SUMMARY – PROJECT 5 Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality after hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) due to reactivation or a new infection in these transplant recipients. Our combined PPG laboratories have shown that HCMV infection of CD34+ hematopoietic progenitor cells (HPCs) the site of HCMV latency and subsequent reactivation alters hematopoietic events to favor HPC differentiation towards the myeloid lineage. To fully understand HCMV reactivation process, we need not only understand the process of how reactivating virus in CD34+ HPC directs differentiation towards the myeloid lineage, but also mechanistically how HCMV reactivation in monocytes directs signaling to promote differentiation towards productive macrophages. Through our collaborative program, we have identified viral genes (e.g., UL135, US28, UL7/8) and miRNAs (e.g. miR-US22) that are required for reactivation. Furthermore, through the use of deletion mutant viruses that fail to reactivate, we have identified the existence of blockades to HCMV reactivation that are associated with differentiation into macrophages. The signaling events important for reactivation of latent virus in tissue macrophages remains poorly understood. We suggest temporally unique roles for UL135, US28 and UL7/8 and a unique intersection of these gene products with HCMV miRNAs expressed during latency in usurping the signaling necessary to promote reactivation in this essential cell type. We hypothesize distinct signaling events in monocyte to macrophage differentiation relieve blockades to reactivation and these reactivation events are specifically driven by modifications to cellular signaling by the combined effort of these viral factors. To test our hypothesis, we propose the following aims. Specific Aim 1. Defining signaling networks for HCMV latency and reactivation in monocytes and macrophages. We hypothesize that functionally distinct host cell responses in monocyte-to-macrophage differentiation are essential for reactivation. Specific Aim 2. Determine checkpoint blockades for viruses that fail to reactivate. We hypothesize that a failure in specific cellular signaling reprogramming during reactivation will result in a cell that phenotypically is unable to support viral replication. Specific Aim 3. Define the signaling nodes modulated by infection for reactivation in monocytes/macrophages. We hypothesize that the EGFR and RhoA signaling pathways are critical for HCMV latency and reactivation in macrophages. IMPACT: These proposed aims will allow us to gain greater insight and as we integrate our collective work, we hope to translate the results into novel molecular signatures designed to improve the outcome of an infection that remains a significant problem in transplant recipients.