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

ASFV infection activates the ATF6 pathway. In response to ER stress, ATF6 exits the ER to the Golgi apparatus, where it is processed by proteases to its active form, which in turn translocates to the nucleus. In order to monitor ATF6 activation by fluorescence microscopy, we used an EGFP (enhanced green fluorescent protein)-ATF6 fusion protein (pCMV short-EGFP-ATF6). 32 This protein was under the control of a shortened CMV promoter, which has a deletion of 430 bp from the 5 0 side in order to prevent overexpression, which would modify the localization of the fusion protein itself. The short promoter shows considerably less activity than the full promoter and GFP-ATF6 expressed under the short CMV promoter localizes exclusively to the ER and translocates to the nucleus, in a similar way to endogenous ATF6. 32 Cells transfected with the pCMV short-EGFP-ATF6 plasmid presented a typical ER pattern that relocated to the Golgi apparatus (Figure 6b ) or to the nucleus (Figure 6c ) in response to tunicamycin treatment. However, this characteristic pattern was modified in infected cells. Cells transfected with this construct and infected with ASFV showed GFP-ATF6 localization either in discrete perinuclear cytoplasmic areas corresponding to viral factories, as indicated by their co-localization with DNA stain (Figures 6d and e) , or in the nucleus of infected cells (Figure 6f ). This finding indicates that ASFV activates the ATF6 pathway of the UPR. We next studied the effect of a serine protease inhibitor that inhibits ER stress-induced proteolysis of ATF6, AEBSF 33 on ASFV infection. Addition of this inhibitor before infection affected the percentages of ASFV-infected cells in a dose-dependent manner as detected by FACS (Figure 7a) . AEBSF also induced a strong inhibition of viral production resulting in 100% reduction of viral titer at 300 mM concentrations of AEBSF (data not shown). We analyzed caspase activation under AEBSF to find that this agent abrogated caspase 3, 9 and 12 activation in infected cells. Inhibition of caspases was dependent of AEBSF effect on infection, given that this serine protease inhibitor did not prevent activation of caspase 3 induced by staurosporine (Figure 7b ).