The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection

Galindo, I; Hernáez, B; Muñoz-Moreno, R; Cuesta-Geijo, M A; Dalmau-Mena, I; Alonso, C

To study the impact of apoptosis on ASFV infection, we infected Vero cells treated with an inhibitor of caspase 3 activation (Ac-DEVD-CHO) and with a specific inhibitor of myosin II ATPase activity (Blebbistatin). Myosin II ATPase activity, together with the GTPase ROCK-I, is necessary for cell membrane fragmentation into multiple apoptotic bodies in the execution phase of apoptosis. Also, we analyzed the effect of Y-27632, a ROCK-I inhibitor that blocks membrane blebbing. We then compared final infective virus production at 48 hpi when these drugs were added to the cells, either 2 h before infection, or at 6 hpi. Our results showed that neither blebbing nor caspase 3 inhibition modified total virus production (extracellular plus intracellular virus production; Figure 2a ). Caspase 3 activation in early stages of the infection was required for virus exit, whereas the inhibition of membrane blebbing reduced the release of virus particles from the cell and this effect was enhanced when the inhibitor was added 6 hpi ( Figure 2b ). In order to asses a possible role of caspase 12 in caspase 3 and 9 activation, we analyzed the effect of caspase 12 inhibition in infected cells using Z-ATD-FMK. Addition of this inhibitor before infection did not affect percentages of ASFV-infected cells as detected by flow cytometry (fluorescence-activated cell sorter (FACS); Figure 3a ). Also, this inhibitor did not cause a decrease in virus production nor in the exit of virus particles ( Figure 3b ). Moreover, our results showed that neither caspase 3 nor caspase 9 were inhibited by Z-ATD-FMK and only caspase 12 activation was specifically inhibited (Figure 3c ).