PK-15 cells and Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) at 37°C in 5% CO₂ atmosphere. The PRV strain HeN1 was isolated and stored in our laboratory. The PRV stock was propagated in PK-15 cells with 2% FBS. After incubation for 3 or 4 days, the culture medium was harvested when the cytopathic effect (CPE) was apparent, and the supernatant was used for virus titration. The titer of the virus was 1.2 × 10⁷ plaque-forming units per ml (PFU/ml).

Rapamycin (R0395), 3-MA (3-Methyladenine, M9281) and Z-VAD-FMK (N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone, V116) were purchased from Sigma-Aldrich (USA). Anti-β-actin (ACTB, A5316), anti-ATG5 (A0731) antibody and polyclonal anti-LC3 (Microtubule-associated protein 1A/1B-light chain 3, L7543) antibody were purchased from Sigma-Aldrich (USA); anti-cleaved caspase3 antibody (AC033) was purchased from Beyotime (China, Shanghai); anti-gE antibody was produced from immunized mice. NAC (N-acetyl-L-cysteine, S0077) and a Reactive Oxygen Species Assay Kit (S30033) were purchased from Beyotime. A Cell Death Detection ELISA PLUS Kit (11774425001) was purchased from Roche (USA).

Cells were infected with PRV at an MOI of 1 or 10, as indicated. After incubation for 1 h at 37°C, the unbound virus was removed by washing the cells three times with PBS. The cells were then cultured in DMEM supplemented with 2% FBS for the indicated times.

Western blot (WB) was performed as previously described (22). Briefly, the cells were collected and incubated in RIPA buffer containing a protease inhibitor cocktail (Roche) and 0.1 mM PMSF for 2 h. The protein concentration of the cell lysate supernatants was determined by BCA assay (Thermo). Equal amounts of protein were diluted in 5 × SDS-PAGE loading buffer and separated on SDS-PAGE gels. The proteins were blotted on polyvinylidene fluoride (PVDF) membranes (Millipore, USA), then blocked with 5% non-fat dry milk in PBST for 2 h and incubated with primary antibodies at 4°C overnight. The membranes were incubated with secondary antibodies in a blocking solution. The reactive bands were detected by using the enhanced chemiluminescence system (ECL).

Flow cytometric analysis was performed as previously described (23). PK-15 cells were infected or mock-infected with PRV, followed by the addition of a chemical inducer or inhibitor. The cells were harvested after having been washed in cold PBS, and stained with FITC-labeled annexin V and PI (556547, BD) at room temperature for 15 min and analyzed by flow cytometer (BD Canto II) within half an hour. The cells in the second quadrant (annexin V-positive/PI-negative) were considered early apoptotic cells, and the cells in the fourth quadrant (annexin V-positive/PI-positive) were considered late apoptotic cells. Both of them were considered to be apoptotic cells.

Ten-fold dilutions of the virus-containing culture supernatants were added to 6-well plates with a confluent monolayer of Vero cells. The plates were then incubated at 37°C for 2 h, with gentle agitation at 30 min intervals. The excess virus was washed away with PBS. Subsequently, 2% low-melting-point agarose and 2 × DMEM containing 2% FBS were mixed and added to each well as an overlay medium. The cells were incubated at 37°C with 5% CO₂ for 3–4 days, then stained with 0.5% crystal violet, and the plaques were counted.

PK-15 cells were grown to 80% confluences in 6-well plates and transiently transfected with the indicated shRNAs (Supplementary Table 1) by using the transfection reagent (Roche) according to the manufacturer's instructions. Western blot was used to detect the silencing efficiency of the shRNAs.

ROS was measured using the non-fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as previously described (24). DCFH-DA is deacetylated by esterase to form non-fluorescent DCFH after diffusing into cells passively. ROS were produced when cells were under oxygen stress, and the fluorescent product DCF was formed from DCFH. PK-15 cells were transfected with shATG5 and shNC and infected with PRV 24 h later. Fifty millimeter NAC was added at the infection time to eliminate ROS. Ten micrometer DCFH-DA was added to cultures and incubated for 20 min at 37°C. After incubation, cells were washed in cold PBS and analyzed by flow cytometry. The fluorescent intensity was calculated by Image J software.

All dates were analyzed in SPSS. One-way ANOVA was used to evaluate the difference among one variable treatment. Two-way ANOVA was used to evaluate the difference among treatment groups. The data is expressed as the mean ± standard deviation (S.D.). P-value < 0.05 was considered statistically significant. The intensities of the immune bands were analyzed using Image J software (HIN).

To detect the apoptotic effects of PRV, we used the PK-15 cell line, which is highly susceptible to infection. PK-15 cells were infected by PRV at an MOI of 1, and then the percentage of apoptotic cells among infected PK-15 cells was determined by flow cytometric analysis at different time points. The results shown in Figure 1A demonstrate that PRV infection causes apoptosis at 12 h post-infection (hpi) and later (P < 0.01), but not at earlier time points. The percentage of apoptotic cells shown in the histogram (Figure 1A) ranged between 25% and 40%. The amount of cleaved caspase-3 and PRV gE protein was detected by Western blot and the ratios of intensity of cleaved caspase-3 to ACTB were determined. Consistent with the results of flow cytometry, PRV-induced cleaved caspase-3 was detectable beginning at 12 hpi, and significant cleavage was found at 24 and 36 hpi (P < 0.01) (Figure 1B). In order to analyze the effect of the infectious dose on virus-induced apoptosis, PK-15 cells were infected by PRV at 0.1, 1 and 5 MOI for 24 h. The levels of cleaved caspase-3 and PRV gE protein were visualized by Western blot, and the ratios of intensity of cleaved caspase-3 to ACTB were determined. The cleavage of caspase-3 was increased along with the infection dosage in a dose-dependent manner, and an MOI of PRV as low as 0.1 can induce significant apoptosis (P < 0.05) (Figure 1C). Taken together, we confirmed that PRV is able to induce apoptosis in PK-15 cells, an event that occurs at the later stages of infection.

To investigate the effect of PRV infection on the level of autophagy, the status of the proteins LC3 and SQSTM1/P62 was analyzed in PRV-infected PK-15 cells by Western blot. The expression of LC3-II was determined up to 36 hpi (MOI = 1), and the autophagy level is represented as the ratio of LC3-II/ACTB band intensities. As shown in Figure 1D, the expression of LC3-II was significantly increased at 6 hpi (P < 0.01). A reduced expression of LC3-II was detected from 12 to 36 hpi in PRV-infected cells compared to that at 6 hpi. These results indicate that PRV-induced autophagy occurs at the early stage of infection in PK-15 cells. Of note, the level of the autophagy marker protein LC3-II decreased progressively along with an increased expression of PRV gE protein during the course of infection time, suggesting that PRV inhibits the level of autophagy at the late stage of infection. To visualize LC3 II associated with the autophagic membrane, LC3 fused with a green fluorescent protein (GFP) was used. The LC3 can redistribute from a diffuse cellar localization to distinctive puncta cytoplasmic pattern during autophagy, revealing the recruitment of LC3 to autophagic vesicles (13). PK-15 cells were infected with PRV at an MOI of 1 following transfection with GFP-LC3 for 24 h and then observed with confocal microscopy at different time points after infection. GFP-LC3 proteins were distributed as foci in most of the PRV-infected PK-15 cells at 4 hpi. In contrast, GFP-LC3 remained diffusely distributed throughout the cytoplasm at 24 hpi both in uninfected cells and in PRV-infected cells (Figure 1E). The number of autophagic cells was quantified as the percentage of GFP-LC3 puncta expressing cells among total GFP-expressing cells. Approximately 32.4 and 14.8% of PRV-infected PK-15 cells had formed GFP-LC3 puncta at 4 and 24 hpi, respectively (Figure 1E). These results provide strong evidence that PRV infection induced autophagy at the early infection stage. In order to determine the autophagic degradation during PRV infection, P62, previously described as autophagosome cargo was analyzed using Western blot. The result showed that the level of P62 decreased at the early stage of infection and then increased at 12 hpi and later time points. Chloroquine (CQ) was used to analyze the turnover of autophagosomes. The PRV-infected and mock-infected cells were pre-treated with CQ for 4 h. The expression of LC3-II and P62 was analyzed and quantified at 2 and 8 hpi. Our results showed that CQ increased the level of LC3-II and P62 in PRV-infected cells when compared to PRV-infected cells in the absence of CQ treatment. These results demonstrate that PRV infection enhances the autophagy flux (Figure 1F). Taken together, our results indicate that PRV infection has a dual effect on autophagy: PRV enhances autophagic degradation as well as the autophagy flux at the early stage of infection and decreased autophagy level at the late stage of infection.

The PK-15 cells were pre-treated with 5 mM 3-MA to inhibit the autophagy levels for 4 h and then infected by PRV. The effects of PRV infection on the regulation of autophagy and apoptosis response were analyzed by Western blot (Figure 2A) and flow cytometric analysis (Figure 2C) at 12 hpi. As shown in Figures 2A, B, PRV infection showed a decreased LC3-II level and enhanced cleaved caspase-3 in 3-MA pre-treated cells when compared to non-3-MA treatment cells. The flow cytometric analysis showed in 3-MA pre-treated cells an increase of apoptosis to 59% in PRV-infected cells (P < 0.05) (Figure 2C). The change in the rate of cell death was also analyzed by ELISA, and the results confirmed that 3-MA pretreatment significantly increases the rate of cell death in PRV-infected cells (P < 0.05) (Figure 2D). Next, short hairpin RNA (shRNA) targeting ATG5 was used to decrease autophagy level in PK-15 cells. The regulation of autophagy and interference efficiency is shown in Figure 2E. Treatment with shATG5 reduced the expression of ATG5 and significantly inhibited autophagy in mock- and PRV-infected cells. The Western blot results indicate that the shRNA-induced decrease of the autophagy level is paralleled by an enhanced expression of viral gE protein (Figure 2E). Subsequently, we determined the apoptosis rate in PRV-infected ATG5 knockdown cells. The flow cytometry results revealed that knockdown of autophagy with shRNA targeting endogenous ATG5 resulted in an increase in the percentage of apoptosis cells during PRV infection (Figure 2F). These results suggest that inhibition of the level of autophagy at the early stage of PRV infection may up-regulate PRV-induced apoptosis and facilitate virus replication.

To further verify the regulation of autophagy and apoptosis at the late stage of PRV infection, the virus-induced apoptosis response was analyzed when cells were treated with rapamycin, an autophagy inducer. At 36 hpi, the cells were collected and analyzed by Western blot and flow cytometry to detect the levels of autophagy and apoptosis. The results showed that infection of PRV in 100 nM rapamycin-pretreated PK-15 cells increased the amount of LC3-II and decreased cleaved caspase-3 when compared with PRV infection of cells that had not been pre-treated (Figures 3A, B). This indicates that by enhancing the autophagy level, rapamycin compromises the effect of PRV infection, inhibiting LC3-II production at 36 hpi. Besides, the expression of PRV gE protein was notably reduced in PRV-infected rapamycin-pretreated PK-15 cells. Then we analyzed the apoptosis rate in PRV-infected rapamycin-pretreated cells by flow cytometry and ELISA. These results were consistent with the results obtained by Western blot, i.e., enhancement of the autophagy level significantly decreased the number of apoptotic cells among PRV-infected cells (P < 0.01) as well as the amount of the dead cells determined by ELISA (P < 0.01) (Figures 3C, D). Taken together, our results indicate that enhancement of the autophagy level interferes with the induction of apoptosis during PRV infection.

It has been reported that apoptosis could regulate autophagy response through caspase or other proteins (25, 26). In order to test the role of apoptosis in regulating autophagy during the late stage of PRV infection, Z-VAD-FMK (30 μM) was used to block caspase-dependent apoptosis. The expression of LC3-II and the cleaved caspase-3 after Z-VAD-FMK treatment were detected by Western blot. Our results showed that Z-VAD-FMK slightly decreased the amount of cleaved caspase-3 in PRV-infected cells. In addition, an increased expression of LC3-II was detected along with the inhibition of apoptosis by Z-VAD-FMK in PRV-infected cells (Figures 4A, B). The localization of LC3 in Z-VAD-FMK-treated PK-15 cells was visualized using GFP-LC3. Cells were treated with Z-VAD-FMK and infected with PRV followed by transfection of GFP-LC3 for 24 h and apoptotic cells were stained with TUNEL. Figure 4C showed that PRV infection induced positive TUNEL staining and diffuse GFP-LC3 distribution at 24 hpi; Z-VAD-FMK treatment decreased the number of apoptotic cells, and GFP-LC3 puncta that is a marker of autophagic vesicle formation were formed in non-apoptotic cells which showed no TUNEL stains. And the number of autophagic cells was quantified as the percentage of GFP-LC3 puncta-positive cells to total GFP-expressing cells in PRV infection and Z-VAD-FMK-treated PRV infection cells. The percentage of GFP-LC3 puncta-positive cells increased from approximately 12.4–26.7% when PRV infect on Z-VAD-FMK pre-treated PK-15 cells (Figure 4D). Our results indicate that apoptosis inhibition can increase autophagy may mediate by caspase.

Both autophagy and apoptosis play a role in viral infection and propagation, and our results suggest that PRV infection regulates these two pathways. To evaluate which process affected viral propagation, the amount of infectious virus in autophagy and/or apoptosis-impaired cells was measured. The autophagy inhibitors, 3-MA and shATG5, increase PRV gE protein expression (as shown in Figures 2A, D). The effect of 3-MA on virus production was analyzed by determining the infectivity (PFU assay). Concentrations of 10 and 30 mM 3-MA increased the infectious PRV in PK-15 cells (Figure 5A), which is consistent with the viral gE protein expression. Next, the effect of rapamycin on PRV replication was determined. Figure 5B showed that when cells were pre-treated with rapamycin, the virus titer was significantly reduced in a dose-dependent manner. The effect of the apoptosis inhibitor Z-VAD-FMK was determined in the same way. The amount of infectious PRV was reduced significantly (Figure 5C) when PK-15 cells were pre-treated with 10, 30, or 50 μM Z-VAD-FMK, respectively. To verify whether the Z-VAD-FMK-induced decrease of the virus titer affects virus release, the virus was collected from the supernatants as well as from cell lysates. The results show that Z-VAD-FMK treatment had no influence on the ratio of PRV titer between supernatants and cell lysates compared to that determined with control cells (Figure 5D), indicating that the apoptosis-induced increase of viral production is not due to an enhanced PRV release.

Drugs that influence either autophagy or apoptosis play roles in the replication of the virus, and autophagy and apoptosis cross-inhibit each other in PRV-infected PK-15 cells. To further verify the interaction of these two processes during PRV replication, autophagy-knockdown cells were pre-treated with Z-VAD-FMK, and the amount of infectious PRV in infected cells was assessed. As shown in Figure 5E, in shNC cells, Z-VAD-FMK treatment decreased the PRV titers, while in autophagy-impaired cells (shAtg5 cells), the Z-VAD-FMK treatment showed little or only a reduced effect on the replication of PRV, which indicates that autophagy may have a more important role in PRV replication when compared with apoptosis. The autophagy and apoptosis levels in these drug-treated cells were also determined by Western blot, as shown in Figure 5F. Based on these data, we conclude that PRV infection can be regulated by apoptosis, which acts mainly via inhibiting autophagy. An increased autophagy level reduces the replication of PRV in the Z-VAD-FMK-treated cells.

It has been reported that a high level of ROS is a primary cause of apoptosis under both physiologic and pathologic conditions, and the oxidized substance is degraded during the autophagy process. Therefore, we analyzed whether ROS are involved in the cross-talk between PRV-induced apoptosis and autophagy. For this purpose, we determined the intracellular ROS levels and the apoptotic rates in autophagy-knockdown cells (ATG5 shRNA) by flow cytometry, as shown in Figures 6A, B. The addition of the ROS inhibitor (NAC) at a concentration of 50 mM abolished the staining confirming the specificity of this assay. The ROS level in shATG5-treated cells was significantly higher than they were in shNC-treated cells at 48 hpi (Figure 6A). The percentage of apoptosis in PRV-infected shATG5 knockdown cells was about 63% which was notably higher than that in virus-infected shNC cells, approximately 42%. Furthermore, the NAC treatment decreased the percentage of apoptotic cells in PRV-infected autophagy-impaired cells. However, no different apoptosis level was detected between PRV-infected shATG5 and shNC cells in the presence of NAC (Figure 6B). These data suggest that ROS accumulation mediates the apoptosis in autophagy-impaired cells.