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

Apoptosis is a genetically controlled cell death mechanism involved in the regulation of tissue homeostasis. We analyzed the mechanism by which ASFV induces apoptosis in infected cells and observed that caspases 3 and 9 are activated with similar kinetics as of 16 hpi. However, ASFV-induced apoptosis was independent of caspase 8 activation, a process involved in the extrinsic apoptotic pathways. These results indicated that the virus induces apoptosis through the mitochondrial pathway rather than the death receptormediated pathway. ASFV triggers apoptosis in an early stage of the infection process. 34 Nevertheless, extensive apoptosis of infected cells occurs only at late post-infection times. Programmed cell death itself might be relevant for the release of virus particles into apoptotic bodies, which would provide a mechanism to facilitate virus spread and evasion of the immune system. Our results show that caspase 3 activation in early stages of the infection, was relevant for virus exit and that membrane blebbing contributes to virus dissemination, given the fact that the inhibition of the effector caspase 3 reduced the release of virus particles from the cell and this effect was enhanced when the inhibitor was added after 6 hpi. Thus, ASFV takes advantage of an early induction of apoptosis, through caspase 3 activity, while the final execution of programmed cell death in apoptotic blebs contribute to ASFV invasion of host cells and evasion of the immune surveillance. In addition to its critical role in apoptosis, caspase 3 participates in other cellular processes. 35 This could also be the case of ASFV infection, where activation of caspase 3 may be required during early stages of infection to ensure subsequent virus exit from the cell. Another example is the influenza virus infection, in which caspase 3 inhibitors prevent the formation of progeny influenza A virus particles. 36 We have also shown that ASFV infection in Vero cells induces caspase 12, although this induction is not required for viral production or for caspases 3 and 9 activation. Caspase 12 is a characteristic ER stress response caspase. Virus assembly begins with the modification of ER membranes, which are subsequently recruited to the viral factories and transformed into viral precursor membranes. 37 Moreover, a large amount of viral proteins are synthesized and accumulated in infected cells, thus overloading the ER-folding capacity. This overload can lead to the activation of the ER stress response of the host cell. Activated caspase 12, a key marker of ER stress-mediated apoptosis, may potentially interact with another pro-apoptotic protein, Bap31, resulting in a feedback mechanism involving the release of cytochrome c elicited through the mitochondrial pathway. 38 However, Bap31 was not activated by the fragmentation of p20 in ASFV-infected cells. This observation indicates the absence of direct pro-apoptotic signals between the ER and mitochondria. Caspase 12 was triggered in ASFV-infected cells but this activation does not necessarily result in apoptosis, because the virus has the capacity to block ER stress at a later step. In general, ER stress starts with the transcriptional induction of ER-localized chaperones as a survival signal. Molecular chaperones are key components of the ER machinery; their main function consists of ensuring the quality of the ER by maintaining newly synthesized proteins in a proper state for folding, and marking misfolded proteins