SUMMARY - PROJECT 2 Human cytomegalovirus (HCMV) reactivation from latency remains a serious complication under conditions of immunosuppression. Latency and reactivation are complex processes regulated by a subset of viral proteins and non-coding RNAs in CD34+ hematopoietic progenitor cells (HPCs). The goal of this project is to define how HCMV microRNAs (miRNAs) refine and shape signaling mediated by viral proteins critical for the entry into and exit from latency. We have previously determined that HCMV miRNAs interfere with multiple pathways in latently- infected cells, including TGF, MEK/ERK and RhoA that have critical implications for HCMV latency. Importantly, we have uncovered a role for HCMV miRNAs acting both in coordination and in opposition to viral proteins. For example, in order to regulate apoptosis early after infection of CD34+ HPCs, miR-US5-1 and miR-UL112-3p act in concert with UL7 (Project 4) to reduce the levels and activity of FOXO3a. In contrast, HCMV miR-US5-2 and miR-US22 target components of the MEK/ERK signaling pathway downstream of EGFR critical for viral reactivation and attenuate US28- and UL138-mediated signaling. We have identified RhoA as a target of HCMV miR-US25-1 that plays a key role in regulating the proliferation of latently-infected CD34+ HPCs through modulating cytokinesis. Intriguingly, HCMV US28 (Project 3) activates RhoA through ligand-dependent signaling and recent proximity labelling experiments identified RhoGEFs and Rho GTPases as components of the US28 signalosome. Mutation of US28 or chemical inhibition of RhoA reduces viral reactivation, highlighting the importance of US28 and Rho signaling in the reactivation process. Additionally, UL8 (Project 4) is essential for reactivation and activates Rho signaling through the Wnt pathway. Moreover, proximity labelling experiments identified Wnt and Rho pathway components as part of the UL8 signalosome. We have identified additional HCMV miRNAs that target key components of the Wnt and Rho GTPase pathways including the Wnt effector DVL2, RhoGEFs, the GTPase CDC42 and downstream effector proteins, indicating that Rho GTPase signaling is intricately regulated by viral miRNAs in addition to being stimulated by viral proteins. We hypothesize that HCMV miRNAs target components of the MEK/ERK pathway to downregulate virus-mediated signaling at the time of reactivation, whereas miRNAs expressed during latency target components of the Rho GTPase pathways to oppose the effects of virus-mediated signaling to maintain the virus in latency.