The interferon (IFN) system is one of the primary vertebrate defenses against virus-infection. A number of IFN-resistant virus have recently been characterized. The long-term objectives of the proposed research are to understand the mechanisms involved in resistance of viruses to IFN. The specific aims are to understand the role of one of the poxvirus inhibitors of the IFN system in replication of these viruses and in their resistance to IFN. Since poxviruses, which are being developed as vectors for human vaccines, can rescue other viruses from the effects of IFN, and since group C rotaviruses, which cause severe diarrhea in humans, encode a similar PKR inhibitor, this research may impact health issues. The role in virus replication and resistance to IFN of the vaccinia virus inhibitor of the IFN system will be evaluated by use of a mutant strain of virus, vPl080, that has been deleted for a gene involved in resistance IFN, the E3L gene. This gene has also been implicated in suppression of apoptosis in vaccinia virus infected cells, and can transform cells in culture. Activation of various components of the IFN system in vp1080- infected cells will be analyzed. Ectromelia virus (another poxvirus) deleted for the E3L homologue will be prepared and pathogenesis of this altered virus will be tested in mice. Structure function relationships for the protein encoded by E3L, p25, will be determined. p25 specifically binds double-stranded (ds)RNA, and shares homology with several other dsRNA-binding proteins, including the IFN-induced protein kinase, the human protein that binds to the HIV transactivation (tar) site, TRBP, and the rotavirus NSP3 protein. Structure/function relationships in this conserved dsRNA-binding domain will be determined by site-directed mutagenesis. Crystal structures for proteins bearing this domain will be determined. This work may provide light into how proteins recognize dsRNA. Other vaccinia virus-genes that may affect viral sensitivity to IFN will be isolated, by selecting for genes that can allow growth in the presence of IFN of vaccinia virus deleted the E3L gene. This work will provide insight into how the poxviruses have evolved resistance to IFN.