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      Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics

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          Abstract

          Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis.

          Author Summary

          As parasites, viruses rely on the cells they infect to provide the energy and building blocks required for their survival and propagation. However, relatively little is known about the extent to which viruses modulate host cell metabolism and the consequences of these disruptions. Here we integrate proteomic and lipidomic profiling with computational modeling approaches to probe the impact of HCV infection on the global metabolism of cultured hepatoma cells, and to understand the potential contribution of such perturbations to viral pathogenesis. Our findings suggest that increases in host catabolic and biosynthetic activities occur early during infection, providing the host macromolecules necessary for viral growth. As infection progresses and cellular stress increases, however, a compensatory metabolic shift occurs in an attempt to maintain energy homeostasis and cell viability. This shift is accompanied by changes in lipid abundance, which has predicted consequences for the viral life cycle and pathogenesis. Computational modeling was then used to identify novel host proteins functioning as key regulators of this HCV-associated metabolic reprogramming. In summary, these studies provide new insights into the marked disruption of cellular metabolic homeostasis that occurs during HCV infection and that may contribute to liver disease progression.

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          Most cited references58

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          The lipid droplet is an important organelle for hepatitis C virus production.

          The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.
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            Chemometrics in metabonomics.

            We provide an overview of how the underlying philosophy of chemometrics is integrated throughout metabonomic studies. Four steps are demonstrated: (1) definition of the aim, (2) selection of objects, (3) sample preparation and characterization, and (4) evaluation of the collected data. This includes the tools applied for linear modeling, for example, Statistical Experimental Design (SED), Principal Component Analysis (PCA), Partial least-squares (PLS), Orthogonal-PLS (OPLS), and dynamic extensions thereof. This is illustrated by examples from the literature.
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              Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.

              Viruses rely on the metabolic network of their cellular hosts to provide energy and building blocks for viral replication. We developed a flux measurement approach based on liquid chromatography-tandem mass spectrometry to quantify changes in metabolic activity induced by human cytomegalovirus (HCMV). This approach reliably elucidated fluxes in cultured mammalian cells by monitoring metabolome labeling kinetics after feeding cells (13)C-labeled forms of glucose and glutamine. Infection with HCMV markedly upregulated flux through much of the central carbon metabolism, including glycolysis. Particularly notable increases occurred in flux through the tricarboxylic acid cycle and its efflux to the fatty acid biosynthesis pathway. Pharmacological inhibition of fatty acid biosynthesis suppressed the replication of both HCMV and influenza A, another enveloped virus. These results show that fatty acid synthesis is essential for the replication of two divergent enveloped viruses and that systems-level metabolic flux profiling can identify metabolic targets for antiviral therapy.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                January 2010
                January 2010
                8 January 2010
                : 6
                : 1
                : e1000719
                Affiliations
                [1 ]Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington, United States of America
                [2 ]Laboratory of Virology & Infectious Disease, Center for the Study of Hepatitis C, Rockefeller University, New York, New York, United States of America
                [3 ]Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, United States of America
                [4 ]Computational Biology & Bioinformatics, Pacific Northwest National Laboratory, Richland, Washington, United States of America
                [5 ]Washington National Primate Research Center, University of Washington Seattle, Washington, United States of America
                University of Kentucky College of Medicine, United States of America
                Author notes
                [¤a]

                Current address: iTherx Pharmaceuticals, San Diego, California, United States of America

                [¤b]

                Current address: Institute for Systems Biology, Seattle, Washington United States of America

                Conceived and designed the experiments: DLD AJS JMJ KAW TOM DGC RDS CMR MGK. Performed the experiments: AJS MG QZ RZ. Analyzed the data: DLD. Contributed reagents/materials/analysis tools: DLD AJS JMJ CMS KAW SCP JEM TOM DGC KMW RDS CMR MGK. Wrote the paper: DLD AJS JMJ CMS JEM TOM CMR MGK. Processing of proteomic data: JMJ. Processing of lipidomic data: CMS TOM. Performed computational modeling: JEM KMW.

                Article
                09-PLPA-RA-1174R2
                10.1371/journal.ppat.1000719
                2796172
                20062526
                a6b769f1-df44-44ed-ba8d-9f61f59662a2
                Diamond et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 10 July 2009
                : 10 December 2009
                Page count
                Pages: 18
                Categories
                Research Article
                Computational Biology
                Gastroenterology and Hepatology/Hepatology
                Infectious Diseases
                Infectious Diseases/Viral Infections

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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