The Roles of eIF2 Kinases PKR and GCN2 during Mouse Adenovirus Type 1 Infection

[摘要] During viral infection, a major innate host defense mechanism is to reduce global protein synthesis by phosphorylation of eukaryotic translation inhibition factor 2a (eIF2a). eIF2a is phosphorylated by four different cellular kinases, protein kinase R (PKR), general control nonderepressible 2 (GCN2), PKR-like endoplasmic reticulum kinase, and heme-regulated inhibitor, each responding to a different cellular stress. GCN2, which phosphorylates eIF2a in response to amino acid starvation, UV irradiation, and oxidative stress, can also phosphorylate eIF2a in response to viral infection. PKR senses double-stranded RNA produced by virus infection. Many viruses have methods of inhibiting PKR activation or its downstream effects, thus circumventing protein synthesis shutdown. These methods include sequestering double-stranded RNA or producing proteins that bind to and inhibit PKR activation. Here we describe our finding that PKR was antiviral in mouse adenovirus type 1 (MAV 1) infection. We also showed that in multiple cell types, PKR was depleted during MAV 1 infection. Inhibiting the proteasome reduced the PKR depletion seen in MAV-1-infected cells, indicating that proteasomal degradation is responsible for PKR degradation during MAV-1 infection. Time course experiments showed that the degradation occurred early after infection. Infecting cells with UV-inactivated virus prevented PKR degradation, whereas inhibiting viral DNA replication did not. Together these results suggest that an early viral gene is responsible for the degradation. Degradation of PKR is a rare mechanism to oppose PKR activity, and it has only been described in six RNA viruses. To our knowledge, this is the first example of a DNA virus counteracting PKR by degrading it. In addition to PKR being degraded by MAV-1 during infection, we tested whether GCN2 plays an antiviral role, as seen for several other viruses. Here we describe that GCN2-/- deficient (atc) mice and peritoneal macrophages were significantly more susceptible to infection by MAV 1. However, atc mouse embryonic fibroblasts had similar viral yields compared to wild type mouse embryonic fibroblasts, suggesting that there is a cell-type specific antiviral effect of GCN2. There were no differences in viral yields in infected organs of atc mice at 8 days post infection compared to wild type organs, except for the cecum, indicating that the difference in survival between atc and wild type mice is not likely a result of increased viremia in atc mice. There was also no significant difference in histology of organs from MAV-1-infected atc and wild type mice. However, cytokine analysis showed that MAV-1-infected atc mice had significantly higher levels of interleukin 1a, interleukin 1b, and interferon y in the brain compared to infected wild type mouse brains at 7 days post infection, suggesting that a difference in inflammatory response could be responsible for the decreased survival of atc mice in response to MAV-1 infection. Determining how GCN2 affects the immune response to MAV 1 infection could provide insight into how GCN2 is playing an antiviral role in DNA virus infection.

[发布机构] University of Michigan

[效力级别] GCN2

[关键词] PKR;GCN2;adenovirus;host-virus interactions;eIF2alpha kinase;proteasome;Microbiology and Immunology;Science;Cellular & Molecular Biology