Human cytomegalovirus (HCMV) establishes a life-long persistent infection by evading the immune system, in part, by direct cell-to-cell viral spread. In solid organ or stem cell transplant recipients, HCMV spread leads to end-organ diseases that can cause death. During pregnancy, HCMV spread causes congenital infection and is a leading cause of congenital disabilities. No HCMV treatment offers a cure, and there is no vaccine. Thus, there is a need for new treatments to limit infection based on novel discoveries in HCMV biology. Viral proteins required for HCMV cell-to-cell spread are known, but the host processes involved in HCMV cell-to-cell spread have received less attention. Clinical strains of HCMV spread most efficiently through cell-to-cell means, but the molecular mechanisms—including host metabolic ones—essential to HCMV cell-to-cell spread are largely unknown. Understanding host mechanisms regulating cell-to-cell spread may lead to new understandings of how to reduce HCMV infection. Our research has uncovered a novel role of metabolite signaling in promoting HCMV spread. This project's overall goal is to mechanistically understand virus-host interactions regulating metabolite signaling essential to HCMV cell-to-cell spread. We found a metabolite in tryptophan metabolism—kynurenine (KYN)—enhances HCMV spread. In addition to its metabolic role, KYN is a signaling messenger. KYN signals through aryl hydrocarbon receptor (AhR). We show that activation of AhR supports HCMV replication. Moreover, we found that hypoxia-inducible factor 1α (HIF1α), through its metabolic regulatory function, limits the production of KYN and suppresses HCMV infection. We hypothesize that metabolite-mediated signaling from infected cells to uninfected cells promotes HCMV cell-to-cell spread, which is attenuated by a HIF1α-dependent cellular response. The proposed research will determine molecular mechanisms involved in the enhancement of HCMV infection by KYN-metabolite signaling (aim 1) and define virus-host interactions regulating HIF1α attenuation of HCMV cell-to-cell spread (aim 2). The experimental approach will integrate virus assays, CRISPR/Cas9 engineering, and untargeted metabolomics to understand HCMV biology. Our findings will provide a mechanistic understanding of metabolite signaling and AhR activity in promoting HCMV cell-to-cell spread and the HIF1α-dependent host-response that targets metabolite signaling to reduce infection. Our studies will advance our knowledge in an understudied area of HCMV research that will provide significant steps-forward in developing novel strategies to treat HCMV infection and limit HCMV-related disease.