SARS-Coronavirus triggers apoptosis via protein kinase R but is resistant to its antiviral activity

In this study, infection of 293/ACE2 cells with SARS-CoV activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2 alpha (eIF2alpha). In addition, two of the three cellular eIF2alpha kinases known to be virus-induced, protein kinase R (PKR) and PKR-like endoplasmatic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control non-derepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. RT-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication, nor abrogated SARS-CoV-induced eIF2alpha phosphorylation. Pretreatment of cells with interferon beta prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2alpha phosphorylation and SARS-CoV replication. The various effects from interferon beta treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2alpha phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV has evolved a mechanism to overcome the inhibitory effects of phosphorylated eIF2alpha on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2alpha phosphorylation.