Characterization of a ubiquitin binding domain in interferon pathway protein RIOK3

Project summary This goal of the research proposed here is to explore the nexus of the well-characterized polyubiquitin signaling network that is activated during the cellular antiviral response and the functional implications of ubiquitin binding by innate immune protein RIOK3. Our laboratory has determined that RIOK3 is essential for initiating a robust interferon response to Rift Valley fever virus infection and other immunogenic stimuli in epithelial cells. We have also demonstrated that the pre mRNA for RIOK3 is subject to alternative splicing following immune stimulation. This alternatively spliced mRNA encodes a truncated version of RIOK3 that contains the N-terminal domain, but lacks the kinase domain. Intriguingly, expression of this truncated protein inhibits the cellular interferon pathway response in epithelial cells, but activates the NFkB pathway. Prior observations from other groups demonstrated that RIOK3 exhibits high affinity for K63-linked polyubiquitin chains, and this putative ubiquitin binding domain (UBD) resides in the N terminal region. Together with our data on the functions of RIOK3 isoforms, we hypothesize that the UBD guides RIOK3 to its essential cellular interaction partners during the cellular immune response. Furthermore, the truncated RIOK3 makes the same protein-protein interactions mediated by the UBD, but cannot fulfill the immune signaling function because it lacks its kinase domain. To test this hypothesis, we propose to precisely map the UBD of RIOK3 and characterize the phenotypic consequences of deletion or mutation of the RIOK3 UBD. We will test UBD mutants in vitro and in cell culture for ubiquitin binding properties and phenotypic effects on innate immune responses. Upon completion of these studies, we will have structurally and functionally mapped a novel protein interaction domain in RIOK3 which will shed light on its roles in the cellular antiviral response, with potential future applications in treatment of viral or immune diseases.