Viruses use many strategies to translate their mRNAs in the face of antiviral responses that would otherwise block viral protein synthesis. The most prominent antiviral mechanism affecting protein synthesis is phosphorylation of eukaryotic initation factor 2? (eIF2?? by protein kinase R (PKR). Human adenoviruses (hAds) overcome this via virus-associated (VA) RNAs that block PKR activation. However, mouse adenovirus type 1 (MAV-1) does not encode VA RNAs; how MAV-1 successfully overcomes PKR is unknown. The role of another eIF2? kinase, GCN2, in antiviral responses is less well appreciated, but it is antiviral against MAV-1. There is a critical need to understand how PKR and GCN2 impact viral replication as part of the antiviral response. The long-term goal is to increase fundamental knowledge about adenovirus-host interactions that contribute to disease. The overall objective of this application is to determine how MAV-1 modulates host eIF2? kinases and successfully replicates in the presence of these host antiviral defenses. The central hypothesis is that MAV-1 overcomes the host antiviral response by reducing PKR levels, whereas MAV-1 activates but does not evade GCN2 in macrophages, target cells of viral replication. This hypothesis is based on preliminary data that PKR is depleted in MAV-1 infections, GCN2-deficient macrophages produce more virus, and GCN2-deficient mice are more susceptible and express more proinflammatory cytokines upon MAV-1 infection than control mice. The rationale is that knowing how MAV-1 induces and counteracts the host antiviral responses will enable manipulation of these responses and testing their effects in a powerful in vivo model, which will increase understanding of disease.
The specific aims are to determine 1) how MAV-1 counters the host PKR response, and 2) how the host GCN2 response protects mice from MAV-1 infection.
These aims will use assays of MAV-1 pathogenesis and molecular virology established in the applicant's laboratory.
In Aim 1, preliminary data showing that during infection PKR is degraded via the proteasome and PKR translation is inhibited will be validated and extended to identify the mechanisms and whether specific viral genes are responsible. Approaches will use viral mutants, PKR-/- mice and cells.
Aim 2 will address how the lack of GCN2 leads to higher mouse mortality and proinflammatory responses, and whether and how MAV-1 induces GCN2 activation, using viral mutants, and wild type and GCN2-/- mice and cells. The approach is innovative because it will elucidate for the first time how a DNA virus induces depletion of PKR, and it will broaden the focus on eIF2? kinase GCN2 as a host antiviral response. The research is significant because it is expected to have fundamental relevance to virus-host interactions and human disease.
The proposed research is relevant to NIH's mission because understanding viral-host interactions at the molecular level is crucial for being able to prevent the disease consequences of viral infections. Host kinase enzymes react to cellular stresses such as viral infection by blocking protein synthesis and thus blocking viral replication. A virus counter-response to one kinase, depletion of PKR, has been identified for the first time for a DNA virus and will be investigated using a tractable virus-host system, mouse adenovirus type 1 (MAV-1). Another kinase, GCN2, is not well appreciated as an antiviral enzyme, but it protects mice from effects of MAV- 1 infection. These kinases are also implicated in development and non-viral disease. Thus the proposed research will provide fundamental knowledge about the virus-host interface and control of disease.
