Stearoyl-CoA desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes

Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virushost interaction machinery. IMPORTANCEHepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

Section: Molecular Interaction Of Neob With Lxrs Is Correlated With Tmentioning

confidence: 53%

Section: Molecular Interaction Of Neob With Lxrs Is Correlated With Tmentioning

confidence: 99%

Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System

Nakajima

Watashi

Ohashi

et al. 2016

“…While the present work was in review, Lyn et al (49) reported that inhibition of SCD1 disrupted the integrity of membranous HCV replication complexes and rendered HCV RNA susceptible to nuclease-mediated degradation. In their report, the authors examined a series of SCD1 inhibitors as probes for HCV-induced membrane alterations.…”

Section: Discussionmentioning

confidence: 99%

Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

Nguyen

Lim

Pham

et al. 2014

The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1 is associated with HCV replication complex and that its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection. IMPORTANCEStearoyl coenzyme A (CoA) desaturase 1 (SCD1), a liver-specific enzyme, regulates hepatitis C virus (HCV) replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes, and SCD1 is enriched on the lipid raft by HCV infection. Therein, SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication. Hepatitis C virus (HCV) is an enveloped virus with a positivesense, single-stranded RNA virus that belongs to the genus Hepacivirus in the family Flaviviridae (1). Approximately 170 million people are chronically infected with HCV worldwide. Three million people are newly infected with HCV annually, and more than 350,000 individuals die from HCV-related liver diseases every year (1, 2). Current standard therapy elicits some side effects and results in a sustained virological response in only certain genotypes of HCV patients. Recently, the U.S. Food and Drug Administration approved various direct-acting antivirals (DAAs), including boceprevir, telaprevir, sofosbuvir, and simeprevir, for triple therapy in combination with pegylated interferon and ribavirin for patients with certain genotypes. Nevertheless, these new DAAs also have had some lim...

“…Phospholipid acyl chain saturation has been implicated in genome replication and virion infectivity of RNA and DNA viruses (11,12). Pharmacological inhibition of stearoyl-coenzyme A desaturase (SCD-1), an enzyme that converts saturated to unsaturated acyl chains, was recently shown to suppress HCV replication and hence proposed to be a potential antiviral strategy (58,59). Likewise, in light of our findings, decreasing the levels of PI(4,5)P 2 with unsaturated acyl chains by using a similar strategy may abrogate Gag membrane binding and/or proper Gag localization with minimal effects on cellular PI(4,5)P 2 -dependent processes.…”

Section: Figmentioning

confidence: 99%

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Olety

Veatch

Ono

2015

HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P 2 -specific pleckstrin homology domain of phospholipase C␦1 (PH PLC␦1 ), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P 2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P 2 with unsaturated or saturated acyl chains affect membrane binding of PH PLC␦ 1 and Gag. Both unsaturated dioleoyl-PI(4,5)P 2 [DO-PI(4,5)P 2 ] and saturated dipalmitoyl-PI(4,5)P 2 [DP-PI(4,5)P 2 ] successfully recruited PH PLC␦ 1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P 2 but not DP-PI(4,5)P 2 recruited Gag to GUVs, indicating that PI(4,5)P 2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P 2 , cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PH PLC␦ 1 regardless of PI(4,5)P 2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P 2 had DO-acyl chains. DP-PI(4,5)P 2 -containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P 2 still promoted recruitment of Gag, but not PH PLC␦ 1 , to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P 2 acyl chains in membrane binding of two PI(4,5)P 2 -binding proteins, Gag and PH PLC␦ 1 . Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P 2 containing an unsaturated acyl chain over DP-PI(4,5)P 2 , suggesting that Gag sensitivity to PI(4,5)P 2 acyl chain saturation is determined directly by the matrix-PI(4,5)P 2 interaction, rather than indirectly by a myristate-dependent mechanism. IMPORTANCEBinding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P 2 . Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P 2 -interacting domains represented by PH PLC␦ 1 . However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P 2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P 2 with an unsaturated acyl chain recruited PH PLC␦ 1 and Gag similarly, whereas PI(4,5)P 2 with saturated acyl chains either recruited PH PLC␦ 1 but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P 2 acyl chains differentially modulate membrane binding of PI(4,5)P 2 -binding proteins. Since Gag membrane binding is essential for progeny...