Poxviruses are large DNA viruses that cause significant human and veterinary diseases. Understanding poxviral pathogenesis and its interface with the host immune system are of importance because of the threat of using smallpox as a bioterror weapon, the need for improved vaccination strategies to minimize vaccine-associated complications, such as eczema vaccinatum, and the reimmergence of orthopoxvirus infections in humans such as monkeypox. Poxviruses have also been studied intensively as vaccine vectors and oncolytic agents. Our long-term goal has been to understand host antiviral defense mechanisms and viral immune evasion strategies. Under the K08 award from NIAID, the Principle Investigator has been focusing on elucidating how poxviruses are sensed in dendritic cells (DCs), macrophages and epithelial cells. We identified that the cytosolic dsRNA sensing pathway mediated by MDA5/MAVS/IRF3 plays an important role in sensing vaccinia virus in epithelial cells, which is antagonized by the dsRNA binding domain of vaccinia virulence factor E3. We found that modified vaccinia Ankara (MVA) infection of conventional DCs triggers type I IFN production through a RNA polymerase III/IRF3/IRF7 dependent pathway, which is antagonized by the Z-DNA binding domain of E3. We plan to assess the role of MDA5/MAVS/IRF3 in host defense against vaccinia infection in an intranasal infection model (Aim 1A). We also plan to test whether innate immune sensors play a role in the development of adaptive immune responses using vacciniation with &#916;E3L virus (with deletion of fulllength of E3) via skin scarification or intranasal infection (Aim 1B). We will perform biochemical studies to dissect the molecular mechanisms through which vaccinia E3 inhibit cytosolic dsRNA and DNA sensing pathways (Aim 1C). We have developed a new line of research into autophagy induction and evasion by poxviruses. We found that attenuated vaccinia or myxoma (a rabbit poxvirus) infection of dendritic cells and macrophages trigger autophagy, whereas wild-type vaccinia infection does not. Poxviruses encode several immunomodulatory proteins that might target autophagy induction. We plan to delineate the signaling pathway mediating autophgay induction by attenuated vaccinia viruses and myxoma (Aim 2A). We plan to elucidate the mechanisms of autophagy inhibition by viral proteins using both genetic and biochemical approaches (Aim 2B) and to assess the role of autophagy machinery in the generation of innate and adaptive immune responses against poxvirus infection in vivo (Aim 2C). The results of the proposed research will contribute to the fundamental knowledge of the dynamics of virus-host interaction and the role of innate immune sensors in pathogen restriction and the development of adaptive immune responses. It will have an important positive impact on the preventive and therapeutic interventions for poxvirus infection, and the design of safe and effective smallpox vaccines and poxvirus-based immunotherapy and oncolytic therapy.