Potential conflict of interest: Nothing to report.
Hepatitis C virus (HCV) is a major human pathogen that causes serious illness, including acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Using a mass spectrometry–based proteomics approach, we have identified 175 proteins from a cell culture supernatant fraction containing the HCV genotype 2a (JFH1) virus, among which fatty acid synthase (FASN), the multifunctional enzyme catalyzing the de novo synthesis of fatty acids, was confirmed to be highly enriched. Subsequent studies showed that FASN expression increased in the human hepatoma cell line, Huh7, or its derivative, upon HCV infection. Blocking FASN activity by C75, a pharmacological FASN inhibitor, led to decreased HCV production. Reduction of FASN by RNA interference suppressed viral replication in both replicon and infection systems. Remarkably, FASN appeared to be selectively required for the expression of claudin-1, a tight junction protein that was recently identified as an entry coreceptor for HCV, but not for the expression of another HCV coreceptor, CD81. The decrease in Claudin-1 expression resulting from FASN inhibition was accompanied by a decrease in transepithelial electric resistance of Huh7 cells, implying a reduction in the relative tightness of the cell monolayer. Consequently, the entry of human immunodeficiency virus–HCV pseudotypes was significantly inhibited in C75-treated Huh7 cells. Conclusion: As far as we know, this is the first line of evidence that demonstrates that HCV infection directly induces FASN expression, and thus suggests a possible mechanism by which HCV infection alters the cellular lipid profile and causes diseases such as steatosis. (HEPATOLOGY 2008.)
Nearly 200 million people worldwide are infected by the hepatitis C virus (HCV), resulting in serious complications such as liver diseases and cancer. This enveloped, positive-stranded RNA virus is classified in the Hepacivirus genus of the Flaviviridae family.1 The 10-kb viral genome comprises a large open reading frame encoding a precursor polyprotein of approximately 3,000 amino acids. Cotranslational and posttranslational cleavages of this polyprotein generate at least 10 viral proteins, including a core protein, two glycoproteins, E1 and E2, p7, and a number of nonstructural proteins.2, 3 Nearly all of these viral proteins form complexes with host factors to carry out the viral life cycle. It is also believed that these viral–host interactions contribute via several complex mechanisms to such maladies as inflammation, steatosis, fibrosis, altered lipid metabolism, insulin resistance, and hepatocellular carcinoma.4
Enveloped viruses can incorporate both viral and host proteins into their membrane(s) or inside the envelope during virus assembly and budding. Infection of host cells by viruses is also likely to alter the profile of secreted proteins (secretomes). Knowledge of the protein composition of the infectious viral particle and the secretome provides important information for functional studies because subsequent characterization can be designed to focus on specific targets. In recent years, liquid chromatography (LC), coupled online with tandem mass spectrometry (MS/MS), has been successfully applied in several cases to analyze compositions of purified virions, leading to the identification of many previously unknown components of viral particles.5 In this study we collected a supernatant fraction containing infectious HCV genotype 2a (JFH1) virions (cell culture–grown HCV [HCVcc]) following ultracentrifugation and profiled the protein content by LC-MS/MS. A total of 175 proteins were identified, including fatty acid synthase (FASN), which was verified to be up-regulated during HCV infection of the Huh7.5.1 cell line. Subsequent functional analysis revealed a critical role of FASN in regulating HCV infection.
ApoE, apolipoprotein E; CLDN1, claudin-1; FA, fatty acid, FASN, fatty acid synthase; HCV, hepatitis C virus; HCVcc, cell culture–grown HCV; HCVpp, HIV particles pseudotyped with HCV envelope proteins; HIV, human immunodeficiency virus; JFH1, HCV genotype 2a; LC, liquid chromatography; MS/MS, tandem mass spectrometry; shRNAi, short-hairpin–based RNA interference; TER, transepithelial electric resistance; TJ, tight junction.
Materials and Methods
Cells and Reagents.
The human kidney epithelial cell line HEK293T (CRL-11268) was purchased from the American Type Culture Collection. The human liver cell line Huh7 was obtained from Apath Inc., with the permission of Dr. Charles Rice (Rockefeller University). The Huh7.5.1 line, which was generated from a cured HCV replicon cell line, was kindly provided by Dr. Francis Chisari (Scripps Research Institute). The full-length HCV genotype 1b-replicon–containing cell line (2−3+) and the cured cell line (2−3c) were generous gifts from Dr. Stanley Lemon (University of Texas Medical Branch, Galveston, TX).6 HCV genotype 2a replicon cells were established by transfecting Huh7 cells with the full-length genomic luciferase-JFH1 RNA (obtained from Dr. Ralf Bartenschlager, University of Heidelberg, Germany) and selected for clones that only supported viral replication and not the production of infectious virus. All cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA), 1% nonessential amino acids, and 10% fetal bovine serum (Hyclone, Logan, UT) and routinely checked to ensure they were free of mycoplasma contamination. Antibodies were obtained from Zymed/Invitrogen (anti-claudin-1 [CLDN1], clones 2H10D10 and JAY.8, anti-ZO-1, clone ZO1-1A12, and fluorescein isothiocyanate–conjugated or rhodamine-conjugated secondary antibodies), Affinity BioReagents (HCV core; Golden, CO), BD Biosciences (FASN; San Diego, CA) and Sigma (β-actin; St. Louis, MO). C75, a pharmacological FASN inhibitor, was purchased from Axxora LLC (San Diego, CA) and dissolved in dimethyl sulfoxide (DMSO). Orlistat and lentiviral-based short-hairpin–based RNA interference (shRNAi) constructs targeting human FASN were purchased from Sigma. Draq5, a far-red fluorescent DNA dye, was purchased from Biostatus (Shepshed, Leicestershire, UK).
Production of Pseudotyped Virus.
To produce the human immunodeficiency virus (HIV) particles pseudotyped with HCV envelope proteins (HCVpp) and HIV particles pseudotyped with vesicular stomatitis virus envelope protein G, HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) with a DNA transfection mixture composed of pNL-4.3-Luc-E−R−, phCMV-HCV E1E2, or pHEF-VSV-G (expressing the vesicular stomatitis virus envelope protein G) using a procedure described elsewhere.7
Quantification of HCV RNA by Real-Time Reverse-Transcription Polymerase Chain Reaction.
RNA was isolated from the cytosol or supernatants of infected cells using the Qiagen RNAeasy kit following the manufacturer's instructions. A total of 5 μL of RNA was analyzed with a real-time reverse-transcription polymerase chain reaction (PCR) assay at the Division of Molecular Diagnostics of the University of Pittsburgh Medical Center using an internally-developed protocol. Random primers were used for initial reverse-transcription, and pairs of gene specific primers covering a 50-bp region in the 5′ internal ribosome entry site were used for real-time PCR. The results are interpreted as international units per milliliter (IU/mL) for RNA derived from supernatant or international units per 1 million cells for cytoplasmic RNA. This number is divided by 1.6, giving the equivalent copy number of HCV genome RNA.
Transepithelial Electric Resistance Measurement.
A total of 1,000 Huh7 or 1,000 Caco-2 cells were seeded on a 24-well transwell plate (Corning; 6.5-mm membrane diameter, 0.4-μm pore size). After treatments with inhibitors, resistance was measured daily following a published procedure.8
Flow Cytometric Analysis.
Cells stained with CD81 monoclonal antibody were analyzed on a Coulter XL flow cytometer. Acquired data were analyzed and plotted using WinMDI 2.9 software.
Identification of Proteins from the HCV Virion-Containing Supernatant Fraction.
To purify HCV virions, we produced virus in large volumes and then passed the virus containing supernatant through a concentrator column to remove proteins smaller than 100 kDa. Concentrated viruses were pelleted by ultracentrifugation over a 25% sucrose cushion. For the negative control, the same amount of culture medium from uninfected cells was prepared in parallel. Virus and control pellets were separately dissolved in Laemmli sample buffer and resolved by one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After staining, a dozen distinct bands were noticed exclusively in the HCV-containing sample (Fig. 1A). These bands were excised and “in-gel” digested by trypsin. Following LC-MS/MS analysis of the extracted peptides, 175 proteins were positively identified by strict criteria, including a requirement of observing two peptides per protein matching the database (Supporting Table 1). Analysis of the gene ontologic classifications of the identified proteins revealed that most were involved in cellular metabolisms, biogenesis, transport, or other cellular processes (Fig. 1B). To validate the mass spectrometry results, we performed western blotting and confirmed that both FASN and apolipoprotein E (ApoE) were indeed highly enriched in the sample containing HCV (Fig. 1C). ApoE is a known virion-associated protein and is required for the production of infectious virus.9 It should be pointed out that these data represent only a partial proteomics analysis. A subtractive analysis comparing the protein profiles of both samples may likely yield more protein identifications.
FASN Was Up-Regulated Following HCV Infection.
Our initial functional characterization was focused on FASN, the critical enzyme that catalyzes de novo lipogenesis. FASN is known to be upregulated in many cancers, implying an interesting role in tumorigenesis.10 The FASN gene encodes a polypeptide that comprises seven functional domains including an acyl-carrier protein that work together to catalyze FA synthesis.11 The primary products of FASN activity are the 16-carbon FA palmitate, as well as the 14-carbon myristate and the 18-carbon stearate FAs. FAs have previously been implicated in the regulation of HCV production.12, 13 Since HCV infection is known to be associated with accumulation of fat in liver cells (i.e., steatosis), we first profiled FASN expression during HCV infection. Infection of Huh7 cells with the JFH1 virus (genotype 2a) for 7 days led to considerable increase in FASN protein abundance (Fig. 2A). The level of FASN expression was also weakly increased in a replicon system that harbors a full-length genotype 1b genome (Fig. 2B). To our knowledge, this is the first evidence that HCV infection directly up-regulates FASN.
FASN Is Required for Effective Viral Replication.
To better understand the role of FASN in the regulation of HCV infection, we used a highly specific β-ketoacyl synthase domain FASN inhibitor C75 to block its function.14 Because C75 treatment at concentrations equal to or lower than 75 μM did not cause significant cell death (Supporting Table 2), we chose 50 μM as the working concentration throughout this study; this dose is comparable to what has been reported to specifically inhibit the FASN activity.15 Treatment of JFH1-infected cells with C75 reduced the cellular HCV core protein production by three-fold to four-fold (Fig. 3A). The inhibitory effect was observed also in HCV genotype 1b and 2a replicon systems at both protein and RNA levels (Fig. 3B,C), indicating that C75 treatment suppresses the viral replication (Fig. 3C). Furthermore, we observed a significant drop in the infectious titer of the released virus from C75-treated Huh7 cells that have been infected by the JFH1 virus (Fig. 3D,E). Together, these results demonstrated that C75 treatment inhibited HCV production in cell culture systems.
Next, we sought to repeat the above experiments using another FASN inhibitor, the U.S. Food and Drug Administration–approved drug Orlistat. When tested, this drug did not lead to cell death at all at the indicated doses (data not shown). Addition of Orlistat to the media was also able to suppress the HCV core protein production in the infection system (Supporting Fig. 1). Surprisingly, Orlistat weakly enhanced the viral replication in the genotype 1b replicon system at either the RNA or the protein level (Supporting Fig. 1). Because the pharmacological inhibitors used could have had side effects, we next knocked down the endogenous FASN expression using shRNAi in both replicon and infection systems and found that both HCV core protein and RNA synthesis were indeed suppressed (Fig. 4).
FASN Regulates CLDN1 Expression and Viral Entry.
To investigate whether FASN may affect steps of viral life cycle other than replication, we performed infection assays and found that C75 treatment had a strong inhibitory effect on the HCVpp entry but not on HIV particles pseudotyped with vesicular stomatitis virus envelope protein G infection (Fig. 5A,B). Again, the same observation could be repeated using shRNAi to knock down endogenous FASN expression (Fig. 5C,D). The inhibition on HCVpp entry is possibly due to the down-regulation of the newly identified entry coreceptor CLDN1 because CLDN1 expression. but not CD81 expression. decreased significantly following C75 treatment (Fig. 6A-C). The same could be said for the FASN-silenced cells (data not shown). By contrast, the expression of another tight junction (TJ) protein, ZO-1, did not appear to decrease as much (Fig. 6C). Since CLDN1 is an integral component forming the TJ strand and we have previously shown that Huh7 cell monolayers exhibited considerable transepithelial electric resistance (TER),7 we then measured the TER in the presence or absence of inhibitors. Not surprisingly, C75-treated Huh7 and Caco-2 cells both displayed marked reductions in TER, implying the loss of the gate function of TJ (Fig. 6D,E; Supporting Fig.2). On the contrary, Orlistat treatment did not exert any inhibitory effect.
Determining the makeup of virions remains very challenging because the purity of the virions is always a major concern. Our proteomics profiling revealed many proteins worthy of further investigation for their functional roles in viral pathogenesis. The fact that a reported virion-associated protein, ApoE, was identified in this study validated the success of the approach. It must be noted, however, that the purification method employed in this study failed to obtain viral particles of high homogeneity. Under the current procedure, it is not possible to distinguish the virion-associated proteins from those that are merely secreted along with HCV virions. In fact, reports have shown cellular vesicles contained in prepared HIV virions.16, 17 Ideally, a purification scheme based on sucrose gradient ultracentrifugation and/or affinity separation would minimize contaminants. Complete proteome coverage would require identifying all the proteins contained in the HCV sample and the negative sample, and conducting a quantitative comparison to identify copurified contaminants. Nevertheless, the enriched proteins in Fig. 1A represent those that were copurified in the HCV virions–containing supernatant fraction under our purification procedure and may therefore play important roles in the regulation of the viral life cycle. In nature, HCV may exist in several forms: enveloped lipoprotein-free virus, enveloped lipoprotein-associated virus, nonenveloped lipoprotein-free virus, and nonenveloped lipoprotein-associated virus.18 It is therefore conceivable that compositions of these different forms of HCV virions may vary greatly.
The finding that FASN was highly enriched in the HCV-containing supernatant was quite surprising, despite the fact that we cannot confirm whether the extracellular FASN is associated with virions. Interestingly, although FASN is an intracellular protein, it was also found in breast cancer cell culture supernatants and in the blood of patients with breast cancer.19, 20 It is unclear what role extracellular FASN plays or the mechanism by which FASN exits cells. It has been speculated that secreted FASN might be connected to tumor metastasis. It will be interesting to measure the extracellular FASN level in HCV patients. If it correlates with disease processes such as steatosis and carcinogenesis, the extracellular FASN level may become a potential diagnostic and prognostic marker.
The exciting discovery of FASN as a HCV-inducible gene directly links viral infection to the lipid metabolic disorder.21 Notably, during the acute infection of chimpanzees, FASN was expressed at higher levels in successfully-cleared and transiently-cleared animals, although the pattern was not well correlated with HCV RNA levels.22 HCV is known to cause the formation of lipid droplets with which the HCV core protein associates and with which it recruits nonstructural proteins for viral assembly.23, 24 Our results now provide the first evidence for a causal relationship between HCV infection and the abundance of FASN. It is tempting to hypothesize that up-regulation of FASN results in increased lipogenesis. If that is the case, HCV infection could directly increase lipogenesis, contributing to the formation of steatosis. Induction of FASN in HCV-infected cells could also contribute to tumorigenesis, as FASN is commonly up-regulated in cancer cells. The molecular mechanism by which viral infection induces FASN expression remains unclear at this point. Two recent reports showed that the overexpression of HCV genotype 3a core protein and genotype 1a NS2 protein was able to up-regulate FASN promoter activity in a luciferase-based assay.25, 26 Clinically, HCV genotype 3 is associated with more pronounced hepatic steatosis.27 It is conceivable that FASN expression may be differentially modulated by individual HCV genotypes.
Previous studies using the replicon system have demonstrated that HCV RNA replication is regulated by host geranylgeranylation and FAs.13, 22 FAs are essential constituents of all biological membrane lipids and are important substrates for energy metabolism. The endogenous biosynthesis of FAs is catalyzed by the 250-kd to 270-kd homodimeric FASN. A number of inhibitors have been reported to block one of the FASN enzymatic activities. Both C75 and Cerulenin (an antifungal antibiotic that inhibits sterol and fatty acid biosynthesis) target the ketoacyl synthase activity of FASN and induce a rapid accumulation of malonyl-coenzyme A. The U.S. Food and Drug Administration–approved drug Orlistat is an inhibitor of FASN thioesterase activity despite that it is designed to inhibit pancreatic lipase activity.11 Cerulenin treatment was shown to block viral RNA synthesis in the replicon system.22 The disruption of membrane integrity by blocking the FA synthesis pathway was thought to account for this effect because HCV replication complex is established on a membranous web.28, 29 In this study, C75 was able to block viral RNA synthesis in both replicon and infection systems, as did FASN knockdown by shRNAi. Treatment of cells with Orlistat, however, had no effect in the replicon system, emphasizing the difference among inhibitors. While it is difficult to explain these differences, the reasons could potentially be attributed to efficacy and secondary targets of the inhibitors or the different cellular capabilities of compensating each individual FASN activity. In addition, varied responses to these inhibitors may also exist among different HCV genotypes.
C75 treatment also selectively inhibited CLDN1 expression. CLDN1 was recently identified by Evans et al.30 as the third entry coreceptor for HCV. Like CD81, CLDN1 spans the plasma membrane four times and is a component of the TJ barrier that restricts the free exchange of ions and aqueous molecules between cells.31, 32 We have demonstrated previously that CLDN1 exhibited a TJ-like distribution pattern in Huh7 cells and disruption of this pattern by tumor necrosis factor α treatment inhibited HCV entry.7 In light of the finding that depletion of cholesterol decreased CD81 cell surface expression but increased scavenger receptor class B type 1 cell-surface expression,33 it is reasonable to assume that C75 treatment does not deplete membrane cholesterol because it did not change the surface level of CD81. Interestingly, regulation of TJ permeability and occludin expression by polyunsaturated FAs has been reported.8 Further study is required to understand the detailed mechanism.
In conclusion, our results highlight the promise of applying functional proteomics in the study of HCV virology. FASN is not only important for productive HCV infection, but elevated FASN expression could contribute to many HCV-associated disease processes. Our findings warrant further investigations to determine the mechanism by which HCV infection induces FASN expression and the resulting impact of that on the production of FAs as well as on the alteration of cell phenotypes.
We thank Drs. C. Rice, F. Chisari, S. Lemon, F. Cosset, R. Bartenschlager, K. Li, C. Coyne, and T. Wakita for providing cell lines and constructs. We thank R. Busch, S. Dami, and M. Melan for help with reverse-transcription PCR. We also thank Dr. Leland Yee for critical reading of the manuscript.