PROJECT SUMMARY/ABSTRACT The more than 40-year cessation in smallpox virus vaccinations after the eradication of variola virus (VARV) has left a growing population in the US and globally vulnerable to orthopoxvirus infection. Concerns of bioterrorism usage of VARV or other species of orthopoxvirus necessitate an in-depth understanding of poxvirus immune evasion of the host. With the current immunity gap identification of new treatment targets for development of novel therapeutics remains a critical task. On the other hand, clinical use of poxviruses to treat cancer requires comprehensive knowledge of poxvirus-induced cellular response that can improve therapeutic outcome. Our long-term goal is to improve understanding of the host intrinsic immunity that not only protects the host from viral infection but also can be stimulated to facilitate the elimination of malignancy. The goal of the proposed study is to understand how additional mechanisms enforce species barrier that can prevent cross-species viral infection and how we discover and investigate new antiviral pathways using poxviruses as model organisms. More importantly, sensing of cytoplasmic DNA leads to induction of type 1 interferon (IFN-I) and while we found new regulation of host DNA sensing pathway we investigate viral strategy to evade it that affects pathogenesis. We will study species barrier function and the antiviral function of a host protein, sterile α motif domain-containing protein 9 (SAMD9). SAMD9 plays a central role in human health and possesses antiviral effect to a broad-spectrum of viral pathogens. However, its function is poorly characterized. To study SAMD9's antiviral properties, we utilize myxoma virus (MYXV) as the model organism. MYXV is a rabbit-specific poxvirus causing lethal infection in European rabbits and a candidate oncolytic virus to treat human cancers. We previously identified MYXV M062 as the viral inhibitor of SAMD9. We thus utilize viral M062 and M062R-null MYXV as probing tools to study SAMD9 function. Our central hypothesis is that SAMD9 detects poxvirus infection and DNA replication, and once it binds to poxvirus DNA it inhibits DNA replication while inducing IFN-I responses that affects viral pathogenesis. To test our hypothesis, we propose the following studies: (1) To understand the mechanism of SAMD9 for the inhibition of viral DNA replication and for induction of IFN-I; (2) To investigate how inhibiting SAMD9 affects pathogenesis; and (3) to study how SAMD9 mediates the host species barrier function. Through the completion of the proposed study, we will fill a critical gap in our knowledge of host regulation linking the inducible antiviral response and the intrinsic immunity. This improves our understanding of the mechanism for immunotherapeutic virotherapy using MYXV. We also characterize a novel class of viral evasion of host immune response.