Project Summary Bats are important reservoirs for diverse viral pathogens affecting humans. However, we have a poor understanding of the key bat innate immunity factors that restrict virus replication. Functional assays that can identify bat factors that are truly relevant to combating viruses are needed to understand the innate immune mechanisms that ultimately define bat susceptibility to viral infection. While historically such functional screens have relied on genome-wide genomic editing (e.g. CRISPR-Cas9)- or RNA interference (RNAi)-based techniques, such platforms are unavailable for most bat species. Thus, new methods for uncovering functionally- relevant components of the bat immune response to virus infection are needed. To address this need, we have developed an innovative arbovirus "rescue" assay wherein immune evasion proteins (IEPs) encoded by mammalian pathogens can be expressed in bat cells and one can assay for changes in bat cell susceptibility to arbovirus infection. Enhancement of arbovirus replication after expression of a candidate IEP indicates that the IEP likely inhibits bat immunity mechanisms that normally restrict arbovirus replication. Using these IEPs as "tools", one can then identify the bat immunity factors these IEPs target. Thus, this screening methodology provides a mechanism to both identify novel IEPs and functionally-relevant components of the bat immune response. To discover IEPs that promote arbovirus replication in bat cells, we will screen an expression library encoding ~200 bacterial effector proteins. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that modulate or inhibit various eukaryotic cellular processes to promote bacterial replication. Many bacterial pathogens that replicate in the cytoplasm of eukaryotic host cells encode effectors that function as IEPs. Thus, we hypothesize that some effectors may suppress immune responses that restrict both bacteria and cytoplasmic viruses such as arboviruses. Indeed, our initial screens have identified four effectors that promote the replication of four different arboviruses when expressed in bat cells. We have characterized one of these effector screen "hits" as a novel ubiquitin ligase that targets an uncharacterized Ring Finger (RNF) Domain-containing protein for degradation in eukaryotic cells. Importantly, RNAi depletion of this RNF factor in human and bat cells promotes arbovirus replication, suggesting that it may be a novel component of human and bat immune responses. These results suggest that we can use bacterial effectors as tools to both inhibit, and identify, functionally-relevant immunity factors in bats. Our study has the following specific aims: 1) Identify bacterial effector proteins that promote arbovirus repli...