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Nuclear Receptors Control Pro-Viral and Antiviral Metabolic Responses to Hepatitis C Virus Infection

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Nuclear Receptors Control Pro-Viral and Antiviral Metabolic Responses to Hepatitis C Virus Infection ARTICLE PUBLISHED ONLINE: 10 OCTOBER 2016 | DOI: 10.1038/NCHEMBIO.2193 Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection Gahl Levy1,2,11, Naomi Habib1–3,11, Maria Angela Guzzardi1,4,11, Daniel Kitsberg1,2, David Bomze1,2, Elishai Ezra1,5, Basak E Uygun6, Korkut Uygun6, Martin Trippler7, Joerg F Schlaak7, Oren Shibolet8, Ella H Sklan9, Merav Cohen1,2, Joerg Timm10, Nir Friedman1,2 & Yaakov Nahmias1,2* Viruses lack the basic machinery needed to replicate and therefore must hijack the host’s metabolism to propagate. Virus- induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host–pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)- responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4a, ketogenesis by PPARa and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4a reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARa or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host’s metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host. iral infection is one of the leading medical challenges of enzymes of oxidative phosphorylation as well6. Enigmatically, sev- the 21st century. Viruses are parasites that lack the basic eral studies have demonstrated upregulation of both lipid peroxida- Vmetabolic machinery needed to replicate or assemble and tion and lipid accumulation in infected Huh7 cells5,6. therefore must hijack host processes to complete their life cycle. Interestingly, host metabolic processes are regulated at the Early research on metabolic changes in infected cells used 13C trac- transcriptional level by a network of ligand-activated transcrip- ing of glucose and glutamine to show that human cytomegalovi- tion factors called nuclear receptors12. Unraveling the connections rus (HCMV) infection induces glycolysis and fatty acid synthesis between transcriptional regulators and the virus-induced meta- in fibroblasts1. Direct pharmacological inhibition of this pathway bolic response would permit systematic modulation of metabolic suppresses HCMV replication2. More recently, lipidomics has been pathways, enabling scientists to probe the complexity of host–virus used to identify fatty acids and lipid mediators critical for influ- interactions. Regretfully, transcriptional control of metabolism is enza replication in A549 cells3,4. Again, reversal of virus-suppressed often obscured by other, massive disease-induced transcriptional ω3-polyunsaturated fatty acid (PUFA) metabolism was shown to responses, such as inflammation and wound healing. Consequently, block viral replication4. System-wide proteomic and transcriptomic attempts to link metabolic fluxes to transcriptional regulation thus analysis of HCV infection in replicating Huh7.5 cells has suggested far have been carried out primarily in single-cell organisms13. that the virus similarly hijacks host metabolism to support its Here we overcame the technical barriers to in vitro study of HCV replication, assembly and egress5,6. Together, these findings sug- infection by using oxygenated cocultures of primary human hepa- gest that viruses evolved as metabolic engineers, modulating host tocytes that support normal gene expression levels and metabolic processes to optimize virus production. function14. We found that these cocultures supported robust HCV Our focus was HCV infection, a pandemic affecting 3% of the infection, which caused metabolic changes that stabilized after 8 d © 2016 Nature America, Inc., part of Springer Nature. All rights reserved. All rights part Nature. of Springer Inc., America, Nature © 2016 world’s population7. This nonintegrating virus replicates in liver of culture (Fig. 1). This stable experimental system allowed us to cells, causing fatty liver disease and type 2 diabetes8,9. The under- use targeted metabolomics to derive a complete metabolic network lying cause of these metabolic changes is difficult to study owing induced by HCV infection. Using this metabolic network, we iden- to the lack of biopsy samples from patients in the early stages of tified differentially activated metabolic fluxes and corresponding infection and difficulties infecting primary hepatocytes in culture10. changes in gene expression. We identified transcriptional regulators, Nevertheless, studies using derivatives of the Huh7 cell line have validated them experimentally using a library of GFP reporters, and shown that HCV strongly depends on host lipid metabolism for systematically perturbed their activity using small-molecule inhibi- its replication, assembly and egress11. Proteomic analysis of HCV tors. Our analysis showed that HCV-infected primary hepatocytes infection in Huh7 cells has shown that, like HCMV and influenza, become dependent on an HNF4α-induced shift from oxidative HCV induces glycolytic enzymes but paradoxically upregulates the phosphorylation to glycolysis. In addition, we clarify the paradoxical 1Grass Center for Bioengineering, Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel. 2Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel. 3Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA. 4Institute of Clinical Physiology, National Research Council (CNR), Pisa, Italy. 5Faculty of Engineering, Jerusalem College of Technology, Jerusalem, Israel. 6Center for Engineering in Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA. 7Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany. 8Liver Unit, Department of Gastroenterology, Tel-Aviv Medical Center and Sackler Faculty of Medicine, Tel Aviv, Israel. 9Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. 10Institute for Virology, Medical Faculty, University of Düsseldorf, Düsseldorf, Germany. 11These authors contributed equally to this work. *e-mail: [email protected] NATURE CHEMICAL BIOLOGY | VOL 12 | DECEMBER 2016 | www.nature.com/naturechemicalbiology 1037 ARTICLE NaTUrE chEMicaL BioLoGY Doi: 10.1038/NchEmbio.2193 disconnect between lipid oxidation and accumulation, and con- was markedly altered by HCV infection. Naive cells displayed a comitantly show that drug metabolism was significantly induced cuboidal morphology, smooth cytoplasm and bright cell borders by HCV infection in this study. Finally, we confirmed our find- indicative of differentiated, healthy cells. In contrast, JFH-1-infected ings using patient serum and biopsy samples. Our work elucidates cells were noticeably smaller and contained numerous lipid droplets the metabolic fingerprint of HCV infection, identifying distinct in their cytoplasm (Fig. 1b). To visualize infection and lipid accu- assemblies of transcriptional–metabolic regulatory circuits in pri- mulation, we infected hepatocytes with a JC1 variant of HCV that mary human hepatocytes. contained an in-frame insertion of RFP in the NS5A viral protein16 (hereinafter called JC1-RFP) and counterstained for intracellular RESULTS triglycerides (Fig. 1c). Fluorescence microscopy showed that 50% Robust HCV infection of primary human hepatocytes ± 15% of the hepatocytes were positive for HCV after 10 d of infec- We cocultured freshly isolated primary human hepatocytes with tion. HCV production stabilized after 8 d of infection, reaching endothelial cells expressing the HCV capture receptor L-SIGN15. 30% of the RNA level of model cells (Huh7.5.1 infected with JFH-1) Cells were seeded in 95% oxygen under serum-free conditions14 and infectivity titers of 400 focus forming units (f.f.u.) per ml and infected with the JFH-1 cell-culture variant of HCV or a mock- (Fig. 1d). Intracellular analysis showed HCV core protein levels of infection control on the first day of culture (Fig. 1a). Hepatocytes 6.2 ± 2 fmol per mg of total protein and a 4.4-fold increase in intra- cocultured with L-SIGN-expressing endothelial cells showed 2.6- cellular triglycerides (P < 0.05); these results confirm steatosis, a fold higher levels of intracellular HCV RNA after 10 d of infection clinical hallmark of infection (Fig. 1d). Interestingly, although JFH- compared with those cultured alone (P < 0.05; Supplementary 1-infected Huh7.5.1 cells showed a marked increase in apoptosis Results, Supplementary Fig. 1). Primary hepatocyte morphology upon infection, no significant cell death was observed in primary a HCV HCV-infected Transcriptional infection cells Primary human regulatory analysis hepatocytes Function Transcriptomics Time and fluxomics Sham control 10 d Naive cells 95% O2 Serum-free b c Naive JFH-1-infected JC1/RFP / triglycerides d 0.5 600 e Naive JFH-1-infected HCV RNA Infectivity (f.f.u./ml) 0.4 1.2 120 50 Infectivity 450 cells) 6 0.3 cells) 40 6 0.9 cells) 90 300 * 6 0.2 30 ** 0.6 60 g/d/10 g/d/10 150
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