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

Abstract

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.

Regulation of Hepatitis C Virus Infection by Cellular Retinoic Acid Binding Proteins through the Modulation of Lipid Droplet Abundance

“…In fact, our results ( Fig. 6C to F) and observations by others support our conclusion well, as these studies demonstrated that the LXR-RXR pathway modulates the efficiency of HCV infection via LD abundance or the formation of virus-associated membrane compartments (46,47). We believe that these observations justify our proposed mechanisms well.…”

The aryl hydrocarbon receptor–cytochrome P450 1A1 pathway controls lipid accumulation and enhances the permissiveness for hepatitis C virus assembly

“…RNAs were synthesized in vitro using MEGAscript T7 (Ambion) and isolated using the RNeasy Mini kit (Qiagen). RNA was transfected to cells as described previously (54).…”

“…HCV was recovered from the medium of Huh-7 cells transfected with HCV JFH-1 RNA as described (36). HCV was used to infect Huh-7 or Huh-7.5.1 cells at a multiplicity of infection of 0.1-0.2 for 4 h. After washing out of the HCV inoculum, the cells were cultured in the presence or absence of various compounds for 48 or 72 h. Production of HCV was quantified by measuring the infectivity of HCV and the amount of HCV core protein in the culture supernatant by the infectious focusforming assay and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse, Fujirebio) (54). For the focus-forming assay, naive Huh-7.5.1 cells were inoculated with HCV (at different dilutions) for 4 h, and then HCV-positive cells were visualized at 48 h post-inoculation by detecting HCV core protein using immunofluorescence analysis to count the foci and calculating focus-forming units/ml of the HCV inoculum (54).…”

“…For the focus-forming assay, naive Huh-7.5.1 cells were inoculated with HCV (at different dilutions) for 4 h, and then HCV-positive cells were visualized at 48 h post-inoculation by detecting HCV core protein using immunofluorescence analysis to count the foci and calculating focus-forming units/ml of the HCV inoculum (54). HCV core protein level was measured by CLEIA according to the manufacturer's protocol (54).…”

“…The viability of cells was quantified using the Cell Proliferation Kit II (XTT) (Roche Diagnostics) as described previously (54).…”