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 determine whether ER stress is involved in ASFVinduced cellular responses, we analyzed several ER stressrelated proteins, including CHOP, GADD34, XBP1, ATF6 and ATF4. Previous results showed that viral infection in Vero cells inhibits the induction of the ATF4-CHOP signaling arm of the UPR. 24, 25 However, we observed expression of ATF4 from 48 hpi when virus is thought to promote programmed cell death in order to spread progeny. The inhibition of this UPR branch results in an attenuation of protein translation. To avoid this, ASFV encodes the DP71L gene, a GADD34 homolog that interacts with the catalytic subunit of PP1, and activates its phosphatase activity 40 and that also promotes the expression of cellular GADD34, as shown here. The activation of the ATF6 branch and its transcriptional activation of chaperone-encoding genes might benefit the virus by assisting the folding of accumulated proteins and preventing protein aggregation. We demonstrated ATF6 translocation to the nucleus. Activated 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. 7 We used a short promoter that has considerably lower 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 manner to endogenous ATF6. 32 The analysis of ATF6 distribution by confocal microscopy in Vero cells infected with ASFV revealed that this protein was translocated into the nucleus. ATF6 was also located in discrete perinuclear cytoplasmic areas that corresponded to viral factories, as indicated by the colocalization of ATF6 with DNA-containing cytoplasmic foci. Furthermore, ASFV viral replication was blocked by inhibition of the ATF6 translocation to the nucleus by AEBSF. To analyze the transcriptional activity of ATF6 in ASFV infection we examined the expression of XBP1 in ASFV-infected cells by qRT-PCR. Surprisingly, ASFV-infected samples showed a decrease in the induction of XBP1 compared with uninfected samples. Activation of ATF6 was necessary for ASFV replication; however, the virus prevented the transcriptional induction of some ATF6-target genes, such as XBP1 and BiP, but not calnexin or calreticulin. These studies have focused interest in ATF6 pathway in this viral infection and have paved the way for further characterization of ASFV viral gene regulation of these molecules in the UPR.