Biology subjects

Purdy, J. G.

Publications and source records attributed to Purdy, J. G..

9 recordsLinked to original sources

bioRxiv2026

Glutamine supports human cytomegalovirus induced lipidome remodeling through reductive carboxylation

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 [~]30% of the carbons from glutamine are delivered to fatty acid synthesis through additional metabolic means. Our current understanding of metabolite utilization during HCMV replication is based on cell culture models where there is an excess amount of glucose, suggesting that deriving carbons from glutamine might be needed when glucose levels are low. 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 still 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. Author SummaryMany viruses hijack cellular metabolic processing to obtain the components needed for replication. Human cytomegalovirus (HCMV) uses several mechanisms to reprogram lipid metabolism and remodel the lipidome of infected cells. HCMV promotes synthesis of very long chain fatty acids that are found in phospholipids and triacylglycerol. Glucose and acetate contribute carbon to fatty acid synthesis and elongation following HCMV infection. In this work, we demonstrate that glutamine is an additional carbon source for fatty acid and lipid synthesis. Phospholipids and neutral lipids are enriched with carbons from glutamine during HCMV infection. Mechanistically, HCMV induces reductive carboxylation to increase glutamine flow to fatty acid synthesis and increased metabolite availability supports additional carbon flow to fatty acids. Overall, this study provides additional insight into virus-induced metabolic remodeling that supplies the molecular building blocks for virus replication.

microbiology↗

bioRxiv2025

Human Cytomegalovirus Infection Reduces an Endogenous Antiviral Fatty Acid by Promoting Host Metabolism

Some viruses, including human cytomegalovirus (HCMV), induce the synthesis of fatty acids and lipids to ensure that the lipid environment of infected cells supports virus replication. HCMV infection broadly reprograms metabolism to ensure central carbon metabolism provides the metabolites required for anabolic synthesis of nucleotides, proteins, and lipids while also meeting the energy demands placed on the infected cells. While HCMV infection increases the levels of most very long chain fatty acids (VLCFA), we found that the levels of erucic acid (EA), a C22:1 monounsaturated VLCFA, are reduced. Treating infected cells with EA disrupted a late step in virus replication, resulting in the release of virions with reduced infectivity. Moreover, we used lipidomics to determine that EA-treated cells had elevated levels of lipids containing a combination of a C22:1 tail and a VLC polyunsaturated fatty acid tail (VLC-PUFA). We demonstrate that fatty acid elongase 5 (ELOVL5) mediated production of VLC-PUFAs is stimulated by HCMV infection. ELOVL5 aided the increase in lipids with C22:1 plus VLC-PUFA tails following EA treatment and reduced the overall level of C22:1 in HCMV-infected cells. Moreover, we found that ELOVL5 mollified EA inhibition of HCMV replication, suggesting ELOVL5 plays a critical role in reducing the level of an endogenous FA with antiviral properties. Our study provides insight into how infection may increase the synthesis of an antiviral metabolite or FA and how the virus may evade their antiviral effect by promoting their metabolism.

microbiology↗

bioRxiv2025

Human cytomegalovirus promotes de novo PC synthesis during early virus replication

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.

microbiology↗

bioRxiv2024

Metabolites that feed upper glycolytic branches support glucose independent human cytomegalovirus replication

The broad tissue distribution and cell tropism of human cytomegalovirus indicates that the virus successfully replicates in tissues with various nutrient environments. HCMV requires and reprograms central carbon metabolism for viral replication. However, many studies focus on reprogramming of metabolism in high nutrient conditions that do not recapitulate physiological nutrient environments in the body. In this study, we investigate how HCMV successfully replicates when nutrients are suboptimal. We limited glucose following HCMV infection to determine how glucose supports virus replication and how nutrients potentially present in the physiological environment contribute to successful glucose independent HCMV replication. Glucose is required for HCMV viral genome synthesis, viral protein production and glycosylation, and virus production. However, supplement of glucose-free cultures with uridine, ribose, or UDP-GlcNAc--metabolites that support upper glycolytic branches--resulted in partially restored viral genome synthesis and subsequent partial restoration of viral protein levels. Low levels of virus production were also restored. Supplementing lower glycolysis in glucose-free cultures using pyruvate had no effect on virus replication. These results indicate nutrients that support upper glycolytic branches like the pentose phosphate pathway and hexosamine pathway can compensate for glucose during HCMV replication to support low levels of virus production. More broadly, our findings suggest that HCMV could successfully replicate in diverse metabolic niches, including those in the body with low levels of glucose, through alternative nutrient usage. IMPORTANCEThe metabolic environment is a determinant in the ability of a virus to successfully replicate. HCMV has broad cell tropism and replicates in various tissues that have diverse and/or limiting metabolic environments. We know that HCMV reprograms host central carbon metabolism to support viral replication, but we have little understanding of HCMV replication in diverse metabolic niches as most studies use high nutrient culture media. Here, we show that glucose limitation suppresses virus production through loss of viral genome synthesis and viral protein glycosylation. However, nutrient compensation by uridine, ribose, and UDP-GlcNAc, metabolites that fuel upper glycolytic branches such as the non-oxidative pentose phosphate pathway support low levels of glucose-independent virus production. Our work indicates that metabolite compensation may facilitate HCMV replication in nutrient limited niches in the body.

microbiology↗

bioRxiv2021

The p97-UBXD8 complex modulates ER-Mitochondria contact sites by modulating membrane lipid saturation and composition

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we show that the p97 AAA-ATPase and its ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulate the prevalence of ER-mitochondria contacts. The p97-UBXD8 complex localizes to contacts and loss of this complex increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics of purified contacts demonstrates alterations in proteins regulating lipid metabolism upon loss of UBXD8. Furthermore, lipidomics studies indicate significant changes in distinct lipid species in UBXD8 knockout cells. We show that loss of p97-UBXD8 results in perturbed contacts due to an increase in membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts in p97-UBXD8 loss of function cells can be rescued by supplementation with unsaturated fatty acids or overexpression of SCD1. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark to a variety of human disorders such as neurodegeneration. Notably, we find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on p97-UBXD8.

cell biology↗

bioRxiv2021

Human Cytomegalovirus uses a Host Stress Response to Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very Long Chain Fatty Acids

Stress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK--and more broadly, ER stress--may be necessary for membrane biogenesis needed to generate infectious HCMV virions. IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

microbiology↗

bioRxiv2021

Hypoxia-inducible factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis

Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1 (HIF1) was previously proposed to support virus replication through its metabolic regulatory function. HIF1 protein levels rise in response to HCMV infection in non-hypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1 in HCMV replication by generating primary human cells with HIF1 knocked out using CRISPR/Cas9. When HIF1 was absent, we found that HCMV replication was enhanced, showing that HIF1 suppresses viral replication. We used untargeted metabolomics to determine if HIF1 regulates metabolite concentrations in HCMV infected cells. We discovered that in HCMV-infected cells, HIF1 suppresses intracellular and extracellular concentrations of kynurenine. HIF1 also suppressed the expression of the indoleamine 2,3-dioxygenase 1 (IDO1) rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication while activating AhR with an exogenous ligand increases HCMV replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1 reduces HCMV replication by regulating metabolism and metabolite signaling. ImportanceViruses, like human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1 (HIF1) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1 suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1-dependent mechanism. Our findings describe a functional connection between HIF1, IDO1, and AhR that allows HIF1 to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.

microbiology↗

bioRxiv2021

Shear-Mediated Platelet Activation is Accompanied by Unique Alterations of Platelet Lipid Profile

Platelet activation by mechanical means such as shear stress, is a vital driver of thrombotic risk in implantable blood-contacting devices used in treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical - shear mediated platelet activation. Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have procoagulant properties. We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices.

cell biology↗

bioRxiv2019

Human Cytomegalovirus Remodeling of Lipid Metabolism Requires pUL37x1

Human cytomegalovirus (HCMV) replication requires remodeling of the host metabolic network, including increasing central carbon metabolism, fatty acid elongation and lipid synthesis. The virus-host interactions regulating HCMV metabolic hijacking are essential to infection. While multiple host factors including kinases and transcription factors have been defined, little is known about the viral factors required. Some host factors involved are dependent on Ca2+ signaling or host stress response. The viral pUL37x1 protein mobilizes Ca2+ and targets the mitochondria. The Ca2+ changes induced by pUL37x1 can activate stress responses. In this study, we tested the hypothesis that the viral pUL37x1 protein is required for HCMV metabolic remodeling. Using a combination of metabolomics, lipidomics, and metabolic tracers, we demonstrate that pUL37x1 protein is necessary for HCMV induced fatty acid elongation and lipid metabolism but not required for most viral hijacking of central carbon metabolism. Additionally, we show that pUL37x1 is required for HCMV to fully induce two host proteins that were previously demonstrated to be important for virus induced lipid metabolism: fatty acid elongase 7 and the ER-stress related kinase PERK. We also demonstrate, for the first time, that HCMV replication results in the increase in phospholipids with very-long chain fatty acyl tails. We conclude that pUL37x1 is required for HCMV metabolic remodeling but is not necessary to for the virus to hijack metabolism on a network-wide scale.

microbiology↗