Identification of Cholesterol 25-Hydroxylase as a Novel Host Restriction Factor and a Part of the Primary Innate Immune Responses against Hepatitis C Virus Infection

Abstract

Hepatitis C virus (HCV), a single-stranded positive-sense RNA virus of the Flaviviridae family, causes chronic liver diseases, including hepatitis, cirrhosis, and cancer. HCV infection is critically dependent on host lipid metabolism, which contributes to all stages of the viral life cycle, including virus entry, replication, assembly, and release. 25-Hydroxycholesterol (25HC) plays a critical role in regulating lipid metabolism, modulating immune responses, and suppressing viral pathogens. In this study, we showed that 25HC and its synthesizing enzyme cholesterol 25-hydroxylase (CH25H) efficiently inhibit HCV infection at a postentry stage. CH25H inhibits HCV infection by suppressing the maturation of SREBPs, critical transcription factors for host lipid biosynthesis. Interestingly, CH25H is upregulated upon poly(I · C) treatment or HCV infection in hepatocytes, which triggers type I and III interferon responses, suggesting that the CH25H induction constitutes a part of host innate immune response. To our surprise, in contrast to studies in mice, CH25H is not induced by interferons in human cells and knockdown of STAT-1 has no effect on the induction of CH25H, suggesting CH25H is not an interferon-stimulated gene in humans but rather represents a primary and direct host response to viral infection. Finally, knockdown of CH25H in human hepatocytes significantly increases HCV infection. In summary, our results demonstrate that CH25H constitutes a primary innate response against HCV infection through regulating host lipid metabolism. Manipulation of CH25H expression and function should provide a new strategy for anti-HCV therapeutics.

Importance: Recent studies have expanded the critical roles of oxysterols in regulating immune response and antagonizing viral pathogens. Here, we showed that one of the oxysterols, 25HC and its synthesizing enzyme CH25H efficiently inhibit HCV infection at a postentry stage via suppressing the maturation of transcription factor SREBPs that regulate lipid biosynthesis. Furthermore, we found that CH25H expression is upregulated upon poly(I·C) stimulation or HCV infection, suggesting CH25H induction constitutes a part of host innate immune response. Interestingly, in contrast to studies in mice showing that ch25h is an interferon-stimulated gene, CH25H cannot be induced by interferons in human cells but rather represents a primary and direct host response to viral infection. Our studies demonstrate that the induction of CH25H represents an important host innate response against virus infection and highlight the role of lipid effectors in host antiviral strategy.

FIG 1

25HC inhibits HCV infection. (A) HCVcc focus reduction assay. JFH1 or PR63 cell culture (PR63cc) were inoculated to Huh7.5.1 cells in the presence of 0.5, 1, 2, or 4 μM 25HC. The infection efficiency was measured by counting the number of HCV-positive foci after immunofluorescence staining. (B) Cell viability assay. Huh7.5.1 cells were treated with 25HC at various concentrations. At 72 h posttreatment, cell viability was determined using the CellTiter-Glo cell viability assay. (C) HCV pseudotyped particles (HCVpp) assay. Huh7.5.1 cells were pretreated with various concentrations of 25HC for 24 h and then inoculated with pseudotyped viruses bearing HCV envelope proteins (JFH1 or H77) glycoproteins. Infection was measured by the luciferase assay at day 2 postinfection. (D) HCVcc assay. 25HC was added to Huh7.5.1 cells at 8 h post-HCV inoculation and remained present through the experiment. At 72 h postinfection, cells were lysed for intracellular HCV RNA quantification by RT-qPCR. The results were presented as the percentage of the mock treatment (0.1% ethanol). The error bars represent standard deviations of triplicates.

FIG 2

CH25H expression inhibits HCV infection. (A) Huh7.5.1 cells were transduced with lentivirus expressing HA-CH25H or empty vector control. The cells were analyzed for the expression of CH25H by Western blotting. (B and C) Huh7.5.1-vector or Huh7.5.1-CH25H cells were infected with HCVcc (JFH1 and PR63cc) (B) or HCVpp (H77 and JFH1) (C). Infection efficiency was measured by counting the number of HCV-positive foci for HCVcc infection or using luciferase assay for HCVpp infection. The results were presented as the percentage of the Huh7.5.1-vector control (ns, P > 0.05). (D) Huh7.5.1 cells were infected with HCVcc (JFH1 or PR63cc) in the presence of culture supernatants from Huh7.5.1-vector or Huh7.5.1-CH25H cells. HCV infection was measured by counting the number of HCV-positive foci, and results are presented as the percentage of the mock treatment control. The error bars represent standard deviations of triplicates.

FIG 3

CH25H expression suppresses SREBP function. (A and B) Huh7 cells stably expressing CH25H (Huh7-CH25H) or Huh7 cells treated with 2.5 μM 25HC were analyzed for SREBP-2, HA-CH25H, and actin expression by Western blotting (A) and analyzed for LDLR and FAS mRNA levels by RT-qPCR (B) (*, P < 0.05). (C) Huh7 and Huh7-CH25H cells were transfected with plasmids expressing N-SREBP-2 or empty vector. At day 2 posttransfection, the cells were infected with JFH1 at an MOI of 0.1. At day 3 postinfection, intracellular HCV RNA levels were analyzed by RT-qPCR and normalized to the cellular GAPDH mRNA levels, and the results are presented as the percentage of the HCV RNA levels in the untreated Huh7 cells. The error bars represent standard deviations of triplicates.

FIG 4

CH25H is upregulated upon poly(I·C) stimulation or HCV infection. (A) CH25H and IFN-β mRNA levels in Huh7 cells transfected with poly(I·C) at 8 h posttransfection. (B) Kinetics of CH25H and IFN-β mRNA levels in PH5CH8 cells transfected with poly(I·C). (C) CH25H, CYP7B1, and CYP27A1 mRNA levels in PH5CH8 cells transfected with poly(I·C) at 8 h posttransfection (ns, P > 0.05). (D) CH25H and IFN-β mRNA levels in primary human hepatocytes transfected with poly(I·C) or HCV 3′UTR RNA at 8 h posttransfection. (E and F) Kinetics of CH25H and IFN-β mRNA levels in Huh7 cells (E) or Huh7-MAVSR cells (F) infected with JFH1 at an MOI of 2. For all of the panels, the results were normalized to the cellular GAPDH levels and are presented as the fold induction compared to mock-treated cells. The error bars represent standard deviations of triplicates.

FIG 5

CH25H is not induced by IFNs. (A) The murine Ch25h, RSAD2, and ISG15 mRNA levels were analyzed in murine monocyte-derived dendritic cells treated with IFN-β (100 IU/ml) at 8 h posttreatment. (B to E) Kinetics of CH25H and MxA or IP-10 mRNA levels in PH5CH8 cells treated with 100 IU of IFN-α/ml (B), 100 IU of IFN-β/ml (C), 200 ng of IFN-λ (IL-28)/ml (D), or 200 ng of IFN-γ/ml (E). (F) PH5CH8 cells were transfected with poly(I·C), and the medium was replaced with fresh medium 6 h later. At 24 h posttransfection, culture supernatants from the transfected cells were transferred to fresh PH5CH8 cells. Both the transfected cells (24 h posttransfection) and the supernatant treated cells (8 h posttreatment) were analyzed for the CH25H and MxA mRNA levels (ns, P > 0.05). (G) CH25H, MxA, and IFN-β mRNA levels in human monocyte-derived macrophages transfected with poly(I·C) or treated with IFN-α (100 IU/ml) at 8 h posttreatment (ns, P > 0.05). (H and I) The kinetics of CH25H and MxA mRNA levels in Huh7 (H) and A549 (I) cells treated with IFN-α (100 IU/ml). (J to L) Kinetics of CH25H and MxA or IP-10 mRNA levels in primary human hepatocytes treated with 100 IU of IFN-α/ml (J), 200 ng of IFN-λ (IL-28)/ml (K), or 200 ng of IFN-γ/ml (L). The results were normalized to the cellular GAPDH levels and are presented as the fold induction compared to mock-treated cells. The error bars represent standard deviations of triplicates.

FIG 6

Poly(I·C)-induced CH25H expression is independent of STAT-1. (A) STAT-1 protein levels in STAT-1 knockdown PH5CH8 cells by Western blotting. (B to D) The ISGs (MxA and ISG15) (B), CH25H (C), and IFN-β (D) mRNA levels in two STAT-1 knockdown PH5CH8 cells upon poly(I·C) transfection. (E) STAT-1 protein levels in STAT-1 knockdown Huh7 cells as determined by Western blotting. (F to H) ISG (MxA and ISG15) (F), CH25H (E), and IFN-β (H) mRNA levels in two STAT-1 knockdown Huh7 cell lines transfected with poly(I·C). The results were normalized to the cellular GAPDH levels and presented as the fold of induction compared to mock-treated cells. The error bars represent standard deviations of triplicates (ns, P > 0.05; *, P < 0.05).

FIG 7

CH25H induction is mediated by MDA5, MAVS, IRF3 and NF-κB.1. (A and B) PH5CH8 cells were transfected with plasmids expressing MAVS, IRF3, NF-κB p65 or empty vector. At 24 h posttransfection, the culture supernatants from the transfected cells were transferred to fresh PH5CH8 cells. Both transfected cells (24 h posttransfection) (A) and supernatant treated cells (24 h posttreatment) (B) were analyzed for CH25H, IFN-β and MxA mRNA levels. (C) RIG-I and MDA5 protein levels in RIG-I or MDA5 knockdown Huh7-MAVSR cells by Western blotting. (D to F) The RIG-I or MDA5 knockdown cells were infected with HCVcc (MOI = 2). The CH25H, IFN-β mRNA and HCV RNA levels were analyzed at the indicated time points. The results were normalized to the cellular GAPDH levels and presented as the fold induction compared to mock-treated cells. The error bars represent standard deviations of triplicates.

FIG 8

CH25H knockdown promotes HCV infection. (A and B) Huh7-MAVSR cells expressing shRNA targeting CH25H or control shRNA were transfected with poly(I·C). At 48 h posttransfection, the CH25H (A) and IFN-β (B) mRNA levels were determined by RT-qPCR. (C) The CH25H knockdown and control cells were infected with JFH1 virus (MOI = 0.1). The cells were collected at the indicated time points for a RT-qPCR assay to analyze the intracellular HCV RNA abundance. The results were normalized to the cellular GAPDH levels and are presented as the fold induction compared to mock-treated cells. The error bars represent standard deviations of triplicates (ns, P > 0.05; *, P < 0.05).