Role of mouse adenovirus type 1 E4orf6-induced degradation of protein kinase R in pathogenesis

Protein kinase R (PKR) is an interferon-induced antiviral protein activated by autophosphorylation in response to double strand DNA (dsRNA) and other stimuli. Activated PKR causes translation inhibition and apoptosis, and it contributes to proinflammatory responses, cell growth, and differentiation. Mouse adenovirus type 1 (MAV-1) counteracts PKR by causing its degradation via a viral protein, early region 4 open reading frame 6 (E4orf6). Degradation is dependent on E4orf6 binding to Cullin 2, a component of the MAV-1 E4orf6 ubiquitin ligase. We investigated the importance of E4orf6 for induction of PKR degradation by exploiting the ability to infect the natural host with the adenovirus MAV-1. First, we used a new PKR-deficient mouse strain, PKR-TKO. PKR-TKO mouse embryo fibroblasts (MEFs) produced higher levels of MAV-1 upon infection than did wild-type (WT) MEFs. PKR-TKO mice had significantly reduced survival, and MAV-1 had a lower LD₅₀ than in WT control mice. However, virus loads in brains and spleens, key organs infected by MAV-1, were similar between PKR-TKO and WT mice. Second, we constructed a virus, E4orf6TMC2, that has three amino acid changes in the E4orf6 domain involved in Cullin 2 binding. In cell culture infection, compared to WT virus, E4orf6TMC2 resulted in reduced PKR degradation, but its growth was equivalent to WT virus. However, E4orf6TMC2 was avirulent in three mouse strains, including the PKR-TKO mice. The results indicate that PKR is an essential antiviral protein that protects against MAV-1 infection. We confirmed that the viral E4orf6 protein is a virulence protein important for PKR degradation during virus infection, and our results suggest its function is not limited to PKR degradation.IMPORTANCEProtein kinase R (PKR) is a host protein that is central to many aspects of the cellular stress response. PKR protects against viral infection by inhibiting viral and host protein synthesis. Most animal viruses have developed ways to circumvent PKR effects by at least one of a variety of means, including inducing its degradation. A new mouse strain knocked out for PKR expression has enabled us to show the importance of PKR for protection from mouse adenovirus type 1 infection in the natural host, which is not possible for human adenoviruses. Mouse adenovirus type 1 induces degradation of PKR through an interaction with host protein Cullin 2. We generated a mutant virus that is defective in its ability to interact with Cullin 2 and showed that the virus does not cause pathogenesis in mice. This work provides critical evidence from mouse studies supporting the importance of PKR for adenovirus pathogenesis.