PROJECT SUMMARY Human cytomegalovirus (HCMV) is a herpesvirus that causes disease and death in the immunocompromised and is a leading cause of congenital disabilities. HCMV replication requires lipids. Since HCMV does not encode a metabolic network, virus replication depends on host lipid metabolism. However, little is known about how HCMV reprograms host metabolism to ensure lipids required for virus replication are made. Our overall goal is to understand the virus-host interactions that regulate lipid synthesis essential for HCMV replication. Recently, we showed that HCMV infection results in an increase in lipid synthesis and a rise in lipid abundances. Here we demonstrate that HCMV infection induces the synthesis of at least 20 previously undescribed lipids unique to infected cells. Most of these unique lipids are phospholipids with very long-chain fatty acid tails (PL-VLCFAs). The PL-VLCFAs discussed in this application are understudied in general and unstudied in HCMV biology beyond our work. While shorter FA tails have been well-studied, we know little about lipids with VLCFAs tails that are as long as those we observe in HCMV infection, including how they will behave in a biological membrane. The molecular mechanisms underlying this HCMV-induced expansion in the host lipidome and the functional roles of the newly generated lipids are largely unknown. We discovered that HCMV pUL37x1 and pUL38 proteins promote PL-VLCFA synthesis, laying the foundation for understanding the mechanisms by which HCMV reprograms lipid synthesis. pUL37x1 and pUL38 induce Ca2+ and mTOR signaling, respectively. We have preliminary data suggesting that stress responses related to these signaling pathways contribute to HCMV remodeling of lipids. We hypothesize that pUL37x1 and pUL38 use Ca2+ and mTOR signaling to promote the synthesis of PC-VLCFAs required for HCMV replication. We will test this hypothesis by determining the mechanisms by which pUL37x1 and pUL38 promote synthesis of PL-VLCFAs (Aim 1) and defining the PL-VLCFA synthesis enzymes required for HCMV replication and the role of PL-VLCFAs in infection (Aim 2). These studies will determine the mechanisms by which HCMV interacts with the host to create a unique lipid environment advancing our knowledge of HCMV reprogramming of metabolism. Furthermore, these studies will define the biological functions of PC-VLCFAs in HCMV replication and further our understanding of lipids required for HCMV infection. Determining the mechanisms involved in HCMV-induced reprogramming of lipid metabolism and functions of PC-VLCFAs will advance knowledge in HCMV biology needed to identify new targets for treating infection.