ABSTRACT Type I interferons (IFN) and IFN stimulated genes (ISGs) provide broad antiviral activities. However, the host protection provided by the IFN and ISGs during bacterial infections is not well defined. This proposal will define new innate immune functions of Oligoadenylate Synthetase (OAS) proteins against intracellular bacterial infections. OASs are a family of ISG that belongs to an evolutionarily ancient family of nucleotidyl-transferases (NTase). The canonical antiviral mechanism of OAS proteins involves the enzymatic synthesis of 2'-5'- oligoadenylates, causing downstream activation of RNase L, leading to RNA degradation and inhibition of protein synthesis. However, the host protective function of multiple enzymatically active and inactive OAS isoforms during bacterial infection is not known. This proposal will address this knowledge gap to define the innate antibacterial functions of two OAS family proteins, OAS1 and OASL. Our preliminary studies suggest that human OAS1 and a mouse ortholog, Oas1b, confer protection from infection with intracellular bacteria by enhancing the expression of several proteins including IRF1, a major antibacterial transcription factor. Furthermore, we found that mice lacking Oasl2 (ortholog of human OASL), another member of OAS family, are highly susceptible to intracellular bacterial infection, and show increased production of host-detrimental inflammatory cytokines. Based on these preliminary findings, we hypothesize that specific members of OAS- family proteins contribute to host defense against intracellular bacteria through unique NTase activity- independent mechanisms. The goal of this proposal is to determine the innate antibacterial functions of two OAS family proteins and the underlying mechanisms in three independent aims: (1) Identify the mechanisms of cell- intrinsic antibacterial activity of OAS1. Here, we will define the molecular mechanism by which OAS1 mediates cell intrinsic antibacterial defense using in vitro biochemical and cell biological approaches. (2) Determine the in vivo role of Oas1b in anti-bacterial innate immunity. We will use a newly generated Oas1b knock-in mouse model to identify the in vivo mediators and cell-type specific antibacterial function of Oas1b. (3) Define the role of OASL in host defense against intracellular bacterial infections and the underlying molecular mechanism(s). Taken together, this proposal is designed to elucidate new NTase activity-independent molecular functions of OAS proteins, and to integrate it with antibacterial cellular defense in an innovative functional-model. Upon completion of this study, we will establish a new paradigm of innate host defense orchestrated by OAS proteins that may lead to new therapeutic strategies.