Abstract Human cytomegalovirus (HCMV) is a leading cause of birth defects and severe illness in immunocompromised individuals. Understanding how HCMV manipulates cellular metabolism will provide insight into an emerging host-pathogen interaction that can potentially lead to novel therapeutic targets, an important goal as many current HCMV therapies have poor bioavailability and elevated toxicity profiles. HCMV induces numerous metabolic activities and modulates the expression and activity of a variety of host metabolic genes. For many of these changes, it is unclear how they contribute to successful infection. One gene that we find to be induced during infection is the neuron-specific Enolase 2. The goal of this proposal is to investigate the role of Enolase 2 during HCMV infection through three specific aims. In the first aim, I will investigate how Enolase 2 expression contributes to viral infection. My preliminary data show that (i) HCMV induces Enolase 2 during infection, and (ii) that shRNA-mediated loss of Enolase 2 attenuates viral infection. shRNA- and CRISPR-Cas9- mediated knockdown/knockout of Enolase 2 will be utilized for all parts of this proposal. The first part of this aim will investigate the mechanism of this attenuation and where in the viral lifecycle is Enolase 2 required. In the second part of this aim, I will investigate the specificity of Enolase 2 for viral infection. There are three well defined isozymes of enolase encoded in the human genome. While these genes are highly conserved, literature has shown they may have biological differences. I will test the ability of Enolase 1 or Enolase 3 or catalytically dead Enolase 2 to restore HCMV infection. In my second aim, I will investigate the role of Enolase 2 in HCMV-mediated metabolic reprogramming. Enolase has not been shown to have a role in regulating glycolysis, yet my preliminary data show that loss of Enolase 2 ablates glucose consumption during infection. Using LC/MS/MS, I will investigate changes in metabolic reprogramming that occur during viral infection when Enolase 2 is present or not. The second part of aim 2 will investigate the requirement of Enolase 2 under different physiological conditions such as temperature. HCMV infection can cause flu-like symptoms including fever. Therefore, Enolase 2 may be required over Enolase 1/3 due to its higher stability. In my third aim, I will elucidate the mechanism behind HCMV induction of Enolase 2. Preliminary data suggest that the UL38 viral protein is required for the induction of Enolase 2. I will investigate the ability of UL38 to include Enolase 2 expression and determine the mechanism. Results from this proposal will provide insight into the role of enolase during HCMV infection and will highlight physiological and biological differences between the enolase isoforms.