Ethan Thomas, Queen's University

Michelle Li, Elise Rougier, Maike Bossert, and Bruce W. Banfield. Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada

Expressing & Multiplying

pUs3 is an alphaherpesvirus serine/threonine kinase that phosphorylates multiple cellular and viral proteins throughout infection. In HSV-1 infected cells, pUs3 functions together with the viral protein VP11/12 to maintain mTORC1 activation and sustain virus replication in the face of amino acid insufficiency. How pUs3 interacts with its substrates and synergizes with VP11/12 to promote virus replication is not completely understood. To advance our knowledge of pUs3 interactions with viral and cellular proteins, we utilized proximity-dependent biotinylation to identify proteins in proximity to kinase-active, and kinase-dead, pUs3. To do this, HSV-2 strains were constructed that express WT pUs3, or kinase-dead pUs3, fused to the promiscuous biotin ligase miniTurbo. Infected human keratinocytes were treated with exogenous biotin from 8-9 hpi to induce biotinylation of pUs3 proximal proteins. Biotinylated proteins were purified and identified by LC MS/MS. The most prominent protein in proximity to both active and kinase-dead pUs3 was VP11/12. Co-immunoprecipitation analyses demonstrated that pUs3 was in complex with VP11/12. Additionally, immunofluorescence microscopy experiments demonstrated that pUs3 and VP11/12 co-localized to discrete small puncta throughout the cytoplasm of WT HSV-2 infected cells. Interestingly, pUs3 kinase activity was not required for cytoplasmic pUs3 and VP11/12 puncta formation. Our finding that pUs3 and VP11/12 are in complex with each other advances our understanding of how these proteins synergize to maintain mTORC1 activity and support virus replication. We hypothesize that pUs3 and VP11/12 co-localize at a signaling hub that serves to concentrate and promote communication between factors that maintain mTORC1 activity and support alphaherpesvirus infection.