Figure 4

Overexpression of eIF2αwt or eIF2α(S51D) in PK15 cells inhibited PRV replication. A PK15 cells were transfected separately with the pCMV-HA, eIF2αwt, eIF2α(S51D), and eIF2α(S51A) constructs for 24 h and were then infected with PRV. Cells were subjected to puromycin labelling for 1 h at 23 hpi and were then harvested at 24 hpi. Western blot analysis was performed to detect eIF2α and PRV proteins. PRV protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes (bottom panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01, *** p < 0.001. B, C PK15 cells were transfected separately with the pCMV-HA, eIF2αwt, eIF2α(S51D), or eIF2α(S51A) constructs for 24 h and were then infected with PRV for another 24 h. Supernatants and cells were harvested. RT–qPCR was performed to determine the relative mRNA level of PRV-gE compared to β-actin (B); the viral titre in the supernatants was determined in PK15 cells (C). The data are presented as the mean ± SD of three independent experiments. *** p < 0.001. D De novo protein synthesis was measured in pCMV-HA-, eIF2αwt-, eIF2α(S51D)-, and eIF2α(S51A)-transfected cells by using a monoclonal antibody against puromycin; the intensities of bands corresponding to puromycin-labelled proteins were quantified by densitometry and normalized to β-actin and are shown as fold changes (right panel). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05. *** p < 0.001.