Eve Simpson, University of Saskatchewan

Eve Simpson (Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan), Valeria Molina Martinez (Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan), and Anil Kumar (Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan)

Suppressing & Conquering

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted pathogen endemic to eastern regions of Canada and the United States. The virus circulates in nature between birds and mosquitoes. During infection in humans, EEEV can cross the blood-brain-barrier and cause fatal encephalitis. No antiviral therapies or vaccines are currently available to manage this disease in humans. EEEV non-structural protein 1 (NSP1) plays a critical role in viral replication by mediating viral RNA capping, thereby promoting efficient viral protein translation and evasion of host immune responses. We mapped the host protein interactome of NSP1 using affinity purification coupled with mass spectrometry. Among the hits identified, we validated an interaction between EEEV NSP1and the host protein ATP6V1B2 through immunoprecipitation and western blot analysis. ATP6V1B2 is a subunit of the vacuolar ATPase (vATPase) complex, which regulates intracellular vesicle acidification and is essential for membrane trafficking, protein processing, and degradation. We hypothesize that NSP1 interaction with the vATPase complex enhances viral replication fitness by modulating vATPase functions in infected cells. To investigate the functional consequences of this interaction, we are examining how NSP1 interaction with vATPase complex modulates endosomal acidification and lysosomal degradation of proteins. Deletion mapping of NSP1 will be performed to identify domains required for ATP6V1B2 binding. Collectively, this work will provide insight into how EEEV manipulates host cellular machinery to promote infection, uncovering novel aspects of EEEV biology.