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While several metabolite-related pathways exhibit altered activity in infected cells, the alteration of lipid-related pathways by HCMV has not been examined beyond fatty acid synthesis and elongation. In this study, we addressed this lack of understanding by focusing on phosphatidylcholine (PC), a class of lipids we previously showed is increased by HCMV infection in human foreskin fibroblasts. Here, we expand upon this finding by demonstrating that HCMV infection increases the abundance of PCs in several different fibroblasts and, similarly, in endothelial and epithelial cells. Additionally, HCMV elevates PC levels regardless of the level of confluency, type of growth medium, and presence of serum. Next, we investigated if HCMV alters the activity in the three PC synthesis pathways. We demonstrate that HCMV infection promotes the activity in the\n                      <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                      PC synthesis pathway using a\n                      <jats:sup>13<\/jats:sup>\n                      C-choline isotopic tracer and liquid chromatography high-resolution tandem mass spectrometry. Infection did not alter the activity in the other two pathways. Moreover, we examined the kinetics of PC remodeling by HCMV and found that PC synthesis was promoted and the PC lipidome shifted after 24 h post-infection. Furthermore, we found that PC remodeling occurred when DNA synthesis and subsequent steps of virus replication were inhibited by phosphonoacetic acid. Overall, this work suggests that the early steps of HCMV replication promote the reprogramming of host lipid metabolism to ensure the synthesis of a lipidome necessary to support HCMV infection.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Human cytomegalovirus (HCMV) is a common herpesvirus that establishes a lifelong and persistent infection in its human host. HCMV infection in most people does not cause overt disease. However, in immunocompromised individuals, severe CMV-associated disease can lead to permanent disabilities and even death. Additionally, congenital CMV is the leading infectious cause of birth defects. Viruses have evolved to hijack host metabolic pathways to facilitate their replication cycle. In this study, we determine that HCMV promotes the activity in the\n                        <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                        pathway of phosphatidylcholine (PC) synthesis. We demonstrate that the activity in the other PC synthesis pathways, the phosphatidylethanolamine N-methyltransferase and Lands cycles, is unaltered by HCMV infection. Moreover, we found that HCMV infection alters metabolic activity to increase the PC lipidome before 48 h post-infection. Additionally, our results suggest that immediate-early and early gene expression promotes changes in PC lipids. Together, our findings demonstrate that infection promotes the\n                        <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                        PC pathway to increase PC lipids during the early stages of virus replication.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.00579-25","type":"journal-article","created":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T13:03:02Z","timestamp":1755608582000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Human cytomegalovirus promotes\n                    <i>de novo<\/i>\n                    PC synthesis during early virus 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