PROJECT SUMMARY (See instructions): Human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic progenitor cells (HPCs) and cells of the myeloid lineage and its reactivation is exquisitely linked to hematopoietic differentiation and stress. Latency is poorly defined for HCMV at the molecular and cellular level. The long-term goal of our work has been to define the mechanisms by which HCMV enters and exits the latent infection. The UL 133-UL 138 locus coordinates the expression of four genes, UL 133, UL 135, UL 136, and UL 138 with pro-latency and pro-reactivation functions. We have defined the cellular pathways modulated by these viral proteins to define mechanisms of latency and reactivation. Our work has shown that accumulation of the reactivation determinant, UL 136p33, is controlled by the host E3 ubiquitin ligase IDOL, which is induced by the liver X receptor in response to sterol levels. Maintenance of low levels of UL 136p33 by IDOL is critical for the establishment of latency and downregulation of IDOL with differentiation is important for reactivation. A host target of IDOL is the low-density lipoprotein receptor (LDLR). We determined that HCMV infection downregulates LDLR and prevents the maturation of LDLR through the ER. We show that UL 138 is important in driving ER-associated degradation (ERAD) of immature LDLR forms in infection. UL 138 further is required for the induction of the unfolded protein response and sterols in infection. We have identified UL 138-host interactors important to ERAD and regulation of the unfolded protein response and ER stress. We hypothesize that UL 138 regulates ER stress, proteostasis, and sterol metabolism important for viral latency through its ERAD-related host interactions. We will determine how UL 138 regulates ERAD by defining virus-host interactions (Aim 1), how UL 138-mediated regulation of ERAD impacts ER stress and lipogenesis/sterol synthesis in infection (Aim 2), and how this regulation of host pathways impacts the regulation of latency and reactivation (Aim 3). We anticipate that UL 138 regulation of ERAD impacts the cellular sterol environment sensed by HCMV to control UL 136p33 levels through changes in IDOL concentration for latency. Our work reveals new mechanisms by which HCMV controls UPR/ERAD with implications for the regulation of ER stress and sterol synthesis to regulate HCMV latency and reactivation. Defining these mechanisms elucidates novel host pathways important for HCMV latency and reactivation.