The BmCPV-derived viral small peptide vsp1S4(-) suppresses viral replication by triggering apoptosis via the ROS-JNK signalling pathway

Figures

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  Fig. 1. vsp1S4(-) induces caspase-dependent apoptosis in BmN cells. (A) Real-time PCR analysis of pro-apoptotic genes (SUR1, SUR2, APAF, and Caspase) in BmN cells transfected with pUC57-OpIE2-rORF1 [vsp1S4(-)] or the empty vector control pUC57-OpIE2 (48 h posttransfection). The data were normalized to actin and are presented as the mean fold change ± SD (n = 3). ****P < 0.0001. (B) Western blotting analysis of cleaved caspase-3 (C-Cas3) in transfected cells (representative blot on the left, quantification on the right). The data are presented as the means ± SDs (n = 3). **P < 0.01, ****P < 0.0001). (C) Flow cytometry analysis of apoptosis via Annexin V/PI staining (representative plots in D, quantification in E). The data are presented as the means ± SDs (n = 3). *P < 0.5. (D) Effects of vsp1S4(-) on the mitochondrial membrane potential. The data are presented as the means ± SDs (n = 3). ***P < 0.001, ****P < 0.0001. (E) Cell cycle distribution analyzed by flow cytometry (PI staining) in BmN cells transfected with vsp1S4(-) or the control vector (48 h posttransfection). Representative histograms (left) and quantification of cell cycle phases (right) are shown. The data are presented as…

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  Fig. 2. vsp1S4(-) triggers mitochondrial ROS-dependent apoptosis in BmN cells. (A) Intracellular ROS levels measured by DCFH-DA fluorescence in BmN cells transfected with vsp1S4(-) or the control vector (48 h posttransfection). The data are presented as the mean fluorescence intensity (MFI) ± SD (n = 3). ****P < 0.0001. (B) Cell viability (CCK-8 assay) of BmN cells treated with H2O2 (0-0.5 mM) for 24 h. Data are presented as the means ± SDs (n = 3). ****P < 0.0001. (C) Viability of BmN cells treated with NAC (0–8 mM) for 24 h. Data are presented as the means ± SDs (n = 3). ****P < 0.0001. (D) Flow cytometry analysis of the effects of H2O2 and NAC on apoptosis induced by vsp1S4(-). The data are presented as the means ± SDs (n = 3). ***P < 0.001, ****P < 0.0001. (E) ROS levels (DCFH-DA fluorescence) in vsp1S4(-) BmN cells treated with 0.2 mM H2O2 or 2 mM NAC for 6 h. Representative histograms (left) and quantification of the MFI (right). The data are presented as the means ± SDs (n = 3). ***P < 0.001, ****P < 0.0001. (F) Western blotting analysis of cleaved caspase-3 (C-Cas3) in the same treatment groups

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  Fig. 3. vsp1S4(-) activates JNK signalling. (A) Transcriptomic analysis (RNA-seq) of MAPK signalling pathway enrichment in vsp1S4(-) -transfected vs. control cells (48 h). (B) Western blot analysis of phosphorylated JNK (p-JNK) and total JNK in transfected cells. The data are presented as the mean p-JNK/JNK ratio ± SD (n = 3). **P < 0.01. (C, D) Cell viability (CCK-8 assay) of vsp1S4(-) -transfected BmN cells treated with anisomycin (JNK agonist, 0–50 µM) or SP600125 (JNK inhibitor, 0–50 µM) for 24 h. Data are presented as the means ± SDs (n = 3). ****P < 0.0001. (E) Western blotting analysis of p-JNK, c-JNK, and C-Cas3 in vsp1S4(-) -transfected cells treated with 5 µM anisomycin or 5 µM SP600125 for 12 h. (F) Flow cytometry analysis of the effects of anisomycin and SP600125 on the degree of apoptosis induced by vsp1S4(-). The data are presented as the means ± SDs (n = 3). ****P < 0.0001

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  Fig. 4. ROS-induced apoptosis requires JNK activation. (A) vsp1S4(-) BmN cells were treated with 0.2 mM H2O2 alone or in combination with 5 µM anisomycin (a JNK agonist) or 5 µM SP600125 (a JNK inhibitor) for 2 h, followed by overnight culture. Apoptosis quantification by flow cytometry (annexin V/PI). The data are presented as the means ± SDs (n = 3). ****P < 0.0001. (C, D) Western blot analysis of p-JNK and C-Cas3 in the same treatment groups is shown in Fig. 4A

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  Fig. 5. vsp1S4(-) suppresses viral replication by triggering apoptosis via the ROS-JNK signaling pathway. (A, B) Western blotting analysis of the viral structural protein VP7 in vsp1S4(-) -transfected BmN cells treated with H2O2 (0.2 mM), anisomycin (5 µM), NAC (5 mM), or SP600125 (10 µM) for 24 h