PROJECT SUMMARY The goal of our Program is to elucidate the molecular mechanisms by which HCMV regulates host signaling in CD34+ hematopoietic progenitor cells (HPCs) for the establishment and maintenance of viral latency and reactivation, and to determine how viral dysregulation of signaling contributes to HCMV myelosuppression. HCMV remains a significant cause of morbidity and mortality via myelosuppression after solid organ and hematopoietic stem cell transplantation despite advances in diagnostics and therapeutics. HCMV latency is complex and the signaling mechanisms for establishment and maintenance of HCMV latency, as well as for reactivation of virus are poorly understood. Our program has identified a number of viral proteins and miRNAs that regulate both latency and hematopoiesis by targeting epidermal growth factor receptor (EGFR) and signaling pathways downstream of EGFR. The complexity of signaling events and approaches to comprehensively address questions on viral latency and hematopoiesis can only be achieved through a collaborative effort. Using state-of-the art in vitro models in primary, human CD34+ HPCs and in vivo models in mice humanized with bone marrow, liver and thymus, our Program will address the individual and combined roles of viral factors modulating host signaling. Our Project (Project 1) will address the roles of proteins encoded within the UL133/8 “latency locus” in modulating host signaling. We have defined roles for the four genes encoded by the UL133/8 locus in either positively or negatively regulating the maintenance of viral latency. Intriguingly, these genes opposingly regulate the trafficking and activation of EGFR. We have shown that EGFR and downstream PI3K signaling is important for the maintenance of latency and inhibitors of EGFR or PI3K stimulate reactivation and replication. We hypothesize that EGFR is a critical signaling axis targeted by UL133/8 proteins to modulate viral latency, reactivation and hematopoiesis. In Aim 1, we will use a systems approach to define the global pathways regulated by UL133/8 proteins in the context of infection in CD34+ HPCs. We will then define the significance of UL133/8-modulation of host signaling to viral latency and reactivation in Aim 2 and to hematopoiesis in Aim 3. Throughout our studies we will integrate our data sets with those of the other Projects to determine how UL133/8 interfaces with other viral proteins (US28 in Project 3; UL7 in Project 4) and viral miRNAs (Project 2) and contributes to the broader regulation of host signaling in regulating latency, reactivation and hematopoiesis. Project 5, focused on defining the hematopoieitic signaling pathways targeted by HCMV, will inform our understanding of pathways targeted by UL133/8 proteins to regulate hematopoiesis. Collectively, our projects will provide the first comprehensive and mechanistic insights into the multi-faceted regulation of host signaling for the control of HCMV latency and myelosuppression. ...