PLoS Pathog. 2026 Aug 12. 22(8):
e1014496
Lipidome remodeling during human cytomegalovirus (HCMV) replication is a complex process that requires induction of lipogenic proteins and altered metabolite flow to support synthesis of fatty acids and lipids. HCMV infection increases the utilization of glucose and acetate to provide enough carbons to support increased demand for lipogenesis during virus replication, but other carbon contributors have not been studied. Here, we identify glutamine as a carbon source for lipogenesis during HCMV infection. Metabolic tracing with 13C-labeled glutamine revealed carbons from glutamine are enriched in phospholipids and neutral lipids during infection, including phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, and triacylglycerol. Additional metabolic tracing demonstrates that HCMV infection promotes glutamine flow to fatty acid synthesis primarily through reductive carboxylation, i.e., conversion of glutamine to citrate through isocitrate. Through the use of two different 13C-labeled forms of glutamine, we found that ~70% of the carbons from glutamine are delivered to fatty acid synthesis through reductive carboxylation. Our current understanding of metabolite utilization during virus replication is based on cell culture models in which glucose is in excess, suggesting that HCMV may encode mechanisms to promote glutamine flow to lipids if glucose levels in vivo are insufficient. To determine if concentrations of glucose and glutamine change their contributions to fatty acid synthesis, we investigated lipogenesis when glucose and glutamine are at physiological levels (5 mM and 0.55 mM, respectively). We determined that physiological levels of glucose and glutamine are sufficient to support the increased demand for fatty acid synthesis caused by HCMV infection, despite a reduction in virus production. Using metabolic tracing with 13C-labeled forms of glucose or glutamine, we determined that both carbon sources contribute to fatty acid synthesis when present at physiological levels. Overall, our results identify viral activation of reductive carboxylation that increases glutamine flow to lipogenesis during infection. This work provides additional insight into metabolic reprogramming that supports HCMV-induced lipidome remodeling.