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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 Yu Xiang,a Jing-Jie Tang,b Wanyin Tao,a Xuezhi Cao,a Bao-Liang Song,c Jin Zhonga Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, Chinaa; The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, Chinab; The College of Life Sciences, Wuhan University, Wuhan, Chinac

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.

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epatitis C virus (HCV), a causative agent of acute and chronic liver diseases, is an enveloped positive-sense single-stranded RNA virus belonging to the family of Flaviviridae. Approximately 170 million people worldwide are infected with HCV. The tremendous progress has been achieved in the therapeutics of chronic hepatitis C thanks to the development of direct antiviral agents (DAAs), but the worldwide application of these highly effective DAAs is limited due to the high cost of these treatments (1). In addition, drug resistance mutations remain a potential problem since DAAs are becoming a standard therapy for chronic hepatitis C. Furthermore, no vaccine is available for preventing new HCV infection. Therefore, HCV still imposes a big threat to human public health. HCV has a 9.6-kb single-stranded RNA genome that contains a single open reading frame encoding both structural proteins (core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The open reading frame is flanked by highly conserved untranslated regions (UTR) at

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the 5= and 3= ends which play important roles in HCV protein translation and genome replication. HCV exploits host lipid metabolism for entire viral life cycle. The virus relies on host lipid transport molecules, LDLR and SRB1, for virus entry (2, 3), creates a lipid-rich microenvironment for viral genome replication (4), utilizes host lipid storage vesicles

Received 4 March 2015 Accepted 10 April 2015 Accepted manuscript posted online 22 April 2015 Citation Xiang Y, Tang J-J, Tao W, Cao X, Song B-L, Zhong J. 2015. 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. J Virol 89:6805–6816. doi:10.1128/JVI.00587-15. Editor: M. S. Diamond Address correspondence to Jin Zhong, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00587-15

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lipid droplet for viral particles assembly (5), and finally releases viral particles in a very-low-density-lipoprotein-dependent manner (6). During infection, HCV upregulates host lipid metabolism through a variety of molecular mechanisms (7–9), which may eventually contribute to the development of hepatic steatosis that occurs in almost 50% of HCV-infected patients (10). In mammals, the lipid biosynthesis is precisely controlled by a family of transcription factors named sterol regulatory element-binding proteins (SREBPs) (11). SREBPs include two genes, SREBP-1 and SREBP-2. SREBP-1 is responsible for cholesterol and fatty acid synthesis, while SREBP-2 mostly mediates cholesterol synthesis (12). Synthesized on endoplasmic reticulum (ER) membranes as the precursor, the activation of SREBPs requires the transport of the precursor to the Golgi complex where SREBPs undergo proteolytic cleavage to release the transcriptionally active fragment capable of translocating into nucleus to turn on the transcription of a wide variety of target genes (11). This activation process is regulated by the levels of cholesterol and oxysterols, which are derivatives of cholesterol with extra hydroxyl or keto groups (11, 13, 14). 25-Hydroxycholesterol (25HC) is a natural occurring oxysterol that is synthesized by cholesterol 25-hydroxylase (CH25H [CH25H in humans and Ch25h in mice]) (15). In addition to its potent inhibitory effects on the SREBPs maturation, recent studies have expanded its roles in immune systems. 25HC has a wide range of immunological effects, including suppressing the production of IgA by B cells, regulating immune cell migration, differentiation, and modulating inflammatory signaling (16). Ch25h is upregulated in murine macrophages in response to Toll-like receptor (TLR) ligand stimulation, suggesting that Ch25h represents a host response against virus infection (17–19). Recent studies have shown that Ch25h is an interferon (IFN)-stimulated gene (ISG) in mice and that its product 25HC exerts antiviral effects (20, 21). However, these studies differ in their conclusions for the antiviral mechanisms. The precise role of CH25H as an antiviral factor remains to be elucidated. We found that 25HC and its catalyzing enzyme CH25H inhibit HCV infection through blocking SREBP maturation to suppress viral genome replication. Interestingly, in contrast to the reports in mice, we found that CH25H is not an ISG in humans and acts as a primary response to virus infection. MATERIALS AND METHODS Cell culture and virus preparation. Hepatic (Huh7 and Huh7.5.1) cells were cultured in complete Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 mg of streptomycin/ml (Invitrogen, Carlsbad, CA). PH5CH8 cells were cultured in DMEM/F-12 (Invitrogen) as described previously (22). Primary human hepatocytes were purchased from Research Institute for Liver Diseases (Shanghai, China) and cultured at a density of 105 cells/cm2 on 48-well plates coated with type 1 collagen using serum-free differentiation medium. Human monocyte-derived macrophages were generated as described previously (23). Human peripheral blood mononuclear cells (PBMCs) were obtained from the Shanghai Blood Center (Shanghai, China). Human monocytes were separated by Percoll density gradient centrifugation (GE Healthcare Bio-Sciences, Sweden) from isolated PBMCs. Monocyte-derived macrophages were generated by incubation of primary monocytes with recombinant M-CSF (20 ng/ml) for a week. All cells were cultured in humidified air containing 5% CO2 at 37°C. The preparation of HCV cell culture (HCVcc) was as previously described (24, 25).

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Reagents and antibodies. Long poly(I·C) was purchased from Invivogen (San Diego, CA). SREBP-2 antibody was obtained from Abcam (Cambridge, MA), STAT-1 antibody was obtained from Cell Signaling (Danvers, MA), and hemagglutinin (HA) and actin antibodies were obtained from Abmart (Shanghai, China). 25HC was from obtained Steraloids, Inc. (Newport, RI), reconstituted in ethanol at a concentration of 1 mg/ml, and stored at ⫺20°C. Plasmid construction. To make the lentiviral vector-based expression constructs pLVX-HA-CH25H-IRES-Puro and pLVX-HA-N-SREBP-2IRES-Puro, the cDNA encoding human CH25H or N-terminal of SREBP-2 (amino acids 1 to 480) was cloned into pLVX-IRES-Puro vector (Clontech Laboratories, Mountain View, CA). All constructs were verified by DNA sequencing analysis. HCV focus reduction assay. HCVcc was inoculated to Huh7.5.1 cells plated at a density of 8,000 cells per well in a 96-well plate in the presence of 25HC. Three days after infection, the cells were fixed with paraformaldehyde and stained with a human monoclonal antibody AR3A against HCV envelope protein E2 (26). Bound primary antibodies were detected by using Alexa 555-conjugated secondary antibody (Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst dye. The HCV-positive foci in each well were counted by fluorescence microscopy. Cytotoxicity assay. Various concentrations of 25HC were added to Huh7.5.1 cells for 3 days and the cell viability was determined using CellTiter-Glo luminescent cell viability kit (Promega, Madison, WI) according to the manufacturer’s instructions. Generation of Huh7.5.1-CH25H and Huh7-CH25H cells. Lentiviruses expressing HA-CH25H were generated by cotransfecting pLVXHA-CH25H-IRES-Puro with the packaging vector psPAX2 and envelope vector pMD2.G into HEK293T cells. Viral supernatants were harvested 48 h posttransfection, passed through a 0.45-␮m-pore-size filter, and used to infect naive Huh7.5.1 or Huh7 cells. At 48 h postinfection, 0.75 ␮g of puromycin/ml was added to the culture medium, and the puromycinresistant cells were pooled and expanded. Pseudotyped virus infection. HCV-pseudotyped particles (HCVpp) were generated as previous described (24). Briefly, HEK293T cells were cotransfected with plasmids expressing HCV ⌭1, ⌭2 glycoproteins, retroviral core packaging component, and luciferase. The medium was refreshed at 6 h posttransfection. Supernatants were collected 72 h later and filtered through 0.45-␮m-pore-size membranes. For infection experiments, 8,000 Huh7.5.1 cells seeded in 96-well plates were pretreated with various concentrations of 25HC and then infected with HCVpp. Three days after infection, the firefly luciferase activity was measured by using luciferase assay system according to the manufacturer’s instructions (Promega). RNA isolation, quantitative RT-PCR (RT-qPCR), and Western blotting. The protocols were as previously described (25, 27). The primer sequences were as follows: human CH25H, 5=-ATCACCACATACGTGG GCTTT-3= and 5=-GTCAGGGTGGATCTTGTAGCG-3=; CYP7B1, 5=-A AAGGTTGGCTTCCTTATCTTGG-3= and 5=-GCAACTGACTGATGCT AAATGCT-3=; CYP27A1, 5=-GGTGCTTTACAAGGCCAAGTA-3= and 5=TCCCGGTGCTCCTTCCATAG-3=; murine Ch25h, 5=-TGCTACAACGGT TCGGAGC-3= and 5=-AGAAGCCCACGTAAGTGATGAT-3=; RSAD2, 5=-T GGGGATGCTGGTGCCCACT-3= and 5=-ACCCCGGACCTGTGGCTGT T-3=; and ISG15, 5=-TGGGACCTAAAGGTGAAGATGCTG-3= and 5=-TGC TTGATCACTGTGCACTGGG-3=. The sequences of primers for human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), IFN-␤, MxA, ISG15, LDLR, and FAS were described previously (27, 28). RNA interference. Vector GV248 (Genechem, Shanghai, China) expressing shRNA targeting CH25H (5=-CTCACTTTAACTGCAACTT-3=) was transfected into HEK293T cells, together with two packaging plasmids psPAX2 and envelope plasmid pMD2.G. The produced lentiviruses were collected at 72 h posttransfection, passed through a 0.45-␮m-poresize filter, and used to infect Huh7-MAVSR cells. Gene knockdown by lenti-CRISPR-Cas9. HEK293T cells were seeded into six-well plates 1 day prior to transfection at a density of 7 ⫻ 105 cells

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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 ␮M 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.

per well. The cells were cotransfected with 1.3 ␮g of VSV-G expressing plasmid, 2.5 ␮g of pCMV-dR8.91 plasmid, and 2.5 ␮g of lenti-Cas9sgRNAs (29) targeting STAT-1 (sg-STAT1#1, 5=-TTCCCTATAGGATGT CTCAG-3=; sg-STAT1#2, 5=-GCTTTTCTAACCACTGTGCC-3=) using Lipofectamine 2000 (Invitrogen). Viral supernatants were harvested at 48 h posttransfection, filtered, and used to infect PH5CH8 cells. After 48 h postinfection, 0.75 ␮g of puromycin/ml was added to the medium for extended culture. Genomic DNA sequencing analysis indicated that the STAT-1 gene is defective in ca. 50% of the cells (data not shown). RIG-Ior MDA5-knockdown Huh7-MAVSR cells were generated as previously described (30). Statistical analysis. Statistical analysis was performed using a Student t test. Differences were considered statistically not significant (ns) when the P value was ⬎0.05 and significant when the P value was ⬍0.05.

RESULTS

25HC inhibits HCV infection. Previous studies showed that 25HC, a naturally occurring oxysterol, inhibits HCV replication using a subgenomic replicon system (31). We sought to verify this phenomenon using the HCV infection cell culture model (HCVcc) (25, 32–34). Two different genotype 2a HCVcc strains were used: JFH1 (25) and PR63cc that was recently constructed directly from a genotype 2a clinical isolate and had a similar infectivity titer and infection kinetics (34). First, we examined the effect of 25HC on HCV infection in HCVcc focus reduction assay. About 50 focus-forming units of HCVcc were inoculated into Huh7.5.1 cells in the presence of serial concentrations of 25HC, and the infection foci were counted at 72 h postinfection. As shown in Fig. 1A, both HCVcc strains were efficiently inhibited by

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25HC treatment in a dose-dependent manner. At a 4 ␮M concentration, 25HC treatment almost completely abolished HCV infection whereas no apparent cytotoxicity was observed (Fig. 1B), confirming that 25HC possesses a potent anti-HCV activity. To investigate at which step HCV life cycle was inhibited by 25HC, we examined the effect of 25HC on viral entry by using HCV-pseudotyped particles (HCVpp) that recapitulate HCV envelope glycoprotein-mediated entry process (35). Huh7.5.1 cells were pretreated with serial concentrations of 25HC for 24 h and then infected with HCVpp. The infection was monitored as the firefly luciferase activity at 48 h postinfection. As shown in Fig. 1C, HCVpp infection was insensitive to 25HC treatment, indicating that HCV entry was not affected and that 25HC likely inhibits a step downstream of HCV glycoprotein-mediated entry. To test this hypothesis, 25HC was added to Huh7.5.1 cells at 8 h postinoculation and remained present throughout the experiment. The intracellular HCV RNA levels were measured by RT-qPCR. As shown in Fig. 1D, even when added after virus inoculation, 25HC still achieved a significantly inhibitory effect on virus infection. Taken together, these results suggested that 25HC is a potent agent against HCV infection at a postentry stage. Expression of CH25H inhibits HCV infection. CH25H is an ER-associated enzyme that catalyzes oxidation of cholesterol to 25HC (15). Having known the potent antiviral effect of 25HC, we speculated that the expression of its synthesizing hydroxylase gene CH25H should also inhibit HCV infection. To evaluate the effect of CH25H on HCV infection, we generated a Huh7.5.1 cell line that stably ex-

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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.

presses the HA-tagged CH25H, designated Huh7.5.1-CH25H, and assessed its susceptibility to HCV infection. The CH25H expression level was confirmed by immunoblotting assays (Fig. 2A). As shown in Fig. 2B, CH25H expression efficiently inhibited HCV infection. Consistently, HCVpp infection was not affected by CH25H expression (Fig. 2C). Since 25HC is a secreted soluble factor, we examined whether CH25H can inhibit HCV infection in a paracrine manner. Naive Huh7.5.1 cells were infected with HCVcc in the presence of the culture supernatants from the Huh7.5.1-vector or Huh7.5.1-CH25H cells. As shown in Fig. 2D, the supernatants from the Huh7.5.1CH25H cells inhibited HCV infection, demonstrating that Huh7.5.1CH25H cells secrete soluble anti-HCV factors. Taken together, these results indicated that CH25H expression can exert its antiviral effect in both autocrine and paracrine ways. CH25H expression disrupts the SREBP function. 25HC is a potent suppressor of the maturation of SREBPs, master transcription factors in lipid biosynthesis (13, 14). HCV infection is tightly depended on host lipid metabolism and reagents that perturb lipid metabolism can suppress HCV replication (31, 36–38). To test whether CH25H inhibits HCV infection through suppressing SREBPs function, we first examined effects of CH25H expression on the SREBP maturation process. SREBP activation requires the cleavage of precursor and the translocation of the nuclear form of SREBP (n-SREBP) from the ER to the nucleus. It has been well studied that 25HC can retain precursor SREBP-2 on ER to prevent

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SREBP-2 activation (39). The levels of n-SREBP-2 in Huh7 cells stably expressing CH25H (designated Huh7-CH25H) and the parental Huh7 cells were analyzed. The wild-type Huh7 cells treated with 25HC were included as a control. As shown in Fig. 3A, the level of n-SREBP-2 was significantly decreased by either CH25H expression or 25HC treatment. We next examined the expression of two SREBP targeted genes, LDLR and FAS (28), in Huh7-CH25H cells. Consistently, these two genes were downregulated in Huh7-CH25H cells, as well as in Huh7 cells treated with 25HC, likely due to the blockade of the SREBP activation (Fig. 3B). Moreover, we found that overexpression of N-terminal domain of SREBP-2, whose transcriptional activity is not affected by 25HC (40), could at least partially rescue HCV infection in the CH25H-expressing cells (Fig. 3C), indicating that SREBP-2 is a key molecule responsible for the antiviral effect of CH25H. Taken together, these results suggested that CH25H inhibits HCV infection through suppressing the function of SREBP. CH25H is upregulated upon poly(I·C) stimulation and HCV infection. Although it is expressed at a low level in most tissues, Ch25h can be induced rapidly upon exposure to TLR ligands in murine macrophages and dendritic cells (17–19), suggesting that it represents a host response against viral infection. HCV is a highly hepatotropic pathogen that predominantly infects human hepatocytes. Therefore, we examined the induction pro-

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FIG 3 CH25H expression suppresses SREBP function. (A and B) Huh7 cells

stably expressing CH25H (Huh7-CH25H) or Huh7 cells treated with 2.5 ␮M 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.

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file of CH25H in human hepatocytes upon pathogen stimulation. First, we transfected Huh7 cells with poly(I·C), a synthetic double-stranded RNA, and measured the CH25H and IFN-␤ mRNA expression levels by RT-qPCR at 8 h posttransfection. As shown in Fig. 4A, CH25H expression level was greatly induced, as well as IFN-␤. We then tested PH5CH8 cells, another hepatic cell line that mounts an efficient innate immune response (22, 41). As shown in Fig. 4B, poly(I·C) stimulation efficiently activates CH25H expression with kinetics similar to those of IFN-␤. Next, we examined whether other oxysterol synthesizing enzymes, CYP7B1 and CYP27A1, could also be upregulated. Interestingly, we found CYP7B1 was not induced, and CYP27A1 was only slightly induced by 2-fold upon poly(I·C) stimulation in PH5CH8 cells (Fig. 4C). We then examined CH25H induction in primary human hepatocytes. Primary human hepatocytes were transfected with poly(I·C) or HCV 3=UTR, which is the HCV PAMP motif recognized by RIG-I to trigger IFN-␤ transcription (42). The mRNA expression levels of CH25H and IFN-␤ were measured by RT-qPCR at 8 h posttransfection. As shown in Fig. 4D, both CH25H and IFN-␤ were dramatically induced after the stimulation. Furthermore, we examined whether CH25H can be induced during natural HCV infection in Huh7 cells. As shown in Fig. 4E, HCV infection did not induce the expression of CH25H and IFN-␤ in Huh7 cells in which HCV NS3/4A protease can cleave the MAVS protein (43, 44). Our data (not shown) also indicated that neither CH25H nor IFN-␤ induction was observed even at early time points (2, 4, 6, and 12 h) after HCVcc infection. We recently generated a Huh7 cell line that stably expresses a MAVS mutant resistant to the NS3/4A protease cleavage, designated Huh7-MAVSR, and HCV infection can induce a robust IFN response in this novel cell culture system (30). We infected Huh7-MAVSR cells with HCVcc (JFH1 strain) at a multiplicity of infection (MOI) of 2. The CH25H, IFN-␤, and HCV RNA levels were determined by RT-qPCR at the indicated time points. As shown in Fig. 4F, both CH25H and IFN-␤ mRNA levels were induced by HCV infection. Taken together, these results demonstrated that CH25H can be specifically induced in hepatocytes in response to poly(I·C), HCV 3=UTR, or HCV infection, suggesting CH25H constitutes a host innate immune response against virus infection in hepatocytes. CH25H is not a typical ISG. Previous studies showed that Ch25h is an interferon-stimulated gene (ISG) and that its expression can be induced by type I or type II IFN treatment in murine dendritic cells and macrophages (19–21, 45, 46). Our results also showed that Ch25h, as well as two well-known ISGs (RSAD2 and ISG15), can be induced in murine bone marrow-derived dendritic cells upon IFN-␤ treatment (Fig. 5A). To investigate whether CH25H can be induced by IFNs in human hepatocytes, PH5CH8 cells were treated with IFN-␣, IFN-␤, IFN-␭, or IFN-␥ for 2, 4, 8, 24, and 48 h. RT-qPCR was performed to determine the CH25H and MxA or IP-10 mRNA levels. As shown in Fig. 5B to E, CH25H mRNA expression levels barely changed upon any IFN treatment, whereas MxA and IP-10, well-known ISG controls, were dramatically induced for over 100-fold by type I or III and type II IFNs, respectively. Our result also indicated that various concentrations of IFN-␣ (20, 200, and 2,000 IU/ml) did not induce the CH25H expression in PH5CH8 cells (data not shown). To examine whether poly(I·C) induced-CH25H expression was dependent upon the production of IFNs, we carried out the

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FIG 4 CH25H is upregulated upon poly(I·C) stimulation or HCV infection. (A) CH25H and IFN-␤ mRNA levels in Huh7 cells transfected with poly(I·C) at 8 h posttransfection. (B) Kinetics of CH25H and IFN-␤ 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-␤ 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-␤ 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.

supernatant transfer assay as performed for Fig. 2D. The culture supernatants from PH5CH8 cells transfected with poly(I·C) were transferred to naive PH5CH8 cells. The CH25H and MxA mRNA levels were measured by RT-qPCR. As shown in Fig. 5F, CH25H was only induced in poly(I·C) transfected but not in supernatanttreated cells, whereas MxA was induced in the both cells, indicating poly(I·C) transfection directly but not the cytokines produced by the poly(I·C) stimulation triggered CH25H induction. Next, we tested whether CH25H can be induced in human monocytederived macrophages. Our results showed that, in contrast to the observation in mice (Fig. 5A), CH25H can be only induced by poly(I·C) stimulation but not by IFN-␣ treatment in human monocyte-derived macrophages (Fig. 5G). Furthermore, our re-

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sults showed that IFN-␣ failed to induce the expression of CH25H in other human cells, including Huh7 and A549 cells (Fig. 5H and I). Finally, we found that IFN-␣, IFN-␭, or IFN-␥ did not induce CH25H expression in primary human hepatocytes (Fig. 5J to L). To further confirm that poly(I·C)-induced CH25H expression in human was independent upon IFNs, we examined the CH25H induction in poly(I·C)-transfected PH5CH8 cells in which STAT-1 expression was knocked down using CRISPR/ Cas9 technology. The transduction with lentiviruses expressing two sgRNAs (sg-STAT1#1 and sg-STAT1#2) efficiently reduced STAT-1 expression in the PH5CH8 cells (Fig. 6A). As expected, the knockdown of STAT-1, a critical transcription factor mediating the IFN signaling, decreased the induction of

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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-␤ (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-␣/ml (B), 100 IU of IFN-␤/ml (C), 200 ng of IFN-␭ (IL-28)/ml (D), or 200 ng of IFN-␥/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-␤ mRNA levels in human monocyte-derived macrophages transfected with poly(I·C) or treated with IFN-␣ (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-␣ (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-␣/ml (J), 200 ng of IFN-␭ (IL-28)/ml (K), or 200 ng of IFN-␥/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.

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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-␤ (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-␤ (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).

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FIG 7 CH25H induction is mediated by MDA5, MAVS, IRF3 and NF-␬B.1. (A and B) PH5CH8 cells were transfected with plasmids expressing MAVS, IRF3, NF-␬B 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-␤ 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-␤ 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.

two classical ISGs MxA and ISG15 stimulated by poly(I·C) but had no significant effect on IFN-␤ and CH25H (Fig. 6B to D). Consistently, the knockdown of STAT-1 had no effect on the CH25H induction upon poly(I·C) treatment in Huh7 cells (Fig. 6E to H). Taken together, these data clearly indicated that, in contrast to our observations in murine macrophages and dendritic cells, CH25H is not a classical ISG in human hepatocytes. Upregulation of CH25H is mediated by MDA5, MAVS, IRF3, and NF-␬B. Poly(I·C) and HCV 3=UTR stimulation can activate

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IRF3 and NF-␬B pathway, which are essential for IFN induction in response to virus infection (27). Therefore, we tested whether CH25H is induced through the similar signaling pathway. We transfected PH5CH8 cells with plasmids expressing MAVS, IRF3, or NF-␬B p65, the key proteins in the IFN induction signaling pathway. The CH25H and IFN-␤ mRNA levels were measured by RT-qPCR. As shown in Fig. 7A, all of these molecules activated CH25H expression as well as IFN-␤. The culture supernatants of these transfected cells were transferred to naive PH5CH8 cells,

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and the CH25H, IFN-␤, and MxA mRNA levels were measured by RT-qPCR. Consistently, MxA, but not CH25H or IFN-␤, was induced by the supernatants of poly(I·C)-transfected cells (Fig. 7B), demonstrating that CH25H, like IFN-␤, directly responds to stimulation in the innate immune response that is mediated by MAVS, IRF3, and NF-␬B. HCV 3=UTR and HCV infection can initiate innate immune cascade via pattern recognition receptor (PRR) RIG-I or MDA5, respectively (30, 42). To determine which PRR is responsible for CH25H induction in response to HCV infection, we used CRISPR/Cas9 technology to knockdown RIG-I or MDA5 in Huh7-MAVSR cells as previously reported (30). Two sgRNA targeting sequences were used for each gene, and the knockdown efficiency was verified by immunoblotting assays (Fig. 7C). Next, we determined the CH25H induction in response to HCV infection. As shown in Fig. 7D and E, the knockdown of MDA5 nearly completely abolished the induction of CH25H and IFN-␤, whereas the knockdown of RIG-I had no apparent effect on CH25H induction. Consistently, the HCV RNA levels negatively correlated with the host antiviral response (Fig. 7F). These results suggested that MDA5 may act as a viral RNA sensor to trigger CH25H induction in HCV infection. Knockdown of CH25H promotes HCV infection. Finally, we sought to determine the functional relevance of CH25H in host antiviral response against HCV infection. Because HCV-encoded NS3/4A protease can effectively shut down innate the immune response in Huh7 cells by cleaving MAVS, we tested the functional role of CH25H in HCV infection using Huh7-MAVSR cells in which the MAVS is resistant to the NS3/4A cleavage (30). First, we generated a Huh7-MAVSR cell line in which CH25H was stably knocked down by shRNA. Induction of CH25H was severely impaired in the knockdown cells upon poly(I·C) stimulation (Fig. 8A), while the induction of IFN-␤ was much less affected (Fig. 8B). We then infected the knockdown cells with HCVcc (JFH1) at an MOI of 0.1 and measured the intracellular HCV RNA level abundance by RT-qPCR at the indicated time points. As shown in Fig. 8C, the CH25H knockdown cells displayed increased HCV infection compared to the control cells, suggesting that knockdown of CH25H leads to increased susceptibility to viral infections. HCV RNA levels dropped in the CH25H knockdown cells at day 3 postinfection, likely due to the antiviral action of IFNs induced by HCV infection in these cells as we previously reported (30). DISCUSSION

25HC has been reported as a potent antiviral mediator against a panel of enveloped and nonenveloped virus (20, 21, 31, 47). However, these studies differ in their conclusions about the exact antiviral mechanism. It has been reported that 25HC can either block virus entry by modifying cells membrane or suppressing virus replication via impeding viral gene expression. It is believed that multiple mechanisms may exist depending on virus-host context. HCV infection is critically dependent on host lipid metabolism. Inhibition of host lipid synthesis can block HCV RNA replication and virus production (31, 36–38). Study has shown that 25HC, as an SREBP inhibitor, can decrease HCV replication within hepatoma cells (31). Here, our study has shown that host can utilize 25HC as an antiviral strategy by upregulating CH25H expression. Our data have shown that CH25H-mediated SREBP antagonism might be responsible for its antiviral activity. It worth

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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-␤ (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).

noting that the SREBP pathway may not account for all of the observed antiviral effect, since overexpression of the constitutive active form of SREBP-2 can only partially restore HCV infection in the CH25H-expressing cells, suggesting the CH25H may pos-

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CH25H in Primary Innate Immunity against HCV

sess additional anti-HCV activity that is independent of the SREBP suppression. Interestingly, a recent study from Genghong Chen’s and Hongyu Deng’s groups suggests that CH25H interacts with HCV NS5A protein to inhibit HCV replication, and this suppression is independent of the hydroxylase activity of CH25H (48). Our study found that CH25H expression is dramatically induced upon poly(I·C) stimulation or HCV infection in human hepatocytes. Interestingly, distinct from the studies in murine macrophages and dendritic cells (19–21, 45, 46), we found that CH25H is not an ISG in human cells. Instead, CH25H induction represents as a direct antiviral response, which shares a similar signaling pathway with IFN-␤ activation via IRF3 and NF-␬B. Our conclusion is supported by several lines of evidence. First, neither type I, type II, nor type III IFN induces CH25H expression in human cells, including human hepatic cell lines (PH5CH8 and Huh7), the human alveolar epithelial cell line (A549), human monocyte-derived macrophages, and primary human hepatocytes. Second, the culture supernatants from the poly(I·C) treated cells can trigger ISG induction but not CH25H. Third, knockdown of STAT-1, the key molecule mediating IFN-activating ISG expression, decreases ISG induction but has no effect on CH25H expression. Consistent with our finding, a microarray analysis by Hemmi et al. found that lipopolysaccharide (LPS) could induce Ch25h expression in TBK1-deficient embryonic fibroblasts in which IFNs and classical ISGs cannot be induced by LPS (49), suggesting that LPS induces Ch25h independent of IFN production. Moreover, microarray analyses in human cells indicate that CH25H is not induced by IFNs (50, 51), and microarray analysis in primary human hepatocytes treated with interferons or poly(I·C) shows that CH25H can be indeed induced by poly(I·C) but not by type I or type III IFNs (52). These different observations on the CH25H induction by IFNs could be due to some differences between mouse and human, which certainly deserves to be further investigated. In conclusion, our study showed CH25H could be induced by intrinsic primary innate response and acts as a host restriction factor against HCV infection. Our results demonstrate that metabolism and innate immunity are cross-regulated and shed more light on the function of CH25H in controlling HCV infection.

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ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (grant 81330039), the Chinese National 973 Program (2015CB554300), the Chinese National Science and Technology Major Project (2012ZX10002007-003), and the Shanghai Pasteur Health Research Foundation (SPHRF2013002). We thank Peishan Li (Shanghai Institutes for Biological Sciences) for technical assistance, and we thank Yongfen Xu, Guangxun Meng, and Mingkuan Chen (Institut Pasteur of Shanghai, Chinese Academy of Sciences) for providing monocyte-derived macrophages and murine bone marrow-derived dendritic cells.

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