PROJECT SUMMARY Smallpox was one of the deadliest diseases in human history, and despite its eradication, orthopoxviruses continue to pose a significant public health threat due to bioterrorism concerns and zoonoses. The virulence of these viruses relies upon their evasion of host immune responses by a large arsenal of immunomodulatory proteins. One of these proteins is the C15 protein of ectromelia virus, the cause of mousepox, which significantly impacts virulence in vivo. This protein is the largest in the viral genome and has homologs in the virulent orthopoxviruses, including the agents of smallpox and monkeypox, but no homology to any protein outside this family. Previous work has shown that C15 inhibits CD4 T cell activation, but I have recently determined that C15 significantly enhances viral replication in vivo at times that precede the onset of adaptive immunity, suggesting the existence of a second innate immune targeting function. The goal of this project is to understand this novel role of C15 early in infection. Preliminary work has shown that C15 maintains its replication-promoting effect in RAG KO but not RAG IL2RG double KO mice, implying the targeting of NK cells. Further analyses indicate C15 targeting of another RAG-dependent component of the innate immune response, most likely gd T cells. In searching for a unifying molecular target, additional preliminary data implicates the NK gene complex;; NKG2D is a receptor encoded within the NK complex that is capable of activating both NK cells and gd T cells. I therefore hypothesize that C15 inhibits NKG2D-mediated recognition of infected cells by NK and gd T cells, thereby inhibiting immune control of viral replication. To test this hypothesis, I propose three independent and complementary specific aims, each investigating the impact of C15 on one of the three components of the hypothesis. The first aim will identify the impact of C15 on NK cell function, using a set of orthogonal in vivo and in vitro approaches. The second aim will test the hypothesis that C15 targets gd T cells using similarly comprehensive approaches. The third aim will test the hypothesis that C15 targets NKG2D-mediated activation of immune cells by interrogating the importance of NKG2D and its adaptors DAP10 and DAP12 for the early impact of C15 in vivo as well as the impact of C15 on expression of NKG2D ligands. The identification of separate innate and adaptive immunomodulatory functions in a single viral protein is highly unique. The proposed studies will provide insight into the exceptional virulence of the orthopoxviruses, including those of particular public health concern, and could lead to the use of this protein family as an immune targeting therapeutic strategy.