Pseudorabies virus gM and its homologous proteins in herpesviruses induce mitochondria-related apoptosis involved in viral pathogenicity
Fig 8
Deletion of caspase-3 impairs PRV and HSV-1 replication in vitro.
(A-B) Detection of cells viability. HeLa cells pretreated with Z-VAD or Z-DEVD, followed by PRV infection at an MOI of 1 for 24 h, were analyzed for the LDH released in the supernatants (A) and cellular ATP enzymatic activity (B). (C-D) Caspase-3 inhibitors reduced PRV replication. HeLa cells were pretreated with Z-VAD or Z-DEVD, followed by PRV infection at MOI of 0.5, 1 for 24 h. The cell pellets were used for the PRV DNA copy number assay, and the cell supernatants were collected for the TCID50 assay. (E) Deletion of caspase-3 eliminated PRV infection-mediated apoptosis. HeLa-WT and HeLa-caspase-3-/- cells infected with PRV were stained with PI/Annexin V for flow cytometry. The percentage of PI- and Annexin V-labeled cells was quantified. (F-J) Deletion of caspase-3 gene impaired PRV replication. The pellets of PRV-infected HeLa-WT and HeLa-caspase-3-/- cells were used for detecting PRV DNA copy numbers using qPCR (F-G) or PRV-encoded UL42 protein levels using western blotting (H). The cell supernatants were collected for the TCID50 assay in Vero cells (I-J). (K) Deletion of caspase-3 eliminated HSV-1 infection-mediated apoptosis. HeLa-WT and HeLa-caspase-3-/- cells infected with HSV-1 were stained with PI/Annexin V for flow cytometry. The percentage of PI- and Annexin V-labeled cells was quantified. (L-O) Deletion of caspase-3 diminished HSV-1 replication. The pellets of HSV-1-infected HeLa-WT and HeLa-caspase-3-/- cells were used for HSV-1 genomic DNA detection using qPCR, and the cell supernatants were collected for TCID50 assay in Vero cells. Results shown are representative of three independent experiments (mean ± SD) or of three independent experiments with similar results (one-way ANOVA in panels A-D; two-way ANOVA in panels E-G, I-O). *, P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.