Potential conflict of interest: T.P. has received consulting fees from Biotest AG and from Janssen Global Services.
Supported by grants from the Helmholtz Association (SO-024) and the Deutsche Forschungsgemeinschaft (PI 734/2-1 and SFB 900, Teilprojekt A6; both to T.P.); by an intramural young investigator award of the Helmholtz Centre for Infection Research and the Deutsche Forschungsgemeinschaft (STE 1954/1-1, to E.S.); by the Swiss National Science Foundation (grant 310030B_147089, to M.H.H.; grant 323530_145255, to T.B.); and by the Deutsche Forschungsgemeinschaft (FOR1202, TP1, to R.B.). L.M.S. is a Burroughs Wellcome Fund (BWF) Investigator in the Pathogenesis of Infectious Disease. This work was supported in part by the BWF and the Canadian Institutes of Health Research (CIHR).
Hepatitis C virus (HCV) is a positive-strand RNA virus that primarily infects human hepatocytes. Infections with HCV constitute a global health problem, with 180 million people currently chronically infected. Recent studies have reported that cholesterol 25-hydroxylase (CH25H) is expressed as an interferon-stimulated gene and mediates antiviral activities against different enveloped viruses through the production of 25-hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remain elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes. In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. Levels of hCH25H messenger RNA were significantly up-regulated both in HCV-positive liver biopsies and in HCV-infected primary human hepatocytes. The expression of hCH25H in primary human hepatocytes was primarily and transiently induced by type I interferon. Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype-independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication, which was observed using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web, the HCV replication factory, independent of RNA replication. Conclusion: Infection with HCV causes up-regulation of interferon-inducible CH25H in vivo, and its product, 25HC, restricts HCV primarily at the level of RNA replication by preventing formation of the viral replication factory. (Hepatology 2015;62:702–714)
Hepatitis C virus (HCV), a member of the Flaviviridae, is a positive-strand RNA virus that primarily infects human hepatocytes. Worldwide, an estimated 80 million people are chronically infected with HCV and are at high risk for developing severe liver damage, including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma.[1, 2] For the past 25 years, therapy has consisted of treatment with interferon (IFN)–alpha and the nucleoside analogue ribavirin. Recently, the licensing of directly acting antivirals targeting HCV nonstructural proteins has improved cure rates profoundly, now reaching levels of over 90%. However, many infected individuals have not been diagnosed, and a prophylactic vaccine is not available, which likely is required when considering global control and even eradication of HCV.
The first line of immune defense against HCV is based on cell-intrinsic innate immunity in the liver cells, which leads to the induction of type I and type III IFN systems. These cytokines induce a plethora of genes that exert a strong antiviral effect. In addition, the IFN system is required for efficient activation of the adaptive immune response.[5, 6] Only in a fraction of patients can HCV be cleared, and it becomes chronic in the majority of individuals. This has been linked primarily to excessive preactivation of the IFN response and dysfunctionality of T-cell responses.
The production of IFN induces the expression of hundreds of IFN-stimulated genes (ISGs). Recent screening approaches have been used to identify a broader range of antiviral effectors against different viruses.[6-10] Although in some cases individual ISGs can have profound effects on the replication of certain viruses, such as the MX protein and influenza virus, in the majority of cases the IFN system works in a combinatorial fashion, with multiple ISGs contributing to the antiviral response. The modes of action of only a few ISGs have been well elucidated, including protein kinase R, the 2-5 OAS/RNaseL system, viperin, IFITM1, IFI6, and MX proteins. Using an overexpression screening system for IFN-induced antiviral genes, Liu et al. discovered cholesterol-25-hydroxylase (CH25H), which converts cholesterol into the oxysterol 25-hydroxycholesterol (25HC), as an ISG with antiviral potency against murine gamma herpesvirus 6 (MHV6) and vesicular stomatitis virus. In addition, it has been reported that macrophages and dendritic cells express CH25H in response to toll-like receptor activation.[17, 18] In a more recent study, Liu et al. also described the role of the hydroxylase enzymatic product, 25HC, as a broad-spectrum antiviral that blocks fusion of virions into target cells independently of its function as regulator of sterol regulatory element binding protein (SREBP). Using a mass-spectrometry approach, 25HC was also shown to be up-regulated in murine macrophages as a result of viral infection or IFN treatment. It had antiviral activities against a panel of enveloped viruses in a liver X receptor–independent, but SREBP-dependent, mechanism. The antiviral activity was reported to act mainly at a postentry step, suggesting that 25HC might interfere with multiple steps in the life cycle of viruses.[6, 20]
So far, the role of CH25H as an ISG and antiviral restriction factor has mainly been studied in murine model systems, and its effects on the HCV replication cycle have not been fully elucidated. Amino acid sequence alignment of murine and human cholesterol 25-hydroxylases shows 78% sequence identity, with the most notable difference being a 26–amino acid residue extension that is only present at the carboxy terminus of the murine enzyme. In this study, we characterized the expression of human CH25H (hCH25H) in liver biopsies of chronic HCV-infected patients and elucidated its IFN dependence in primary human cells. The antiviral properties of hCH25H and the enzymatic product 25HC were also characterized against all HCV genotypes, in hepatoma cells and primary human hepatocytes (PHHs). Collectively, hCH25H was identified as an up-regulated ISG in human liver cells that, through its product 25HC, primarily targets early biogenesis of HCV-induced membranous replication factories.
Materials and Methods
Liver Biopsies and Informed Consent
The viral load, HCV genotype, and liver biopsies from patients with chronic hepatitis C (CHC, n = 34) and 10 HCV-negative liver biopsies were obtained in the context of routine diagnostic workup. Several liver biopsies have been reported. Grading and staging of CHC were performed according to the METAVIR classification. All patients gave written informed consent in accord with local ethical committees.
Human macrophages were obtained from human peripheral blood mononucleated cells which were plated in six-well plates at a density of 8 × 106 cells/well and kept in 10% AB-human serum and 10% fetal bovine serum containing Roswell Park Memorial Institute medium. At 10-17 days postseeding, undifferentiated cells and remaining lymphocytes were removed by thorough washing with prewarmed medium. The PHHs were isolated from liver specimens obtained after partial hepatectomy, plated at a density of 1.3 × 106 on collagen in P6 dishes, and kept in hepatocyte culture medium (Lonza) as described. Huh7-Lunet cells containing persistent, selectable reporter subgenomic replicons (SGRs) of genotype 2a (LucUbiNeo_JFH1) have been described. Huh7 cells containing a stable, hygromycin-selectable dengue virus SGR with Renilla luciferase reporter (dengue virus R2H; details of cloning and generation of the cell line will be described elsewhere) were used. Huh7.5 cells were cultured as described.
Transmission Electron Microscopy
Cells were examined by transmission electron microscopy (TEM) as described. Briefly, Huh7-Lunet/T7 or Huh7-Lunet cells transfected either with an HCV NS3-5B expression construct or with a full-length Jc1 genome were treated with three different concentrations of 25HC, chemically fixed, dehydrated, and resin-embedded. Ultrathin sections (70 nm) were contrasted with lead citrate and examined by TEM. Double membrane vesicle (DMV) numbers were counted in five cell profiles per condition, and a total of 90 DMVs were measured to obtain an estimation of their amount and diameter.
Additional material and methods are posted as Supporting Information.
Human CH25H Messenger RNA is Up-Regulated in Liver Biopsies of CHC Patients and HCV-Infected PHHs
To study the regulation of hCH25H in response to HCV infection in humans, we compared messenger RNA (mRNA) expression levels in CHC liver biopsies from 34 patients with 10 liver biopsies from patients with non-HCV chronic liver diseases (Supporting Table S1 for patient data). Using this approach, we observed significantly higher mRNA expression levels of hCH25H in CHC liver biopsies compared to HCV-negative samples (Fig. 1A). Similar results were obtained for hIFIT3, a common marker for ISG induction (Fig. 1B). Induction of hCH25H mRNA was independent of patient gender, HCV genotype, degree of liver inflammation and fibrosis, or viral load (Supporting Fig. S1). To confirm these findings in an ex vivo HCV infection model, PHHs were infected with cell culture–derived Jc1 virus and 48 hours later infection levels were determined by quantification of newly produced infectious virus with the nucleoside polymerase inhibitor 2′C-methyl-adenosine (2′CMA) serving as control (Fig. 1C). To analyze mRNA expression levels of hCH25H, total cellular RNA was extracted for quantitative real-time polymerase chain reaction analysis (Fig 1D,E). De novo produced viruses yielded titers of about 103 50% tissue culture infective dose (TCID50) per milliliter, whereas the 2′CMA treatment completely blocked HCV production (Fig. 1C). A significant up-regulation of hCH25H mRNA expression was detected and completely dependent on viral replication as 2′CMA treatment abrogated this effect (Fig. 1D). A similar phenomenon was again noted for hIFIT3 and hIFNβ (Fig. 1E,F).
CH25H Is an ISG in PHHs
The expression of CH25H is up-regulated in response to type I and type II IFNs in murine macrophages and dendritic cells[16, 17, 19, 20, 27] and in murine liver cells infected with vesicular stomatitis virus. To validate and extend these findings to humans, we incubated PHHs and macrophages with types I, II, and III IFNs for different lengths of time. Human CH25H mRNA was strongly and significantly induced by type I IFN in PHHs (Fig. 2A) and macrophages (Fig. 2B) and to a lesser extent by types II and III IFN treatment. To demonstrate that the cells were in general responsive to types I, II, and III IFN treatment, hIFIT3 mRNA was quantified in parallel and found to be highly induced in hepatocytes (Fig. 2C) and macrophages (Fig. 2D). The induction of other ISGs known to be specifically stimulated by different types of IFN, like hMX1 (types I and III), hIFIT1 (types I and III), hIRF1 (type II), hCXCL10 (type II), and hPLSCR1 (types I, II, and III), was also observed to control the efficiency of each IFN treatment (Supporting Fig. S2).[28, 29] Of note, the type I IFN-dependent induction of hCH25H occurred rapidly already after 4 hours of IFN treatment, followed by a decrease of mRNA expression with longer treatment duration in both cell types (Fig. 2A,B). These results demonstrate that hCH25H is transiently induced by type I IFN in PHHs and macrophages.
Expression and Enzymatic Activity of hCH25H Exerts Antiviral Activity Against HCV Infection
To assess if hCH25H exerts an antiviral effect against HCV, we delivered and transiently expressed N-terminally hemagglutinin (HA)–tagged hCH25H or green fluorescent protein (GFP) as control through lentiviral gene transfer into permissive Huh7.5 cells and subsequently infected these cells with HCV (Fig. 3A). Infection with HCV and lentiviral transduction were evaluated at the single-cell level by monitoring HCV NS5A expression and GFP or HA-hCH25H expression using fluorescence microscopy. Treatment with the human immunodeficiency virus reverse transcriptase inhibitor efavirenz (EFV) served as a control for transduction and transgene expression. The analysis revealed that HCV Jc1 efficiently infected the Huh7.5 cells with transient GFP expression (first row of Fig. 3B). However, transient expression of hCH25H rendered the cells nonpermissive for viral infection (third row of Fig. 3B). This “viral exclusion” phenotype was specific for hCH25H-expressing cells as Huh7.5 cells treated with EFV could efficiently be infected by HCV Jc1 (fourth row of Fig. 3B). The EFV treatment itself had no effect on HCV infection as the compound-treated GFP-transduced cells could still be robustly infected by Jc1 despite the absence of GFP expression (second row of Fig. 3B). Transgene expression of hCH25H had no detectable effect on cell viability (Supporting Fig. S3A). Antiviral activity of hCH25H was also observed at the level of viral protein (NS3) accumulation (Fig. 3C) or in HCV infection experiments with reporter viruses upon multiplicity of infection–dependent transduction of hCH25H (Fig. 3D).
To test if the hCH25H enzymatic activity was responsible for the antiviral property of hCH25H, an inactive form of the hydroxylase, in which two clustered histidine residues were replaced by glutamine (H242Q and H243Q or QQ mutant), was generated and expressed in Huh7.5 cells. The detection of closely spaced double bands by western blot, particularly for the QQ mutant, is probably caused by differentially glycosylated forms of the hydroxylase protein as described (Fig. 3E). Although the transient expression of the QQ mutant still could significantly prevent HCV infection (∼50% HCV inhibition), the loss of enzymatic activity severely attenuated the antiviral property of hCH25H (Fig. 3E,F). Thus, the expression of active hCH25H protects human liver cells against HCV infection.
The Enzymatic Product of hCH25H, 25HC, Exhibits Antiviral Activity Against All HCV Genotypes
The enzyme CH25H catalyzes the conversion of cholesterol into 25HC with the presence of molecular oxygen as an additional substrate and nicotinamide adenine dinucleotide phosphate hydrogen as a cofactor (Fig. 4A). To investigate if hCH25H indeed produced a soluble antiviral factor against HCV, HEK 293T cells were transfected with either GFP, HA-hCH25H, or HA-hCH25H QQ mutant and the supernatant was used to cotreat HCV particles in an infection assay (Fig. 4B). The conditioned media from hCH25H-expressing 293T cells inhibited HCV infection by ∼80% compared to the GFP-control and QQ mutant (Fig. 4C), indicating that expression of hCH25H induces a soluble antiviral factor that is released into the culture supernatant and that this phenomenon requires the enzymatic function of the protein. To further analyze if 25HC is the effector molecule responsible for the antiviral activity against HCV, we incubated chimeric HCV reporter virus particles from all seven genotypes with increasing doses of 25HC for 4 hours, followed by a medium change without 25HC. Viral infectivity was measured 48 hours later. The addition of 25HC during infection of naive Huh7.5 cells with HCV resulted in a dose-dependent inhibition of infectivity with no overt cytotoxicity during these 4 hours (Fig. 4D; Supporting Fig. S2B). Of note, the presence of 25HC for a longer time period (48 hours) influenced cell viability at the highest doses of 25HC (Supporting Fig. S3B), which is in line with a previous report showing cytotoxicity and apoptosis induction of the oxysterol at higher concentrations. The antiviral activity of 25HC against all major HCV genotypes was comparable, indicating that 25HC inhibits HCV independently of the genotype of the virus particles (Fig. 4D). The same inhibition was observed with HCV wild-type viruses without reporter in an immunofluorescence-based infection assay using HCV Jc1 and different concentrations of 25HC (Fig. 4E).
As PHHs more closely resemble the natural host for HCV infection in humans, we next performed HCV infection experiments in PHHs. Cells were infected for 6 hours with HCV Jc1 in the presence of 25HC, and newly released infectious virus particles were quantified by TCID50 assay 24 and 48 hours later. At both time points, 25HC reduced de novo production of infectious particles by ∼10-fold (Fig. 4F). The NS5B polymerase inhibitor 2′CMA was used as a control for blocking HCV RNA replication and thus infectious virus particle production.
25HC Acts on the Target Cells but Does not Affect Virion Integrity
To analyze whether the inhibitory effect of 25HC is caused by an alteration of the target cells rendering it HCV-resistant, by an effect on the virus particles, or by a postentry effect, we administered 25HC before (pretreatment), during (cotreatment), or after (posttreatment) infection of Huh7.5 cells with HCV reporter viruses. Pretreatment of cells inhibited HCV infection efficiently, and the same was found with co- or postadministration in a dose-dependent manner (Fig. 5A), indicating that 25HC acts on the host cell and presumably inhibits HCV primarily at a postentry step. We next corroborated this conclusion using HCV pseudoparticles, which are retroviral cores harboring the HCV glycoproteins in their envelope. Using this approach, 25HC showed only a slight inhibition of HCV pseudoparticle cell entry, which was, however, not dose-dependent and much less efficient compared to anti-CD81 antibodies serving as a positive control (Fig. 5B). To test the effects of 25HC solely on virus envelope–cell membrane fusion, R18-labeled HCV particles were exposed to 25HC or vehicle prior to mixing with Huh7.5 cells. Fusion was evaluated by fluorescence dequenching of R18. The 25HC did not inhibit HCV fusion when the virions were preexposed, but fusion was partially inhibited when the cells were preexposed to 25HC (Fig. 5C). Fusion was more strongly inhibited when the cells were treated after virion binding (Fig. 5C), suggesting that 25HC acts on cellular, and not viral, membranes.
25HC Inhibits HCV RNA Replication by Blocking Membranous Web Biogenesis
To investigate the postentry effect of 25HC and its influence on HCV RNA replication, Huh7.5 cells were transfected with a reporter HCV-Con1 (genotype 1b) or -JFH1 (genotype 2a) SGR, followed by 48-hour incubation in the presence of increasing but nontoxic concentrations of 25HC. Replication efficiency was assessed using luciferase activity assays at the end time point. The 25HC efficiently inhibited HCV RNA replication in a dose-dependent manner (Fig. 6A,B). As HCV-Con1 and -JFH1 SGR replicate in different kinetics, we also performed a time-dependent experiment with 25HC against each SGR RNA replication, which was then assessed using direct quantitative real-time polymerase chain reaction on the RNA in comparison to the luciferase assay results (Supporting Fig. S4). Interestingly, HCV replication in stable cell lines was several orders of magnitude more sensitive to 25HC compared to dengue virus replicon–containing cells and hepatitis E virus replication (Supporting Fig. S5). In line with reduced RNA replication, 25HC treatment inhibited the production of released and cell-associated infectious HCV particles in a similar manner (data not shown).
It is known that 25HC is a ligand of the oxysterol-binding protein 1 (OSBP1),[32, 33] which, together with HCV NS5A and the host cell phosphatidylinositol-4 kinase IIIα,[34, 35] is a crucial component for the formation of the membranous HCV replication factory, termed the membranous web (MW). Therefore, using TEM we analyzed the effect of 25HC on the biogenesis of the MW, which is mainly composed of DMVs. In the initial set of experiments (Fig. 6C), we used Jc1-infected Huh7.5 cells that were treated with 25HC for time periods that did not affect the abundance of viral proteins (Fig. 6D). In mock-treated and HCV-infected hepatoma cells, high numbers of DMVs could be observed (Fig. 6E, left panel). However in HCV-infected cells treated with 25HC (Fig. 6E, middle and right panel), the amounts and diameter of DMVs were decreased in a dose-dependent manner (Fig. 6F, upper and lower panel). These results suggested that 25HC affects the biogenesis and/or integrity of the MW.
We have shown that expression of an NS3-5B polyprotein induces an MW independent of RNA replication with a morphology indistinguishable from the one found in HCV-infected cells. To exclude that the effect on the MW we observed in Jc1-infected Huh7 cells was caused by replication inhibition rather than by a direct effect on web integrity, we treated NS3-5B expressing cells with 25HC and analyzed them by TEM (Fig. 7A). The 25HC treatment did not alter the HCV protein levels (Fig. 7B). In agreement with our assumption, 25HC treatment profoundly altered MW morphology, causing a significant reduction in number and size of DMVs and clustering these vesicles in confined areas (Fig. 7C,D). In summary, these data suggest that 25HC inhibits HCV by blocking MW formation in a replication-independent manner.
In this study, we characterized the ISG hCH25H and its enzymatic product 25HC in liver cells as an antiviral restriction factor against HCV. By investigating the role of this enzyme in human liver biopsies, we found a significant elevation of CH25H gene expression in CHC patients compared to HCV-negative controls (Fig. 1A). Because hCH25H's main biological role is to catalyze the production of the oxysterol 25HC, these findings are supported by a recent report showing increasing serum oxysterol concentrations, particularly 25HC, in patients with CHC infection that, however, were significantly reduced during antiviral therapy. Human CH25H is normally poorly expressed in the healthy liver, but the elevation of hCH25H during HCV infection observed in the liver biopsies could be also seen in HCV-infected PHH cultures (Fig. 1B). Interferons are the central cytokines responsible for the induction of an antiviral state in virus-infected cells and for the activation and regulation of the cellular components of innate immunity. The rapid IFN induction of CH25H was initially identified in murine bone marrow– and lung-derived dendritic cells and macrophages in which toll-like receptor–mediated induction of this enzyme was observed through a mechanism that is dependent on signaling through the IFN-α/-β receptor and STAT1.18 We observed in primary human macrophages and hepatocytes an early and transient induction of hCH25H primarily after type I IFN stimulation (Fig. 2), in line with data from murine systems demonstrating that CH25H was more potently activated by type I IFN.[16, 19, 20] The short duration of this transcriptional stimulation upon IFN treatment, together with the significantly higher levels in CHC patients, argues for a constant stimulation in the infected liver. Moreover, the transient nature of hCH25H stimulation in PHH indicates the presence of a negative feedback regulation. Future studies are required to understand the underlying mechanism in more detail.
Ectopic expression of hCH25H by lentiviral gene transfer protected human hepatoma cells against HCV infection, and the enzymatic activity was crucial for the antiviral activity (Figs. 3A-D, 4). Another recent report described similar findings with hCH25H suppressing HCV infection and suggested that the mutant form of hCH25H could still inhibit HCV replication independently of its enzyme activity by directly interacting with and inhibiting NS5A dimerization. As we observed blockage of MW formation by treatment with 25HC without hCH25H gene expression, the binding and disruption of NS5A dimerization by hCH25H seem to be an independent and additional mechanism of antiviral restriction. We also found a significant reduction of HCV replication by the hCH25H mutant, which was, however, significantly lower compared to enzymatically active hCH25H (Fig. 3E-F), suggesting that the 25HC plays the more important role in antiviral activity against HCV. Very recently, Xiang et al. also demonstrated that the induction of hCH25H represents an important host innate response against HCV infection and highlighted the role of 25HC as the host lipid regulator responsible for the anti-HCV activity.
The product 25HC belongs to a diverse class of endogenous oxysterols that possess complex biological roles including inflammation.[42, 43] It regulates the activation of genes under the control of the SREBP transcription factor and the nuclear hormone receptor liver X receptor. Down-regulation of low-density lipoprotein receptor as one of the proteins regulated through the SREBP and liver X receptor pathway has been suggested to be the reason for the antiviral activity of 25HC against HCV. However, the strong inhibitory effect observed under posttreatment conditions (Fig. 5A) and on SGRs (Fig. 6 and Chen et al.) and the minor effect on HCV entry (Fig. 5B) suggest that the main mode of action is the inhibition of HCV RNA replication. Similar observations were initially reported by Pezacki et al., who described a down-regulation of many key genes involved in the mevalonate pathway by 25HC, leading to cholesterol depletion and an antiviral state against HCV Con1 replicons. Using electron microscopy, in this study we observed a collapse of HCV-induced DMV formation upon 25HC treatment (Figs. 6, 7). It is known that HCV extensively remodels intracellular membranes, giving rise to an MW that is composed predominantly of endoplasmic reticulum–derived DMVs with an average diameter of ±150 nm. Induction of DMVs does not require viral RNA replication but can be triggered by the sole expression of the HCV replicase proteins NS3-5B.[35, 36] It has been shown that NS5A and NS5B recruit phosphatidylinositol-4 kinase IIIα to endoplasmic reticulum–derived membranes, leading to a massive accumulation of phosphatidylinositol-4–phosphate at these sites.[46, 47] Phosphatidylinositol-4–phosphate enrichment likely facilitates the recruitment of cellular lipid transporters such as FAPP-2 and OSBP1, delivering sphingolipids and cholesterol to these membranes, respectively.[36, 48-50] Cholesterol in particular is thought to provide membrane stability to DMVs as cholesterol depletion induces shrinking of these membranes whereas knock-down of phosphatidylinositol-4 kinase IIIα causes in addition DMV clustering.[36, 51, 52] Interestingly, Strating et al. recently identified an inhibition of OSBP1-dependent cholesterol transfer activity caused by 25HC in in vitro liposomal assays. These results suggest that 25HC acts as an OSBP ligand, preventing the delivery of cholesterol to DMVs and thus biogenesis and integrity of the MW, reminiscent to what has been described for poliovirus replication.[36, 49, 54] This hypothesis is also supported by the fact that 25HC showed only a minor inhibitory effect on dengue virus replication (Supporting Fig. S3), another plus strand RNA virus that requires host cholesterol but does not require OSBP1 for membrane rearrangements.[49, 55, 56]
In summary, we show that HCV infection causes up-regulation of the ISG hCH25H in PHHs and CHC patients. The product produced by this enzyme, 25HC, profoundly blocks HCV RNA replication by affecting the biogenesis of the membranous HCV replication factory.
We are grateful to Takaji Wakita and Jens Bukh for JFH1 and J6CF isolates, respectively; to Charles Rice for Huh-7.5 cells and the 9E10 monoclonal antibody; to Suzanne Emerson for the hepatitis E virus p6 clone; and to Wolfgang Fischl for establishing the subgenomic dengue virus replicon. Moreover, we thank Stephanie Pfaender for critical reading of the manuscript and all members of the Institute of Experimental Virology, Twincore, for helpful support, suggestions, and discussions.