Metabolomic analysis by liquid chromatography–high-resolution mass spectrometry results in data sets with thousands of features arising from metabolites, fragments, isotopes, and adducts. Here we describe a software package, Metabolomic Analysis and Visualization ENgine (MAVEN), designed for efficient interactive analysis of LC–MS data, including in the presence of isotope labeling. The software contains tools for all aspects of the data analysis process, from feature extraction to pathway-based graphical data display. To facilitate data validation, a machine learning algorithm automatically assesses peak quality. Users interact with raw data primarily in the form of extracted ion chromatograms, which are displayed with overlaid circles indicating peak quality, and bar graphs of peak intensities for both unlabeled and isotope-labeled metabolite forms. Click-based navigation leads to additional information, such as raw data for specific isotopic forms or for metabolites changing significantly between conditions. Fast data processing algorithms result in nearly delay-free browsing. Drop-down menus provide tools for the overlay of data onto pathway maps. These tools enable animating series of pathway graphs, e.g., to show propagation of labeled forms through a metabolic network. MAVEN is released under an open source license at http://maven.princeton.edu.

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.

Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.herpesvirus | siRNA V iruses are dependent on host cell signaling pathways for replication and spread. Infection with the prevalent β-herpes virus human cytomegalovirus (HCMV) induces increased levels of protein phosphorylation and markedly alters host cell signal transduction pathways (1). A portion of the phosphorylation changes induced by HCMV infection are attributed to the Ser/ Thr kinase encoded by the viral genome, pUL97 (2), and others may derive from cellular kinase(s) packaged into virions (3) or from cellular kinases known to be activated by HCMV (4).Here we sought to more completely delineate the effects of HCMV infection on kinase signaling by performing an siRNA screen of the entire cellular kinome. The screen identified 106 kinases predicted to influence the production of virus. The hits included the 5′-AMP-activated protein kinase (AMPK), a sensor of cellular energy homeostasis. AMPK is composed of three subunits: a catalytic subunit, AMPKα, and two regulatory subunits, AMPKβ and AMPKγ. Activation of the kinase requires cooperative AMP binding to AMPKγ, which occurs stochastically with shifts in the AMP:ATP ratio, and phosphorylation of AMPKα at Thr172 (5, 6). At least three different kinases are reported to phosphorylate Thr172 of AMPKα: Ca 2+ /calmodulindependent kinase kinase (CaMKK), TGF-β-activated kinase 1 (TAK1), and liver kinase B1 (LKB1) (7). Activated AMPK phosphorylates a number of substrates to effect changes in central carbon metabolism, lipid metabolism, physiological homeostasis, cell growth, apoptosis, and gene expression (5).HCMV induces glycolysis (8-10) and also causes increased levels of the glucose transporter GLUT4 at the plasma membrane increasing glucose uptake (11).AMPK controls GLUT4 relocalization to the plasma membrane (5), and this regulation likely links the kinase to altered metabolism in HCMV-infected cells. However, previous work indicates that pharmacological activation of AMPK during the early phase of HCMV infection can be deleterious to viral replication (12), yet CaMKK activity is required for ...

A rapid series of synchronous cell divisions initiates embryogenesis in many animal species, including the frog Xenopus laevis. After many of these cleavage cycles, the nuclear to cytoplasmic ratio increases sufficiently to somehow cause cell cycles to elongate and become asynchronous at the mid-blastula transition (MBT). We have discovered that an unanticipated remodeling of core metabolic pathways occurs during the cleavage cycles and the MBT in X.laevis, as evidenced by widespread changes in metabolite abundance. While many of the changes in metabolite abundance were consistently observed, it was also evident that different female frogs laid eggs with different levels of at least some metabolites. Metabolite tracing with heavy isotopes demonstrated that alanine is consumed to generate energy for the early embryo. dATP pools were found to decline during the MBT and we have confirmed that maternal pools of dNTPs are functionally exhausted at the onset of the MBT. Our results support an alternative hypothesis that the cell cycle lengthening at the MBT is triggered not by a limiting maternal protein, as is usually proposed, but by a decline in dNTP pools brought about by the exponentially increasing demands of DNA synthesis.

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