Human cytomegalovirus (HCMV) is a wide-spread pathogen, infecting the majority of the population in the United States. This virus poses a significant threat to developing fetuses as well as to children and adults who lack a competent immune system, often causing severe disease and mortality. Once individuals acquire an HCMV infection, the virus remains with the host for life, in a latent or quiescent state in the hematopoietic compartment. During times of severe immunological stress, the virus reactivates to its active state, allowing for dissemination and subsequent disease. With the exception of the immuno-naïve and sero-negative organ transplant recipients, primary infection with HCMV rarely causes disease, but rather it is reactivation that leads to significant complications. Thus, to prevent HCMV-associated disease, we must gain a complete understanding of viral latency and reactivation. During latency, HCMV encodes three of the four viral G protein-coupled receptors (vGPCRs): US28, UL33, and UL78. We previously showed US28-mediated signaling is required for establishing and maintaining latency and modulates the expression of specific cellular targets that regulate the Major Immediate Early (MIE) locus, a master regulator in the latent-to-lytic switch. Our findings also reveal a requirement for UL33 and UL78 for efficient viral reactivation from latency. Therefore, we hypothesize the vGPCRs modulate specific host signaling pathways to regulate the balance between latency and reactivation. To explore this hypothesis, we will take advantage of novel approaches coupled with our arsenal of viral recombinants, as well as both in vitro and ex vivo latency model systems. In Aim 1, we will define mechanisms underlying US28-mediated signaling and how this impacts the recruitment of silencing factors to the MIE locus during latency. In Aim 2, we will determine how UL33 signaling impacts the transactivation of the MIE locus and subsequent reactivation, as well as delineate how the interaction between US28 and UL78 tips the balance from latent-to-lytic replication. In sum, the experiments proposed herein will lead to a greater understanding of the vGPCRs’ biological functions during latency and reactivation, laying the foundation for future studies to develop novel therapeutics to prevent HCMV reactivation and disease.