receptor mediated pathways are respectively induced by caspase-9 and caspase-8 (19). The contribution of caspases to PSV-induced apoptosis in ST and HEK293A cells were detected. As shown in Figures 2A, B , PSV infection promoted the cleavage of caspase-3 and caspase-9 at 24 hpi. In addition, the cleaved fragment of DNA repair enzyme PARP1, a substrate for caspase-3, increased with the dose of PSV infection. Furtherly, ST cells were pretreated with broad-spectrum caspase inhibitor (Z-VAD-FMK), specific inhibitor of caspase-8 (Z-IETD-FMK), or specific inhibitor of caspase-9 (Z-LEHD-FMK), and then infected with PSV for 24 h. The immunoblot results showed that pretreatment with Z-VAD-FMK and Z-LEHD-FMK significantly decreased PSV-induced cleavage of PARP1 ( Figure 3A ). In addition, the percentages of Annexin V positive cells were significantly reduced after pretreatment with Z-VAD-FMK or Z-LEHD-FMK, compared with PSV-infected cells alone ( Figures 3B, C ). These results suggest that caspase-3 and caspase-9 were activated by PSV infection.

To explore the linkage between apoptosis and PSV replication. ST cells were pretreated with apoptosis inhibitors, followed by PSV infected at MOI = 1. The immunofluorescence and VP1 protein expression were detected at 24 hpi, and the results indicated that Z-VAD-FMK and Z-LEHD-FMK inhibited the viral replication ( Figures 4A, C, E ). Moreover, the viral titers were determined by TCID₅₀ at 48 hpi, and caspase-3 and caspase-9 inhibitors caused significant reduction in viral load ( Figures 4B, D, F ). These results reveal that PSV replication is decreased after pretreatment with caspase-3 and caspase-9 inhibitors.

After confirming that PSV infection resulted in apoptosis, the putative PSV-encoded protein(s) that may be responsible for the induction of apoptosis were tried to identify. The expression plasmids of all PSV-encoded proteins were constructed and transfected them into HEK293A. The expression of viral proteins were examined by Western blotting using anti-Flag antibody, and all genes were expressed as anticipated ( Figure 5A ). The cleaved PARP1 in 2A-transfected cells was detected by Western blotting ( Figure 5A ). Furthermore, the cell apoptosis induced by individual viral proteins was detected by flow cytometry. 2A and 3C produced a significant level of apoptosis, the percentage of Annexin V positive reached 36.7% and 25.4%, respectively ( Figures 5B, C ). The results indicated that 2A appeared to be one of the most potent inducer of apoptosis among the viral proteins.

To further assess the pathway of 2A-induced apoptosis, the TUNEL, Annexin V/PI, and immunoblotting assays were used to detect apoptosis in PSV-2A transfected cells. The results showed that TUNEL fluorescence intensity of the pCAGGS-Flag-2A transfected cells was enhanced, and Annexin V positive cells also significantly increased in the 2A-transfected cells ( Figures 6A-C ). In addition, the abundance of the cleaved fragments of caspase-3, caspase-9, and PARP1 detected by Western blotting was increased with the transfection dose in the 2A-transfected cells at 24 h ( Figure 6D ).

It is well known that Bax is one of the major pro-apoptotic factors which increases the mitochondrial outer membrane permeability, while Bcl-2 is an anti-apoptotic member promoting cell survival (20). The balance between Bax and Bcl-2 is necessary for the integrity of the mitochondrial membrane permeability. Bax and Bcl-2 were detected after pCAGGS-Flag-2A transfection. The results showed that 2A activated Bax and suppressed the Bcl-2 expression at 24 h ( Figure 6E ). The gray density of Bax/Bcl-2 was analyzed and the results showed that the ratio of Bax/Bcl-2 was increased after pCAGGS-Flag-2A transfection ( Figure 6F ).

Furthermore, release of cyto C into the cytosol is critical for intrinsic apoptotic stimulation (21). We also observed that cyto C was decreased in the mitochondrial fraction and concomitantly increased dramatically in the cytosol ( Figure 7A ). Meanwhile, significant loss of the mitochondrial membrane potential in the 2A-transfected cells was observed ( Figures 7B, C ). These were further confirmed that 2A triggered the mitochondrial apoptotic pathway.

To further investigate the key residues of the PSV 2A protein, the amino acid sequences of 2A in representative members of picornaviruses were aligned via the software program DNASTAR ( Figure 8A ). Based on the comparisons, a catalytic triad comprising H48, D91, and C164 amino acids was identified in the PSV 2A (marked with red arrow in Figure 8A ). These were also conserved residues. In addition, we selected two amino acids, H58 and H125, as controls (marked with blue arrow in Figure 8A ). Among the 2A mutants, 2A (H58A) and 2A (H125A) appeared to induce apoptosis and PARP1 cleavage. The H48, D91, and C164 mutated proteins suppressed the 2A-induced Annexin V positive cells and PARP1 cleavage ( Figures 8B-D ). These results suggested that the conserved residues H48, D91, and C164 in PSV 2A were crucial for the induction of apoptosis.