Investigating the Protein-Protein Interactions of RIOK3 During Viral Infection

In response to viral infection, cells initiate a series of innate immune signaling cascades that result in inflammation. This process is highly regulated to prevent a damaging hyper-inflammatory response while still fighting off the infection. Infection with Rift Valley fever virus (RVFV), an RNA virus endemic to Sub-Saharan Africa, can result in severe disease in humans and livestock. Infection in pregnant livestock can result in abortive pregnancies and high mortality rates. RIOK3 is an understudied protein expressed in most mammalian cells that has an unclear role in the response to viral infection. Recent work in our laboratory has shown that during RVFV infection in cell culture, the absence of RIOK3 results in a decreased innate inflammatory response and increased viral replication. Our goal was to investigate RIOK3’s protein-protein interactions during viral infection to help determine its function. We used a proximity labeling strategy to tag RIOK3’s interacting partners in cells expressing a chimera of RIOK3 and biotin ligase. In this way, proteins interacting with RIOK3 during immune activation would be labeled with biotin tags. After stimulating the cells with either RVFV or the RNA viral mimetic poly (I:C), biotin was added to induce proximity labeling. At several time points post-stimulation, biotinylated proteins were captured from lysed cells and analyzed using western blots. The experiment was then scaled up and samples were shared with our collaborators to get a more detailed understanding of interacting partners through mass spectrometry analysis. Our current data suggest that RIOK3 interacts with both MDA5 and TBK1, which are known innate immune modulators, during early timepoints in the immune response. We expect that the mass spectrometry data will identify novel interaction with other immune modulators. These results will provide insight into the role(s) of RIOK3 in the innate inflammatory response to viral infection and inform future studies on its potential as a drug target in inflammatory diseases