Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively-cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19’s substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery.
Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with13C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.
Herpes viruses modulate cellular pathways to generate the building blocks that are necessary for their replication. Human cytomegalovirus alters metabolism of infected cells and causes a dramatic increase in lipid biosynthesis. We have investigated the role of lipid pathways in the viral life cycle and discovered that the virus requires several host enzymes that are responsible for the synthesis of very long chain fatty acids. Interestingly, very long chain fatty acids are substantially increased in the lipids of infected cells and saturated forms of these fatty acids are selectively incorporated into the envelope of the virus. Drugs that inhibit the synthesis of very long chain fatty acids generate virus particles with reduced infectivity. The discovery that human cytomegalovirus depends on the production of particular fatty acids furthers our understanding of virus-host cell interaction and suggests potential novel strategies for antiviral therapies.
We present a liquid chromatography-mass spectrometry (LC-MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water-methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 – 800 m/z at 1 Hz and 100,000 resolution in negative ion mode on a stand alone orbitrap (“Exactive”). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with linear range typically ≥ 100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker’s yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.
We find that the metabolome of nutrient-limited yeast varies dramatically with the limiting nutrient's identity. Low glutamine is a hallmark of nitrogen limitation, ATP of phosphorus limitation, and pyruvate of carbon limitation. The availability of these metabolites can quantitatively account for the nutrient-limited yeast's growth rate.
Microbes tailor their growth rate to nutrient availability. Here, we measured, using liquid chromatography-mass spectrometry, >100 intracellular metabolites in steady-state cultures of Saccharomyces cerevisiae growing at five different rates and in each of five different limiting nutrients. In contrast to gene transcripts, where ∼25% correlated with growth rate irrespective of the nature of the limiting nutrient, metabolite concentrations were highly sensitive to the limiting nutrient's identity. Nitrogen (ammonium) and carbon (glucose) limitation were characterized by low intracellular amino acid and high nucleotide levels, whereas phosphorus (phosphate) limitation resulted in the converse. Low adenylate energy charge was found selectively in phosphorus limitation, suggesting the energy charge may actually measure phosphorus availability. Particularly strong concentration responses occurred in metabolites closely linked to the limiting nutrient, e.g., glutamine in nitrogen limitation, ATP in phosphorus limitation, and pyruvate in carbon limitation. A simple but physically realistic model involving the availability of these metabolites was adequate to account for cellular growth rate. The complete data can be accessed at the interactive website http://growthrate.princeton.edu/metabolome.
Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use liquid chromatography-tandem mass spectrometry to quantify more than 100 metabolite concentrations in aerobic, exponentially growing E. coli with glucose, glycerol, or acetate as the carbon source. The total observed intracellular metabolite pool is approximately 300 mM. A small number of metabolites dominate the metabolome on a molar basis, with glutamate most abundant. Metabolite concentration exceeds Km for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the Km of their consuming enzymes and all reactions are near equilibrium. This may facilitate efficient flux reversibility given thermodynamic and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data.
Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple-quadruple mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass-resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.
Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD+. In addition to its role as a cofactor in reduction-oxidation reactions, NAD+ is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD+, which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD+ levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD+ levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD+ metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.
Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using −75°C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly 13C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.
Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, it would accordingly seem that the best method for extracting a particular metabolite is the one giving the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that ≥ 50% water gave increased yield of nucleosides and bases compared to ≤ 20% water, as determined by liquid chromatography-tandem mass spectrometry analysis of the resulting extracts. Spiking of the extracts with isotope-labeled nucleotides revealed, however, that the high yield of nucleosides and bases occurred due to decomposition of nucleotides in the water-rich condition, not good extraction. Spiking combined with isotope labeling provides a general approach to detecting decomposition products in extracts of cellular metabolites. For extraction of E. coli with methanol:water, cold temperature and a high methanol fraction minimizes artifacts due to metabolite decomposition.
Metabolomics; metabolism; extraction; bacteria; sampling; stability; LC-MS/MS; triple quadrupole; small molecule
We present a liquid chromatography – mass spectrometry (LC-MS) method for long-chain and very-long-chain fatty acid analysis, and its application to 13C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C8 reversed-phase chromatography using a water-methanol gradient with tributylamine as ion pairing agent, ionized by electrospray, and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/ml with a linear dynamic range of 100-fold. Ratios of unlabeled to 13C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of 13C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC-MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.
elongase; exactive; fatty acids; high resolution mass spectrometry; lipids; liquid chromatography-mass spectrometry; mass isotopomer distribution analysis; tracer studies; very-long-chain fatty acids
Anapleurosis is the filling of the TCA cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite, phosphoenolpyruvate (PEP). Here we show that E. coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting build-up of PEP is used to quickly import glucose if it becomes re-available. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally on steady glucose. However, they fail to build-up PEP upon glucose removal, grow poorly on oscillating glucose, and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.
Itaconic acid, or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR based metabolomics and 13C-labeling, we have detected itaconic acid in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased itaconic acid production and secretion. Crude cell extracts synthesize itaconic acid via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.
metabolomics; NMR; LC-MS; itaconic acid; tumor cells; macrophages
Gluconeogenesis converts three carbon units into glucose. Here we identify an analogous pathway in Saccharomyces cerevisiae for converting three carbon units into ribose, a component of nucleic acids and nucleotides. This riboneogenic pathway involves the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity was identified based on accumulation of sedoheptulose-1,7-bisphosphate in the corresponding knockout strain. We determined the crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate, and found that the sugar is bound in the closed furan form in the active site. Like fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate is produced by aldolase, in this case from erythrose 4-phosphate and dihydroxyacetone phosphate. Hydrolysis of sedoheptulose-1,7-bisphosphate by SHB17 provides an energetically favorable input to the non-oxidative pentose phosphate pathway to drive ribose production. Flux through SHB17 is enhanced under conditions when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells. Thus, riboneogenesis provides a thermodynamically-driven route of ribose production uncoupled from formation of NADPH.
Microbes survive in a variety of nutrient environments by modulating their intracellular metabolism. Balanced growth requires coordinated uptake of carbon and nitrogen, the primary substrates for biomass production. The mechanisms that balance carbon and nitrogen uptake are, however, poorly understood. We find in Escherichia coli that a sudden increase in nitrogen availability results in an almost immediate increase in glucose uptake. The concentrations of known glycolytic intermediates and regulators, however, remain homeostatic. Instead, we find that α-ketoglutarate, which accumulates in nitrogen limitation, directly blocks glucose uptake by inhibiting Enzyme I, the first step of the phosphotransferase system (PTS). This enables rapid modulation of glycolytic flux without marked concentration changes in glycolytic intermediates by simultaneously accelerating glucose import and consumption of the terminal glycolytic intermediate phosphoenolpyruvate. Quantitative modeling shows that this previously unidentified regulatory connection is in principle sufficient to coordinate carbon and nitrogen utilization.
The fermentation carried out by the biofuel producer Clostridium acetobutylicum is characterized by two distinct phases. Acidogenesis occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). Solventogenesis initiates as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol). Using metabolomics, isotope tracers, and quantitative flux modeling, we have mapped the metabolic changes associated with the acidogenic-solventogenic transition. We observed a remarkably ordered series of metabolite concentration changes, involving almost all of the 114 measured metabolites, as the fermentation progresses from acidogenesis to solventogenesis. The intracellular levels of highly abundant amino acids and upper glycolytic intermediates decrease sharply during this transition. NAD(P)H and nucleotide triphosphates levels also decrease during solventogenesis, while low-energy nucleotides accumulate. These changes in metabolite concentrations are accompanied by large changes in intracellular metabolic fluxes. During solventogenesis, carbon flux into amino acids, as well as flux from pyruvate (the last metabolite in glycolysis) into oxaloacetate, decreases by more than 10-fold. This redirects carbon into acetyl coenzyme A, which cascades into solventogenesis. In addition, the electron-consuming reductive tricarboxylic acid (TCA) cycle is shutdown, while the electron-producing oxidative (clockwise) right side of the TCA cycle remains active. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources (carbon and reducing power) from biomass production into solvent production.
The phosphotransferase system (PTS), encompassing EI, HPr, and assorted EII proteins, uses phosphoenolpyruvate to import and phosphorylate sugars. A paralog of EIIA of the sugar PTS system known as ptsN has been purported to regulate organic nitrogen source utilization in Escherichia coli K-12. Its known biochemical function, however, relates to potassium homeostasis. The evidence for regulation of organic nitrogen source utilization by ptsN is based primarily on the defective growth of ΔptsN mutants on amino acid nitrogen sources and other nutrient combinations. These observations were made with E. coli strains MG1655 and W3110, which carry a nonfunctional version of ilvG. There are three isozymes that effectively catalyze the first committed step of branched-chain amino acid biosynthesis, but ilvG is unique for doing so effectively across a range of potassium concentrations. Here we show that all of the nutrient utilization phenotypes attributed to ptsN are manifested selectively in strains lacking functional ilvG. We conclude that the ptsN gene product does not regulate organic nitrogen source utilization as previously proposed.
Due to the importance of microbes as model organisms, biotechnology tools, and contributors to mammalian and ecosystem metabolism, there has been longstanding interest in measuring their metabolite levels. Current metabolomic methods, involving mass spectrometry-based measurement of cell extracts, enable routine quantitation of most central metabolites. Metabolomics alone, however, is inadequate to understand cellular metabolic activity: Flux measurement and proteomic, genetic, and biochemical approaches with a metabolomics bent are all needed. Here we highlight examples where these integrated methods have contributed to discovery of metabolic pathways, regulatory interactions, and homeostasis mechanisms. We also indicate enduring challenges concerning unstable and low abundance compounds, subcellular compartmentalization, and quantitative amalgamation of different data types.
Autophagy is a process of self-cannibalization. Cells capture their own cytoplasm and organelles and consume them in lysosomes. The resulting breakdown products are inputs to cellular metabolism, through which they are used to generate energy and to build new proteins and membranes. Autophagy preserves the health of cells and tissues by replacing outdated and damaged cellular components with fresh ones. In starvation, it provides an internal source of nutrients for energy generation and, thus, survival. A powerful promoter of metabolic homeostasis at both the cellular and whole-animal level, autophagy prevents degenerative diseases. It does have a downside, however—cancer cells exploit it to survive in nutrient-poor tumors.
Mutation at the R132 residue of IDH1, frequently found in gliomas and acute myelogenous leukemia, creates a neo-enzyme that produces 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). We sought to therapeutically exploit this neo-reaction in mutant IDH1 cells which requires α-KG derived from glutamine. Glutamine is converted to glutamate by glutaminase (GLS) and further metabolized to α-KG. Therefore, we inhibited GLS with siRNA or the small molecule inhibitor BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide) and found slowed growth of glioblastoma cells expressing mutant IDH1 compared to those expressing wild-type IDH1. Growth suppression of mutant IDH1 cells by BPTES was rescued by adding exogenous α-KG. BPTES inhibited GLS activity, lowered glutamate and α-KG levels, and increased glycolytic intermediates while leaving total 2-HG levels unaffected. The ability to selectively slow growth in cells with IDH1 mutations by inhibiting glutaminase suggests a unique re-programming of intermediary metabolism and a potential therapeutic strategy.
Glioma; IDH1; α-ketoglutarate; 2-hydroxyglutarate; cancer metabolism; glutamine
Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells as PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2 expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His-11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1, and may provide an alternate glycolytic pathway that decouples ATP production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support anabolic metabolism observed in many proliferating cells.
Despite the therapeutic importance of antifolates, the links between their direct antimetabolite activity and downstream consequences remain incompletely understood. Here we employ metabolomics to examine the complete metabolic effects of the antibiotic trimethoprim in E. coli. In rich media, trimethoprim treatment causes thymineless death. In minimal media, in contrast, trimethoprim addition results in rapid stoppage of cell growth and stable cell stasis. We show that initial impairment of cell growth is due to rapid depletion of glycine and associated activation of the stringent response. Long-term stasis is due to purine insufficiency. Thus, E. coli has dual systems for surviving folate depletion and avoiding thymineless death: a short-term response based on sensing of amino acids and a long-term response based on sensing of nucleotides.
Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.
Enveloped viruses draw on cellular machinery and materials to generate copies of their genome, structural proteins, and membrane. These biosynthetic processes use the host metabolic network to provide energy and small-molecule precursors. We have investigated how two important enveloped viruses, human cytomegalovirus and herpes simplex virus-1, alter host metabolism to provide materials for viral replication. We show that rather than passively relying on basal host cell metabolic activity, both viruses actively redirect host cell metabolism, implementing divergent metabolic programs that are robust to host cell type and virus strain. Human cytomegalovirus enhances lipid biosynthesis, while herpes simplex-1 gears central carbon metabolism toward the synthesis of pyrimidine nucleotides. Consistent with these changes, human cytomegalovirus is more sensitive to inhibition of fatty acid synthesis and herpes simplex virus-1 to inhibition of central metabolic reactions leading towards pyrimidine synthesis. As these two closely related viruses have divergent metabolic strategies, and since the metabolic perturbations point to potential drug targets, an important priority is defining the metabolic programs of other viruses.