Glutamine Metabolism Is Essential for Human Cytomegalovirus Infection

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to α-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates α-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.Glucose (Glc) and glutamine are the two most abundant nutrients used by mammalian cells. They are necessary for the generation of energy, macromolecules, and second messengers (1, 5-7, 9-12, 16). Glucose has long been considered absolutely essential for the viability of mammalian cells because of its contribution to energy homeostasis through glycolysis and the tricarboxylic acid (TCA) cycle (Fig. ​(Fig.1).1). Recent studies demonstrated that human diploid fibroblasts are killed by glucose deprivation by a mechanism different from apoptosis (20).Open in a separate windowFIG. 1.Glycolysis and the citric acid cycle showing glucose and glutamine utilization. The aspects of the cytoplasmic (Cyto) and mitochondrial (Mito) metabolism of glucose and glutamine discussed in the text are outlined. Dashed lines indicate that there are several intermediates formed (several reactions) between the ones shown. PEPCK, phosphoenolpyruvate carboxykinase; ME: malic enzyme; GDH, glutamate dehydrogenase; GLS, glutaminase; ACL, ATP citrate lyase; OAA, oxaloacetic acid; AcCoA, acetyl coenzyme A.In 1924, Warburg observed that cancer cells metabolize glucose very differently than normal cells (18). Cancer cells converted glucose into lactate even in the presence of sufficient oxygen to support mitochondrial oxidative phosphorylation (Fig. ​(Fig.1).1). This utilization of glucose, called the Warburg effect, results in only 2 ATP molecules produced per molecule of glucose, whereas if it had proceeded through the TCA cycle and mitochondrial oxidative phosphorylation, an additional 36 ATP molecules would have been produced per molecule of glucose. Recently reported data provide an explanation for what appeared to be an inefficient utilization of glucose (7, 8, 19). In cancer cells, exogenous glutamine is used as a carbon source, which facilitates the cell''s ability to use glucose biosynthetically instead of breaking it down completely for energy. This is accomplished by glutamine being converted to α-ketoglutarate via glutaminase (GLS) and glutamate dehydrogenase (GDH) (Fig. ​(Fig.1).1). This process of replenishing TCA cycle intermediates is called anaplerosis. Thus, glutamine anaplerotically fills the TCA cycle (Fig. ​(Fig.1),1), providing NADH for oxidative phosphorylation as well as TCA cycle intermediates, which serve as important biosynthetic precursors (7, 8). In contrast, normal cells are believed to use only a small amount of consumed glutamine for macromolecular biosynthesis and energy; thus, glucose and glutamine metabolism are dramatically altered in tumor cells (8, 16).While glutamine starvation in many cell types has little impact on cell viability, it has been shown to induce cell death in cancer cell lines that overexpress the oncogene c-myc (20). These cells also showed decreased levels of ATP production correlating with decreased concentrations of TCA cycle intermediates; both are predictable consequences of glutamine starvation if glutamine is being used anaplerotically. In agreement with this finding, the effects of glutamine starvation could be reversed by the addition of the TCA cycle intermediates pyruvate (Pyr) and oxaloacetate (OAA) (Fig. ​(Fig.11).Human cytomegalovirus (HCMV) is a slow-growing betaherpesvirus that exerts a large energetic and biosynthetic demand on cells to ensure successful viral replication. Recent mass spectrometry-based metabolic flux studies indicated global metabolic upregulation in infected cells (14, 15). This included greatly increased glycolysis in which the vast majority of glucose-derived acetyl coenzyme A (AcCoA) went to support fatty acid synthesis (Fig. ​(Fig.1)1) to make membranes needed by the virus. Thus, there is a great decrease in the amount of glucose-derived carbon entering the TCA cycle. In other words, the virus induces a modified Warburg effect so that glucose-derived carbon can be used biosynthetically. These metabolomic data also suggest that glutamine may be used to anaplerotically fill the TCA cycle.We have investigated the impact of glucose and glutamine on HCMV replication. We have found that under conditions of glucose deprivation, infected cells are more viable than mock-infected cells. Thus, we hypothesized that the infected cells use glutamine anaplerotically. In agreement with this prediction, glutamine was found to be necessary for ATP production in infected cells but not in uninfected cells. Furthermore, cells starved of glutamine beginning 24 h postinfection (hpi) failed to produce infectious virions. HCMV-induced glutaminolysis was indicated by increased glutamine uptake and ammonia production corresponding to increased activities of glutaminase and glutamate dehydrogenase. These enzymes convert glutamine to α-ketoglutarate (α-KG) for anaplerotic use in the TCA cycle. The anaplerotic use of glutamine in the TCA cycle was also demonstrated by the finding that both ATP production and viral growth could be rescued by replacing glutamine with the TCA cycle intermediate α-ketoglutarate, oxaloacetate, or pyruvate. Thus, our data suggest that in HCMV-infected cells, as in many tumor cells, a program is activated whereby glutamine utilization increases specifically to maintain the TCA cycle, allowing glucose to be used biosynthetically.