Investigating the Role of Ubiquitin-Binding Domain of RIOK3 in Viral Immune Defense

Understanding virus-immune system interactions is critical for preventing future outbreaks in both livestock and humans. This study could help develop treatment strategies to reduce the risk of Rift Valley fever virus (RVFV) becoming a widespread public health crisis. My research involves RVFV, a pathogen regulated by the NIH and USDA due to its ability to cause severe diseases leading to economic losses. By studying RVFV, we identified RIOK3 as a key protein in the immune response. When the body detects a virus, cell surface receptors identify the danger and send alarm signals triggering a chain reaction, activating antiviral proteins like interferons, which alert nearby cells and activate the immune system to fight the infection. RIOK3 plays a key role in regulating interferon production during viral infections, but its precise mechanism remains unclear. This led to investigating RIOK3’s interaction with ubiquitin, a protein that modifies others to control their behavior. Ubiquitin chains serve different functions depending on their linkage type: K48 linkages signal protein destruction, while K63 linkages promote immune signaling. We hypothesize that RIOK3 contains a ubiquitin-binding domain (UBD) that specifically binds K63-linkages to activate cellular immune signaling. This interaction may explain how RIOK3 regulates interferon production. The goal is to confirm the identified UBDs and observe the role K63-linkage bound RIOK3 plays in the production of interferon. We produced fragments of RIOK3 containing either the wild-type (WT)-UBD or mutated (QUAD)UBD and incubated these fragments with K63-linkage. Compared to WT, we observed a decrease in the binding affinity of QUAD-UBD to K63 linkage, confirming that the identified UBD recognizes and binds to ubiquitin. This study’s novelty is that we identified a UBD potentially responsible for binding to the ubiquitin linkages attached to other known proteins. Future work will be to observe how the QUAD mutation changes the interferon production.