Fig 1.
Apoptosis detection induced by PAstV infection.
(A, B) PK-15 cells were infected with PAstV1-GX1 at an MOI of 0.1. The cells were subsequently stained using an Annexin V-FITC/PI Apoptosis Detection Kit, and apoptosis was assessed via fluorescence microscopy (A) and flow cytometry (B). (C) The proportions of early apoptotic (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells identified through flow cytometry were quantified. Data are from three independent experiments (n = 3) and are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA. Statistically significant differences compared to the mock-infected group are indicated by ****, p < 0.0001. (D) Transmission electron microscopy was employed to observe the morphological features of apoptotic cells induced by PAstV infection. The green arrow indicates intact cell membranes and organelles, while the yellow and red arrows denote apoptotic bodies containing damaged organelles and viral particles, respectively.
Fig 2.
PAstV infection causes mitochondrial damage and induces apoptosis through the mitochondrial pathway.
(A) PK-15 cells were either mock-infected or infected with PAstV1-GX1 (MOI = 0.1). Cell lysates were collected at 6, 12, and 24 hpi and then subjected to Western blot analysis using antibodies specific for caspase-3, caspase-9, caspase-8, PAstV nsP1a/4, and β-actin. The relative expression ratios of cleaved caspase-3 to pro-caspase-3 and cleaved caspase-9 to pro-caspase-9 were quantified by grayscale analysis using ImageJ software. (B, C) PK-15 cells infected with PAstV1-GX1 were subjected to subcellular fractionation to isolate cytoplasmic and mitochondrial fractions. The cytochrome c expression levels in the cytoplasmic (B) and mitochondrial (C) fractions were detected by Western blot analysis; β-actin and VDAC1 served as loading controls for the cytoplasmic and mitochondrial fractions, respectively. (D, E) Mitochondrial membrane potential (ΔΨm) was assessed in PAstV1-GX1-infected and mock-infected cells by using the JC-1 probe, with CCCP treatment (10 µM) serving as a positive control (D). The relative fluorescence intensity ratio of green to red fluorescence (JC-1 monomer to JC-1 aggregate) was quantified using ImageJ software (E). Bar graphs display the mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA, with significant differences versus the mock-infected group are indicated by ****, p < 0.0001. (F) Representative transmission electron microscopy (TEM) images showing cellular and mitochondrial morphology in cells of the PAstV1-GX1-infected and mock-infected groups. The green and red arrows denote normal and damaged mitochondria, respectively.
Fig 3.
Analysis of apoptosis modulation on PAstV replication.
(A, B) PK-15 cells were infected with PAstV1-GX1 (MOI = 0.1) and treated with or without Z-VAD-FMK (10 μM) (A) or ABT-263 (5 μM) (B). Cell lysates were collected at 6, 12, and 24 hpi and subjected to Western blot analysis using antibodies against caspase-3, PAstV nsP1a/4, and β-actin. The relative expression ratios of nsP1a/4 to β-actin and cleaved caspase-3 to pro-caspase-3 were quantified by grayscale analysis using ImageJ software. (C, D) PK-15 cells were infected with PAstV1-GX1 (MOI = 0.1) and treated with or without Z-VAD-FMK (10 μM). Intracellular (C) and extracellular (D) viral RNA levels at the indicated time points post-infection were quantified by RT-qPCR. (E, F) PK-15 cells infected with PAstV1-GX1 (MOI = 0.1) were treated with ABT-263 (5 μM) at 6, 12, or 18 hpi. Intracellular (E) and extracellular (F) viral RNA copies were measured at 24 hpi using RT-qPCR. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analyses were conducted using two-way ANOVA. Statistical significance compared to the untreated control is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 4.
Identification of key viral proteins involved in PAstV-induced apoptosis.
(A, B) PK-15 cells were transfected with eukaryotic expression plasmids encoding nsP1a/1, nsP1a/3, nsP1a/4, capsid protein, or an empty vector control. Apoptosis was assessed at 24 hours post-transfection by Annexin V-FITC/PI staining and flow cytometry. Representative flow cytometry plots are shown in (A). The proportions of early apoptotic (Annexin V ⁺ /PI⁻) and late apoptotic (Annexin V ⁺ /PI⁺) cells were quantified in (B). Data are from three independent experiments (n = 3) and are presented as mean ± SD. Statistical analyses were conducted using one-way ANOVA. Statistically significant differences compared to the empty vector group are indicated as **p < 0.01 and ****p < 0.0001. (C) PK-15 cells were transfected with the indicated plasmids. Cell lysates were collected at 24 hours post-transfection and subjected to Western blot analysis using antibodies specific for caspase-3, caspase-9, and β-actin. The relative expression ratios of cleaved caspase-3 to pro-caspase-3 and cleaved caspase-9 to pro-caspase-9 were quantified by grayscale analysis using ImageJ software. (D) PK-15 cells were transfected with increasing amounts (1, 2.5, or 5 µg) of pCAGGS-Flag-nsP1a/3 or empty vector for 24 hours. Western blot analysis was performed on cell lysates using antibodies against caspase-3, caspase-9, Flag, and β-actin. The relative expression ratios of cleaved caspase-3 to pro-caspase-3 and cleaved caspase-9 to pro-caspase-9 were quantified by grayscale analysis using ImageJ software. (E) BHK-21 cells were subjected to co-transfection with pDsRed2-Mito and the specified viral protein expression plasmids. At 24 hours post-transfection, confocal microscopy was utilized to evaluate the co-localization of PAstV proteins (green), tagged with an anti-Flag antibody and a CoraLite Plus 488-conjugated secondary antibody, with mitochondria (red), marked by pDsRed2-Mito. The fluorescence intensity profiles of viral proteins (green) and mitochondria (red) along a defined line were quantified using ImageJ. Scale bar, 5 μm.
Fig 5.
Identification of nsP1a/3 protein interaction with MAVS.
(A, B) HEK-293T cells were co-transfected with pCAGGS-Flag-nsP1a/3 and pCAGGS-HA-MAVS plasmids for 24 hours. Cell lysates were subjected to co-IP with anti-Flag (A) or anti-HA beads (B). The precipitated proteins, along with whole-cell lysates (WCL), were analyzed by Western blot using anti-HA and anti-Flag antibodies. (C, D) PK-15 cells were transfected with plasmid pCAGGS-Flag-nsP1a/3 for 24 hours. Lysates were subjected to co-IP with anti-Flag (C) or anti-MAVS (D) beads. The precipitated proteins and WCL were then analyzed by Western blot using anti-MAVS and anti-Flag antibodies. (E, F) PK-15 cells were infected with PAstV1-GX1 at an MOI of 0.1 for 24 hours. The lysates were subjected to co-IP with anti-nsP1a/3 (E) or anti-MAVS (F) beads. The precipitated proteins and WCL were analyzed by Western blot using anti-MAVS and anti-nsP1a/3 antibodies. β-actin was used as a loading control. (G) BHK-21 cells were co-transfected with pCAGGS-HA-MAVS and either pCAGGS-Flag-nsP1a/3 or pCAGGS-Flag-nsP1a/1. After 24 hours, confocal immunofluorescence was conducted using anti-Flag and anti-HA antibodies, with nuclei stained by DAPI. The right panels display fluorescence intensity profiles of Flag-tagged nsP1a/1 or nsP1a/3 (green) and HA-tagged MAVS (red) measured using ImageJ. Scale bar: 5 μm.
Fig 6.
Effect of MAVS knockdown on PAstV1-GX1-induced apoptosis and viral replication in PK-15 cells.
(A, B) PK-15 cells were transfected with MAVS-targeting siRNA (siMAVS) or control siRNA (siNC) and then infected with PAstV1-GX1 (MOI = 0.1). Apoptosis was assessed at 6, 12, and 24 hpi using Annexin V-FITC/PI staining and flow cytometry. Representative flow cytometry plots are shown in (A). The proportions of early apoptotic (Annexin V ⁺ /PI⁻) and late apoptotic (Annexin V ⁺ /PI⁺) cells were quantified in (B). Data are from three independent experiments (n = 3) and are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA. Statistically significant differences compared to the siNC group are indicated as ****, p < 0.0001. (C) PK-15 cells transfected with siMAVS or siNC were infected with PAstV1-GX1 (MOI = 0.1). Cell lysates were harvested at 24 hpi and subjected to Western blot analysis using antibodies against MAVS, caspase-3, caspase-9, nsP1a/4, and β-actin. The relative expression ratios of MAVS to β-actin, cleaved caspase-3 to pro-caspase-3, cleaved caspase-9 to pro-caspase-9, and nsP1a/4 to β-actin were quantified using ImageJ software for grayscale analysis. (D) Viral RNA levels in the cell lysates (upper panel) and supernatants (lower panel) of cells transfected with siMAVS or siNC were measured by RT-qPCR at 6, 12, and 24 hpi. Data are presented as mean ± SD from three experiments (n = 3). Statistical analysis was performed using two-way ANOVA, with significant differences from the siNC group noted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) PK-15 cells transfected with siMAVS or siNC were infected with PAstV1-GX1 (MOI = 1). At 6, 12, and 24 hpi, cells were processed for IFA using a mouse anti-nsP1a/4 primary antibody and an FITC-conjugated goat anti-mouse IgG secondary antibody, and then observed by fluorescence microscopy. (F) Viral growth kinetics were determined by TCID₅₀ assay using cell and supernatant samples collected from siMAVS- or siNC-transfected PK-15 cells at the indicated times after infection with PAstV1-GX1 (MOI = 0.1). Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA. Statistically significant differences compared to the siNC group are indicated as ** p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 7.
Effect of MAVS overexpression on PAstV1-GX1-induced apoptosis and viral replication in PK-15 cells.
(A) PK-15 cells were transfected with increasing amounts (1, 2.5, and 5 μg) of pCAGGS-HA-MAVS or empty vector prior to infection with PAstV1-GX1 (MOI = 0.1). At 24 hpi, cell lysates were analyzed by Western blot using antibodies against caspase-3, caspase-9, MAVS, nsP1a/4, and β-actin. The relative expression ratios of cleaved caspase-3 to pro-caspase-3, cleaved caspase-9 to pro-caspase-9, cleaved MAVS to β-actin, and nsP1a/4 to β-actin were quantified using ImageJ software for grayscale analysis. (B) RT-qPCR quantification of PAstV1-GX1 viral RNA levels in cell lysates (upper panel) and supernatants (lower panel) from PK-15 cells transfected with pCAGGS-HA-MAVS or empty vector. Data represent mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA, with statistically significant differences compared to the empty vector control group denoted by ****p < 0.0001. (C) PK-15 cells transfected with pCAGGS-HA-MAVS or empty vector were infected with PAstV1-GX1 (MOI = 1). At 6, 12, and 24 hpi, cells were processed for IFA using a mouse anti-nsP1a/4 primary antibody and an FITC-conjugated goat anti-mouse IgG secondary antibody, followed by observation under a fluorescence microscope.
Fig 8.
PAstV nsP1a/3 cleaves MAVS and inhibits type I IFN production.
(A) PK-15 cells were co-transfected with 1 µg of pCAGGS-HA-MAVS and increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control. Cell lysates were harvested 24 h post-transfection and analyzed by Western blot using antibodies against HA, Flag, and β-actin. The relative ratio of cleaved exogenous MAVS to β-actin was quantified via grayscale analysis using ImageJ software. (B) PK-15 cells were transfected with increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control. Cell lysates were collected at 24 h post-transfection and subjected to Western blot analysis using antibodies targeting MAVS, Flag, and β-actin. The relative expression level of cleaved endogenous MAVS to β-actin was quantified by grayscale analysis with ImageJ software. (C, D, E) PK-15 cells were transfected with increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control, followed by infection with SeV (10 HAU). The relative mRNA levels of porcine IFN-β (C), ISG15 (D), and ISG56 (E) were assessed by RT-qPCR and normalized to porcine β-actin. (F) PK-15 cells were co-transfected with pCAGGS-Flag-nsP1a/3 or empty vector control, along with pIFN-β-Luc and pRL-TK reporter plasmids, and then infected with SeV. Luciferase activities were measured at 6, 12, and 24 hpi. (G) PK-15 cells were transfected with increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control, followed by infection with SeV (10 HAU). The relative mRNA levels of SeV were assessed by RT-qPCR and normalized to porcine β-actin. (H, I, J) PK-15 cells were transfected with increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control, followed by infection with PAstV1-GX1 at an MOI of 3. The relative mRNA levels of porcine IFN-β (H), ISG15 (I), and ISG56 (J) were assessed by RT-qPCR and normalized to porcine β-actin. (K) PK-15 cells were transfected with increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control and subsequently infected with PAstV1-GX1 at an MOI of 3. PAstV mRNA copy numbers were measured by RT-qPCR at 24 hpi. (L, M, N, O) HEK 293T cells were transfected with pCAGGS-Flag-nsP1a/3 or empty vector control, followed by infection with SeV (10 HAU). The mRNA expression levels of human IFN-β (L), ISG15 (M), ISG56 (N), and SeV (O) were determined by RT-qPCR and normalized to human β-actin. (P) PK-15 cells were transfected with increasing amounts of pCAGGS-Flag-nsP1a/3 or empty vector control. At 24 hours post-transfection, the cells were infected with SeV for 12 hours, and the culture supernatants were collected. Following UV irradiation, the supernatants were transferred to fresh PK-15 cells. After 24 hours of incubation, these recipient cells were infected with VSV-GFP. Viral replication was assessed 12 hours later by fluorescence microscopy. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA. Significant differences compared to the empty vector control group are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 9.
Inhibition of apoptosis does not affect nsP1a/3-induced MAVS cleavage and type I IFN suppression.
(A) PK-15 cells were transfected with pCAGGS-Flag-nsP1a/3 and treated with or without 10 μM Z-VAD-FMK. Cell lysates were harvested at 24 hours post-transfection and subjected to Western blot analysis using antibodies against MAVS, caspase-3, Flag, and β-actin. The relative expression ratios of cleaved MAVS to pro-MAVS and cleaved caspase-3 to pro-caspase-3 were quantified by grayscale analysis using ImageJ software. (B) PK-15 cells transfected with pCAGGS-Flag-nsP1a/3 were treated with increasing concentrations of Z-VAD-FMK (5, 10, 15 µM) or DMSO as a control. Lysates were collected 24 hours post-transfection and analyzed by Western blot with antibodies specific for MAVS, caspase-3, Flag, and β-actin. The relative expression ratios of cleaved MAVS to pro-MAVS and cleaved caspase-3 to pro-caspase-3 were quantified based on grayscale values using ImageJ. (C, D, E, F) PK-15 cells transfected with either pCAGGS-Flag-nsP1a/3 or an empty vector were infected with SeV. The relative mRNA levels of porcine IFN-β (C), ISG15 (D), ISG56 (E), and SeV (F) were measured by RT–qPCR at 6, 12, and 24 hpi, normalized to porcine β-actin mRNA. (G) PK-15 cells were mock-transfected or transfected with pCAGGS-Flag-nsP1a/3 and treated with or without Z-VAD-FMK. At 24 hours post-transfection, cells were infected with SeV for 12 hours. Supernatants were then collected, inactivated by UV irradiation, and applied to fresh PK-15 cells. After 24 hours of incubation, the recipient cells were infected with VSV-GFP, and viral replication was assessed by monitoring GFP fluorescence at 12 hpi. Data are presented as the mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA, and significant differences compared to the empty vector control group are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 10.
Effect of catalytic triad mutation in nsP1a/3 on MAVS cleavage, apoptosis and IFN-β induction.
(A) Schematic representation of the mutagenesis of the serine protease catalytic triad (His459, Asp487, Ser549) in nsP1a/3 to alanine. The resulting mutant and wild-type plasmids were designated as pCAGGS-Flag-nsP1a/3-3C-/- and pCAGGS-Flag-nsP1a/3WT, respectively. (B) PK-15 cells were transfected with either pCAGGS-Flag-nsP1a/3WT or pCAGGS-Flag-nsP1a/3-3C-/-. Cell lysates were collected at 6, 12, and 24 hours post-transfection and subjected to Western blot analysis using antibodies against MAVS, caspase-3, Flag, and β-actin. The relative expression levels of cleaved MAVS to pro-MAVS and cleaved caspase-3 to pro-caspase-3 were quantified by grayscale analysis using ImageJ. (C) PK-15 cells were transfected with increasing doses (1, 2.5, and 5 μg) of pCAGGS-Flag-nsP1a/3WT or pCAGGS-Flag-nsP1a/3-3C-/-. Lysates were harvested at 24 hours post-transfection and analyzed by Western blot with antibodies specific for MAVS, caspase-3, Flag, and β-actin. The ratios of cleaved MAVS to pro-MAVS and cleaved caspase-3 to pro-caspase-3 were quantified based on grayscale values using ImageJ. (D, E, F, G) PK-15 cells transfected with pCAGGS-Flag-nsP1a/3WT or pCAGGS-Flag-nsP1a/3-3C-/- were infected with SeV. The relative mRNA levels of porcine IFN-β (D), ISG15 (E), ISG56 (F), and SeV (G) were measured by RT–qPCR at 6, 12, and 24 hpi and normalized to porcine β-actin mRNA. (H) PK-15 cells were either mock-transfected or transfected with pCAGGS-Flag-nsP1a/3WT, pCAGGS-Flag-nsP1a/3-3C-/-, or empty vector plasmid. At 24 hours post-transfection, cells were infected with SeV for 12 hours. Culture supernatants were collected, inactivated by UV irradiation, and applied to fresh PK-15 cells. After 24 hours, recipient cells were infected with VSV-GFP. Viral replication was assessed by monitoring GFP fluorescence at 12 hpi. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA. Statistical significance compared to the empty vector control group is indicated as ****p < 0.0001.
Fig 11.
Serine protease inhibitor treatment suppresses PAstV replication.
(A) PK-15 cells infected with PAstV1-GX1 (MOI = 0.1) were treated with increasing concentrations (5, 10, and 15 μM) of SBTI. Viral RNA copy numbers in cell lysates were quantified by RT-qPCR at 24 hpi. (B) PK-15 cells were either mock-infected or infected with PAstV1-GX1 (MOI = 0.1), followed by treatment with 15 μM SBTI or DMSO (control). Viral replication was assessed via IFA using a mouse anti-nsP1a/4 polyclonal antibody and an FITC-conjugated goat anti-mouse IgG secondary antibody. (C) Cell viability of PK-15 cells treated with SBTI (5, 10, and 15 μM) was evaluated using a CCK-8 assay. The data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA. ****p < 0.0001 indicates a statistically significant difference compared to the control group.
Fig 12.
The model illustrates the dual function of PAstV nsP1a/3 in regulating apoptosis and innate immunity.
(By Figdraw.com). Upon invasion of host cells by PAstV, the nsP1a/3 protein plays a dual role. Firstly, it activates caspase 9 and caspase 3, leading to apoptosis and facilitating viral replication through the mitochondrial pathway. Secondly, the nsP1a/3 protein cleaves MAVS, thereby inhibiting the production of type I IFN. Mutation of the serine protease catalytic triad in nsP1a/3 to alanine significantly reduces its ability to induce apoptosis and suppress type I IFN production. Additionally, treatment with soybean trypsin inhibitor (SBTI) has been shown to inhibit PAstV replication.
Table 1.
Sequences of the PCR primers used in this study.