The scavenger receptor class B type I (SR-BI) has recently been shown to interact with hepatitis C virus (HCV) envelope glycoprotein E2, suggesting that it might be involved at some step of HCV entry into host cells. However, due to the absence of a cell culture system to efficiently amplify HCV, it is not clear how SR-BI contributes to HCV entry. Here, we sought to determine how high density lipoproteins (HDLs), the natural ligand of SR-BI, affect HCV entry. By using the recently described infectious HCV pseudotyped particles (HCVpps) that display functional E1E2 glycoprotein complexes, we showed that HDLs are able to markedly enhance HCVpp entry. We did not find any evidence of HDL association with HCVpps, suggesting that HCVpps do not enter into target cells using HDL as a carrier to bind to its receptor. Interestingly, lipid-free apoA-I and apoA-II, the major HDL apolipoproteins, were unable to enhance HCVpp infectivity. In addition, drugs inhibit- Approximately 170 million individuals are infected worldwide by hepatitis C virus (HCV).1 HCV infection causes acute hepatitis, which has a high probability of becoming chronic and in the long term can lead to cirrhosis and hepatocellular carcinoma (1). Because of the serious consequences of its infection in humans, much effort is being made to understand the basic mechanisms of HCV lifecycle. Because of the absence of an efficient cell culture system to replicate HCV, several laboratories have tried to develop surrogate models to study HCV entry (2). These models are based on the expression of HCV envelope glycoproteins E1 and E2, which form a noncovalent heterodimer. Several difficulties have been encountered in these approaches because HCV envelope glycoproteins are located in the endoplasmic reticulum (3), and their folding and assembly is very sensitive to mutations or deletions affecting the endoplasmic reticulum retention domains (4). Recently, infectious pseudotyped particles (HCVpps) that are assembled by displaying unmodified HCV envelope glycoproteins onto retroviral core particles have successfully been generated and now enable studies of HCV entry (5, 6).The cellular tropism of enveloped viruses is largely determined by selective interactions of viral envelope glycoproteins with specific cell-surface receptors. Several candidate receptors for HCV have recently been proposed. Molecules like the CD81 tetraspanin (7), the scavenger receptor class B type I (SR-BI) (8), the LDL receptor (9, 10), and the asialoglycoprotein receptor (11) are potential candidate receptors for HCV, whereas the mannose binding lectins DC-SIGN and L-SIGN have been shown to function as HCV capture receptors but do not mediate viral entry into target cells (12-16). Among these molecules there is mounting evidence that CD81 and SR-BI are necessary for HCV entry (5,6,17). Whereas the role of CD81 in HCV entry is now well documented (5,6,(17)(18)(19), the contribution of SR-BI in HCV entry needs to be further characterized. SR-BI is a 509-amino acid glycoprotein with two cytoplasmic ...

IntroductionEpidemiological and transgenic animal studies have implicated apo C-IlI as a major determinant of plasma triglyceride metabolism. Since

Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum. Hepatitis C virus (HCV),7 a member of the Flaviviridae family, is transmitted during parenteral exposures to infected material, such as contaminated blood or needles. Its genome encodes a precursor polyprotein of ϳ3,000 amino acids (1). Cleavage of this polyprotein generates 10 polypeptides, including a core protein, two surface glycoproteins, E1 and E2, and the nonstructural proteins.HCV is an enveloped virus, implying that specific cell surface molecules mediate the capture of viral particles and their penetration inside the infected cells. Several molecules have been proposed as cell entry receptors of HCV, and most of them have been isolated based on binding studies with soluble recombinant E2 protein or HCV-like particles. Potential receptors include the CD81 tetraspanin (2), the low density lipoproteins (LDL) receptor (3), the scavenger receptor BI (SR-BI) (4) that binds HDL, native, or modified LDL and very low density LDL (vLDL) (5), and several "capture" molecules that induce concentration of viral particles at the cell surface, hence allowing virions to find the cell entry receptors (6). Experimental data using infectious HCV pseudoparticles (HCVpp) harboring authentic E1E2 glycoproteins (7, 8) have substantiated the functional roles of CD81 and SR-BI in HCV entry (6). The requirement for CD81 in cell entry ha...

In view of the evidence linking plasma high density lipoprotein (HDL)-cholesterol levels to a protective effect against coronary artery disease and the widespread use

Fibrates are widely used hypolipidemic drugs which activate the nuclear peroxisome proliferator-activated receptor (PPAR) ␣ and thereby alter the transcription of genes controlling lipoprotein metabolism. Fibrates influence plasma high density lipoprotein and its major protein, apolipoprotein (apo) A-I, in an opposite manner in man (increase) versus rodents (decrease). In the present study we studied the molecular mechanisms of this species-specific regulation of apoA-I expression by fibrates. In primary rat and human hepatocytes fenofibric acid, respectively, decreased and increased apoA-I mRNA levels. The absence of induction of rat apoA-I gene expression by fibrates is due to 3 nucleotide differences between the rat and the human apoA-I promoter A site, rendering a positive PPAR-response element in the human apoA-I promoter nonfunctional in rats. In contrast, rat, but not human, apoA-I transcription is repressed by the nuclear receptor Rev-erb␣, which binds to a negative response element adjacent to the TATA box of the rat apoA-I promoter. In rats fibrates increase liver Rev-erb␣ mRNA levels >10-fold. In conclusion, the opposite regulation of rat and human apoA-I gene expression by fibrates is linked to differences in cis-elements in their respective promoters leading to repression by Rev-erb␣ of rat apoA-I and activation by PPAR␣ of human apoA-I. Finally, Rev-erb␣ is identified as a novel fibrate target gene, suggesting a role for this nuclear receptor in lipid and lipoprotein metabolism.

Rev-Erb␣ (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor ␣ gene. Rev-Erb␣ mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor ␥ (PPAR␥) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erb␣ expression is induced by PPAR␥ activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Adipocyte differentiation is a complex biological process, which is reflected at the molecular level by the transcriptional activation of a number of adipocyte-specific genes and by the acquisition of the ability to accumulate cytoplasmic lipid droplets (1-3). The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥, 1 NR1C3) (4, 5) and members of the CCAAT enhancer-binding protein (C/EBP) family (6 -12) play key roles in this adipogenic process. In addition, the adipocyte differentiation and determination factor-1 (SREBP-1/ADD1) appears to promote adipocyte differentiation by activating the expression of PPAR␥ and increasing the synthesis of endogenous PPAR␥ ligands (13-15). Members of the PPAR family bind as heterodimers with the retinoid X receptors (RXR) to specific response elements termed peroxisome proliferator response elements (PPRE) (for review see Ref. 16). These PPREs usually consist of a direct repeat of the PuGGTCA motif spaced by one nucleotide (DR1). The transcriptional activity of the PPARs is activated by a number of different fatty acid metabolites, most notably products of the cycloxygenase and lipoxygenase pathways. In addition, a large number of synthetic compounds are known to be potent and subtype specific PPAR ligands. For example, thiazolidinedione compounds used as insulin sensitizers in the treatment of type II diabetes are high affinity PPAR␥ ligands (17).Rev-Erb␣ (NR1D1) is another nuclear receptor, the expression of which is induced during adipocyte differentiation (18). Rev-Erb␣ is highly expressed in adipose tissue but also in skeletal muscle, liver and brain (18 -21). Since no ligand has been identified so far, Rev-Erb␣ is considered as an orphan member of the nuclear receptor superfamily. Rev-Erb␣ has been shown to act as a negative regulator of transcription (22) binding either as monomer on nuclear receptor half-site motifs flanked 5Ј by an A/T rich sequence (A/T PuGGTCA), or as a homodimer to a direct repeat of the PuGGTCA motif spaced by two nucleotides (DR2).We have previously shown that PPAR␣ activates the expression of Rev-Erb␣ through an atypical PPRE, a DR-2 element, in the Rev-Erb␣ promoter (23). Transcriptional activation by PPAR␥ through a DR-2 element has so far not been reported. However, since Rev-Erb␣ is induced during the course of adipocyte differentiation, we decided to investigate whether PPAR␥ could be involved in transcriptional induction of RevErb␣ expression in adipocytes. Furth...

The recently discovered APOA5 gene has been shown in humans and mice to be important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. apoAV represents the first described apolipoprotein where overexpression lowers triglyceride levels. Since fibrates represent a commonly used therapy for lowering plasma triglycerides in humans, we investigated their ability to modulate APOA5 gene expression and consequently influence plasma triglyceride levels. Human primary hepatocytes treated with Wy 14,643 or fenofibrate displayed a strong induction of APOA5 mRNA. Deletion and mutagenesis analyses of the proximal APOA5 promoter firmly demonstrate the presence of a functional peroxisome proliferator-activated receptor response element. These findings demonstrate that APOA5 is a highly responsive peroxisome proliferator-activated receptor ␣ target gene and support its role as a major mediator for how fibrates reduce plasma triglycerides in humans.Coronary heart disease continues to be a major cause of morbidity and mortality worldwide. Several epidemiological studies established that, in addition to elevated low density lipoprotein and reduced high density lipoprotein level, elevated triglycerides (TGs) 1 constitute an independent risk factor for coronary heart disease (1, 2). In addition, hypertriglyceridemia is often associated with the metabolic syndrome that characterizes diabetes and obesity (3, 4). Therefore, the identification of factors or genes affecting triglyceride metabolism is of significant medical importance for the correction of hypertriglyceridemia and associated coronary heart disease.Apolipoproteins play a determinant role in lipoprotein metabolism and in lipid homeostasis. More specifically, the APOA1/C3/A4 apolipoprotein gene cluster is tightly linked to plasma lipid profiles. Indeed, mutations in members of this cluster have been shown to cause severe dyslipidemia and heightened atherosclerosis susceptibility (5-8). A comparative genomic characterization of the APOA1/C3/A4 gene cluster flanking regions led to the recent identification of a new apolipoprotein gene, apolipoprotein A5 (APOA5), present in both mice and humans (9). apoAV shares homology with several apolipoproteins, most prominently apoAIV, and is 368 and 366 amino acids long in mice and human, respectively. This gene appears to be predominantly expressed in the liver and resides on high density lipoprotein and very low density lipoprotein particles (9, 10).The properties of apoAV were investigated by creating human APOA5 transgenic and knock-out mice and by searching for associations between human APOA5 polymorphisms and plasma lipid parameters. The Apoa5 knock-out mice display a 400% increase in plasma triglycerides compared with wild-type mice, while APOA5 transgenics exhibit triglyceride levels corresponding to one-third of those in control mice. This determinant link between APOA5 and triglycerides was supported in several separate human studies through the consistent demonstration of associations betwe...

Inhibition of viruses at the stage of viral entry provides a route for therapeutic intervention. Because of difficulties in propagating hepatitis C virus (HCV) in cell culture, entry inhibitors have not yet been reported for this virus. However, with the development of retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recent progress in amplification of HCV in cell culture (HCVcc), studying HCV entry is now possible. In addition, these systems are essential for the identification and the characterization of molecules that block HCV entry. The lectin cyanovirin-N (CV-N) has initially been discovered based on its potent activity against human immunodeficiency virus. Because HCV envelope glycoproteins are highly glycosylated, we sought to determine whether CV-N has an antiviral activity against this virus. CV-N inhibited the infectivity of HCVcc and HCVpp at low nanomolar concentrations. This inhibition is attributed to the interaction of CV-N with HCV envelope glycoproteins. In addition, we showed that the carbohydrate binding property of CV-N is involved in the anti-HCV activity. Finally, CV-N bound to HCV envelope glycoproteins and blocked the interaction between the envelope protein E2 and CD81, a cell surface molecule involved in HCV entry. These data demonstrate that targeting the glycans of HCV envelope proteins is a promising approach in the development of antiviral therapies to combat a virus that is a major cause of chronic liver diseases. Furthermore, CV-N is a new invaluable tool to further dissect the early steps of HCV entry into host cells.More than 170 million people worldwide are chronically infected by hepatitis C virus (HCV) 4 (1). This virus is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (2). In addition, chronic HCV infection has become the most common indication for liver transplantation. Current antiviral therapy is based on the use of polyethylene glycolmodified interferon-␣ in combination with ribavirin. However, this treatment is expensive, relatively toxic, and effective in only half of the treated patients (3). Furthermore, there is as yet no vaccine against HCV. Therefore, more efficacious and better tolerated anti-HCV treatments are sorely needed to combat this major pathogen. HCV is an enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (4). Because of difficulties in propagating HCV in cell culture, many gaps remain in our understanding of the HCV life cycle. A major advance in the investigation of HCV entry was the development of pseudoparticles (HCVpp), consisting of native HCV envelope glycoproteins E1 and E2 assembled onto retroviral core particles (5-7). This system is potentially very powerful to identify and characterize molecules that block HCV entry. Furthermore, data obtained with HCVpp can also now be confirmed with the help of the recently developed cell culture system that allows efficient amplification of HCV (HCVcc) (8 -10).During their biogenesis, the two envelope glycoproteins...