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. 2010 Feb;84(4):1867-73.
doi: 10.1128/JVI.02123-09. Epub 2009 Nov 25.

Glutamine metabolism is essential for human cytomegalovirus infection

Jeremy W Chambers  1 , Tobi G Maguire, James C Alwine
Affiliations

Glutamine metabolism is essential for human cytomegalovirus infection

Jeremy W Chambers et al. J Virol. 2010 Feb.

Abstract

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 alpha-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 alpha-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.

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Figures

FIG. 1.
FIG. 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.
FIG. 2.
FIG. 2.
Glutamine is necessary for virus replication and cell viability of HCMV-infected cells. (A) HF cells were plated and grown to 50% confluence in replete medium and then mock (black bars) or HCMV infected (MOI of 3) (gray bars) for 24 h in replete medium. At this point (24/0), one set of cells was counted, and the viability was determined by using trypan blue exclusion staining. Other sets of cells had the medium changed to glucose-free (−G), glutamine-free (−GT), or replete (R) medium. Forty-eight hours after the medium change (24/48), cell counts and viability were determined. Error bars are based on standard deviations of the means for 3 determinations for three replicate experiments. The standard deviation of the mean of cell viability determinations was no greater than ±5% based on three determinations for three replicate experiments. (B) Confluent monolayers of HF cells were infected and grown as described above (A). At 24 hpi (vertical arrow), the medium was changed to replete (R), glutamine-free (−glutamine), or glucose-free (−glucose) medium. Cells were harvested every 24 h up to 120 hpi, and titers of infectious virions in each sample were determined by using the TCID50 method.
FIG. 3.
FIG. 3.
Glutamine metabolism is altered during HCMV infection. (A) Cells were mock (black bars) or HCMV (gray bars) infected for 24 h in replete medium, at which time the medium was removed and analyzed for glutamine consumption and ammonia production by using a Nova Biomedical Flex analyzer. Error bars are based on standard deviations of the means for 5 separate measurements. (B) Cells were mock (black bars) or HCMV (gray bars) infected for 24 h and then extracted and assayed for glutaminase (GLS), glutamate dehydrogenase (GDH), malic enzyme (ME), and phosphoenolpyruvate carboxykinase (PEPCK), as described in Materials and Methods. Error bars are standard deviations of the means for 3 determinations from 3 separate experiments. (C) Mock-infected (black bars) or HCMV-infected (gray bars) cells were grown for 24 h in replete medium and then placed into either fresh replete, glutamine-free (−Gln), or glucose-free (−Glc) medium for 12 h, at which time intracellular ATP levels (nm/cell) were measured by a luciferase assay as described in Materials and Methods. Error bars are standard deviations of the means for 5 separate determinations.
FIG. 4.
FIG. 4.
Citric acid cycle intermediates rescue ATP production and viral growth in HCMV-infected cells deprived of glutamine. (A) Cells were mock (black bars) or HCMV (gray bars) infected for 24 h in replete medium and then placed into either fresh replete (R) or glutamine-free (None) medium or glutamine-free medium supplemented with glucose (+Glc) (an additional 5 mM), 4 mM pyruvate (+Pyr), 4 mM oxaloacetic acid (+OAA), or 7 mM dimethyl-α-ketoglutaric acid (+αKG). Twenty-four hours after the medium change, ATP levels were determined. Error bars are standard deviations of the means for 5 separate determinations. (B) Confluent monolayers of HF cells were infected (MOI of 3) for 24 hpi, and the medium was then changed to replete medium, glutamine-free medium (None), or glutamine-free medium supplemented with 4 mM pyruvate (+Pyr), 4 mM oxaloacetic acid (+OAA), or 7 mM dimethyl-α-ketoglutaric acid (+αKG). At 120 hpi viruses were harvested, and titers were determined as described in Materials and Methods. Error bars are standard deviations of the means for 3 parallel determinations.
FIG. 5.
FIG. 5.
HCMV infection alters the regulation of cellular glutamine metabolism in a temporal manner. (A) Infection time course done using replete medium (solid line) or where glutamine was removed 24 h prior to harvest (dashed lines, 48 to 72 hpi, 72 to 96 hpi, and 96 to 120 hpi). The titers of infections virions were determined by the TCID50 method. (B to E) cells in replete medium were mock or HCMV infected. To ensure that nutrients were not limiting during the course of this experiment, the replete medium was changed 24 h prior to each measurement. At various time points, glutamine consumption (B), glutaminase activity (C), and glutamate dehydrogenase (GDH) activity were determined as described in Materials and Methods. (E) Western analysis of the levels of glutaminase (GLS), glutamate dehydrogenase (GDH), and actin at each time point during infection.
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