Human cytomegalovirus promotes de novo PC synthesis during early virus replication

Abstract

Human cytomegalovirus (HCMV) infection reprograms metabolism, including lipid synthesis. While several metabolite-related pathways have been demonstrated to have 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 found 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 de novo PC synthesis pathway using a 13C-choline isotopic tracer and liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS). Infection did not alter the activity in the other two pathways. Moreover, we examined the kinetics of PC remodeling by HCMV and found that the de novo synthesis pathway is promoted and the PC lipidome shifts 24 hours post infection. That led us to examining if the early stages of replication are sufficient to alter PC levels. After inhibiting late virus replication, we found that HCMV alters the PC lipidome independent of late gene expression. Overall, this work suggests that an immediate-early or early viral protein promotes the reprogramming of host lipid metabolism to ensure the synthesis of a lipidome necessary to support HCMV replication. IMPORTANCEHuman cytomegalovirus (HCMV) is a common herpesvirus that establishes a lifelong and persistent infection in its human host (1). HCMV infection in most people does not cause overt disease (1). However, in immunocompromised individuals, severe CMV-associated disease can lead to permanent disabilities and even death (1, 2). Additionally, congenital CMV is the leading infectious cause of birth defects (3, 4). 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 de novo pathway of phosphatidylcholine (PC) synthesis. We demonstrate that the activity in the other PC synthesis pathways, the PEMT and Lands cycle, is unaltered by HCMV infection. Moreover, we found that HCMV infection alters metabolic activity to increase the PC lipidome before 48 hpi. Additionally, we demonstrate that changes in PC lipids during virus replication is independent of late gene expression. Together, our findings demonstrate that infection promotes the de novo PC pathway to increase PC lipids during the early stages of virus replication.