SUMMARY - PROJECT 3 Human cytomegalovirus (HCMV) is a b-herpesvirus infecting 44-100% of the population and remains a significant cause of morbidity and mortality in solid organ transplant (SOT) and allogeneic hematopoietic stem cell transplant (SCT) recipients. Infection in SCT patients is often associated with myelosuppression and graft failure due to virus reactivation from latency, but the associated mechanisms are still largely unknown. HCMV encodes multiple latency-associated gene products including the chemokine receptor US28, which binds CC- chemokines as well as the CX3C-chemokine Fractalkine. We have demonstrated that US28 ligand-dependent signaling is required to maintain viral latency in vitro in CD34+ hematopoietic progenitor cells (HPCs) and in vivo in humanized mice. US28 was required in both systems for reactivation of latent HCMV and promotes CD34+ HPC differentiation into myeloid lineage cells, indicating that US28 plays a dynamic role in both processes. While we have identified that ligand binding activity for US28 is required for HCMV latency, we do not yet know whether this effect is ligand-specific. In addition, we have recently demonstrated that HCMV-encoded chemokine ligands modulate US28 signaling. We hypothesize that both the host CC chemokines and viral chemokines UL146/UL147 promote latency by activating specific signaling pathways while other US28 ligands help direct reactivation by modifying signaling. We hypothesize that US28 ligand binding activity (ligand-specific) is required to maintain latency by interfacing with EGFR signaling and during reactivation US28 antagonizes EGFR signaling and shifts to activating RhoA pathways to enhance productive replication. In Specific Aim 1, we will determine which US28 ligands reprogram cells during latency and reactivation using neutralizing antibodies against host and viral chemokines, viral mutants that impact the ability of US28 to selectively bind ligands, and viral mutants lacking the viral chemokines. In SA2, we will utilize our US28 interactome data generated using the BirA-Turbo proximity sensor technology to identify signaling factors that play a role in latency and reactivation. Through mutational analyses we have identified specific regions of US28, in the C’terminal tail and intracellular loop 3 domains, that modify US28 signaling. We will determine what effect these mutations have on both the US28 interactome and latency and reactivation using in vitro and in vivo models. Lastly, in SA3, we will define how US28 signaling influences other HCMV proteins (Projects 1 and 4) and miRNAs (Project 2) with specific focus on how US28 intersects with the EGFR pathway, to contribute to the combined HCMV-manipulation of EGFR signaling (Project 5) and therefore control of latency and reactivation within the overarching goals of this proposal. Results of this study will generate new virus latency and reactivation paradigms promoting the development of novel therapies to prevent ...