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Originally published In Press as doi:10.1074/jbc.M705358200 on August 30, 2007

J. Biol. Chem., Vol. 282, Issue 44, 32357-32369, November 2, 2007
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The Exchangeable Apolipoprotein ApoC-I Promotes Membrane Fusion of Hepatitis C Virus*Formula

Marlène Dreux{ddagger}1, Bertrand Boson{ddagger}2, Sylvie Ricard-Blum§2, Jennifer Molle§, Dimitri Lavillette{ddagger}, Birke Bartosch{ddagger}, Eve-Isabelle Pécheur§, and Francois-Loïc Cosset{ddagger}3

From the {ddagger}Université de Lyon, (UCB-Lyon1), IFR128, INSERM, U758, and Ecole Normale Supérieure de Lyon, Lyon, F-69007, France and §IBCP, UMR5086 CNRS-UCB, Lyon-I, F-69007, France

Received for publication, June 29, 2007 , and in revised form, August 29, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell entry of hepatitis C virus (HCV) is strikingly linked to lipoproteins and their receptors. Particularly, high density lipoprotein (HDL) enhances infectivity of HCV by involving the lipid-transfer function of the scavenger receptor BI, a receptor for both HDL and HCV. Here, we demonstrate that apoC-I, an exchangeable apolipoprotein that predominantly resides in HDL, specifically enhances HCVcc and HCVpp infectivity and increases the fusion rates between viral and target membranes via a direct interaction with the HCV surface. We identify the hypervariable region 1, located at the N terminus of the HCV E2 glycoprotein, as an essential viral component that modulates apoC-I-mediated enhancement of HCV fusion properties. The affinity of apoC-I for the HCV membrane may predispose it for fusion with a target membrane via alterations of its outer phospholipid layer. Conversely, we found that excess apoC-I provided as lipoprotein-free protein induces the disruption of the HCV membrane and subsequent loss of infectivity. Furthermore, our data indicate that HDL neither interacts nor spontaneously exchanges apoC-I with HCV virions. Because apoC-I is not present in serum as a lipoprotein-free form, our results suggest that HDL-embedded apoC-I could be released from the lipoprotein particle through a fine-tuned mechanism regulated via a triple interplay between hypervariable region 1, HDL, and scavenger receptor BI, resulting in optimal apoC-I recruitment on the viral membrane. These results provide the first description of a host serum factor helping the fusion process of an enveloped virus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
With an estimated 170 million infected individuals, hepatitis C virus (HCV)4 has a major impact on public health (2). HCV is an enveloped, positive-stranded RNA virus of the Flaviviridae family. Its genome encodes a single polyprotein processed by viral and cellular proteases into three structural (core, E1 and E2 glycoproteins) and seven non-structural proteins (3, 4).

For a long time the study of HCV cell entry has remained limited because the ex vivo characterization of HCV derived from plasma has proven extremely difficult. This is due in large part to the low infectivity of the virus in cultures of primary hepatocytes, to its high genetic heterogeneity, and to its association through different forms with lipoproteins. Thus, to overcome these severe limitations toward the molecular characterization of HCV infection, several surrogate assays have been developed. Two relevant and complementary in vitro cell entry assays consist of cell culture-grown genuine HCV (HCVcc) derived from a fulminant hepatitis C, JFH-1 (5-7), and of HCV pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins, which are particularly amenable to mutagenesis analysis (8-10). Although HCVcc further permit investigation of the late infection steps, HCVpp, which can be produced in non-hepatic cells that can be readily complemented with hepatic factors, offers a particularly flexible plate form to study the structure/function relationship of HCV glycoproteins both in cell culture and in liposome fusion assays in vitro (11). Thus, both HCVcc and HCVpp infection assays reproduce some cell entry features of native HCV and allow a precise dissection of the cellular and viral factors involved in the early events of HCV infection (for review, see Refs. 12-14).

The viral surface glycoproteins, E1E2, and their receptors mediate the cell entry processes of HCV. At least three receptors for HCV E2 have been identified that mediate concentration, binding, and cell entry of viral particles. They include glycosaminoglycans, the CD81 tetraspanin, and the scavenger receptor B-I (SR-BI), a major receptor of high density lipoprotein (HDL). Using HCVpp and HCVcc infection assays as well as in vitro membrane fusion assays, HCV entry has been shown to occur in a pH-dependent manner (10, 11, 15-17) through endocytosis of the viral particles (18, 19). As for other Flaviviridae (20), the low endosomal pH may induce conformational rearrangement of HCV glycoproteins, leading to fusion of the viral membrane with that of the endosome.

The steps after the initial encounter of the HCV glycoproteins with the target cell surface remain ill-defined. Glycosaminoglycans such as highly sulfated heparan sulfate allow viral particles to adhere to target cells before specific receptors induce cell entry (21). As shown by genetic complementation, down-regulation, and blocking experiments (5, 6, 10, 16, 22-25), CD81 appears an essential receptor in both HCVpp and HCVcc infection assays, yet its role in cell entry remains elusive. Through its down-regulation and blocking, SR-BI has been shown as an important cell entry factor that can boost cell entry of HCVpp and HCVcc cooperatively with CD81 (15, 16, 26, 27). Additional host components contribute to cell entry, as recently highlighted by the finding that claudin-1, a tight junction protein, is required for HCV infection of human hepatoma cell lines (28).

The involvement of SR-BI during cell entry of HCV seems closely related to its physiological function and to its natural ligands. SR-BI mediates binding and lipid transfer from different classes of lipoproteins (29), particularly HDL, accounting for its multiple functions in cholesterol metabolism such as removal of peripheral unesterified cholesterol, steroidogenesis, and bile acid synthesis and secretion. SR-BI mediates direct binding of E2 (30, 31) and, as a multiligand lipoprotein receptor, can also induce binding of HCV associated to beta-lipoproteins (32). Intriguingly, we and others have demonstrated that HDL enhances infectivity of HCVpp and HCVcc (26, 33-37). HDL-mediated enhancement of infection does not occur through a direct binding of HDL to HCV particles but, rather, involves the lipid-transfer function of SR-BI (26, 33, 36). This original mechanism is controlled by the HCV glycoproteins, and more particularly by conserved residues of the hypervariable region-1 (HVR1) (33, 36), a 27-amino acid peptide located at the N terminus of E2. As SR-BI-mediated lipid transfer from HDL locally increases cholesterol content of the lipid membrane (38, 39), it may enhance internalization, membrane rearrangement of components of the HCV receptor complex, and/or membrane fusion of HCV (11, 26, 27). On the other hand, an essential component of HDL that seems responsible for infection enhancement is the apolipoprotein C-I (apoC-I) (35). ApoC-I is a small plasma protein (57 amino-acids) composed of two amphipathic {alpha}-helices. It is the smallest of the exchangeable apolipoproteins (A-I, A-II, A-IV, C-I, C-II, C-III, and E) and circulates in the bloodstream associated with HDL, mainly and with very low density lipoprotein (VLDL) and chylomicron particles (40, 41). Its capacity to interact with lipid surfaces underlies a number of its functional properties and its important role in regulating plasma lipoprotein metabolism (42, 43).

Here we have investigated the mechanisms underlying the enhancement of the early steps of HCV infection by HDL. We demonstrate that apoC-I increases HCVcc and HCVpp membrane fusion via a direct and specific interaction with HCV particles. We show that the HVR1 region is an essential viral component that modulates this interaction and enhancement of HCV fusogenicity. Our data indicate that lipoprotein-associated apoC-I induces HCV infection enhancement through a mechanism that is regulated via a triple interplay between HVR1, HDL, and SR-BI, which results in optimal apoC-I recruitment on the viral membrane. These results provide the first description of a host-soluble factor helping the fusion process of an enveloped virus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Constructs and Production of HCVpp—Expression vectors for the E1E2 glycoproteins of HCV strain H77 (AF009606 [GenBank] ), for the HVR1 deletion mutant ({Delta}G384-N411), and for the HVR1 point mutants were described previously (8, 15, 33). The murine leukemia virus (MLV) packaging and green fluorescent protein transfer vectors and the phCMV-RD114 expression plasmid encoding glycoproteins of cat endogenous virus RD114 were described elsewhere (8). The phCMV-VSV-G, phCMV-MLV-A, and phCMV-HA expression plasmids encoding the glycoprotein of vesicular stomatitis virus, amphotropic MLV, and an avian influenza virus hemagglutinin (HA H7N1) respectively, were described previously (44). The phCMV-712-HIV expression plasmids encoding the glycoprotein of human immunodeficient virus (HIV) was described previously (45).

Viral pseudo-particles named HCVpp, RD114pp, VSV-Gpp MLVpp, HApp, and HIVpp harbored the glycoproteins of HCV, RD114, VSV, influenza virus, and HIV. They were produced (8) by transfection in 293T cells of vectors encoding viral glycoproteins, packaging proteins, and green fluorescent protein-transfer vector. Before harvesting viral particle-containing supernatants, producer cells were incubated in Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum for 24 h. Viral particle-containing supernatants were used to infect Huh-7 hepatoma cells directly or upon purification by ultracentrifugation through a 20% sucrose cushion.

Expression Constructs and Production of HCVcc—The pFK-venus-Jc1 is a chimeric J6CF/JFH1 HCV genome consisting of codons 1-846, derived from J6CF (AF177036 [GenBank] ) and codons 847-3033, derived from JFH1 (AB047639 [GenBank] ) (46, 47). HCVcc were produced by electroporation of Huh7-Lunet cells (6, 47) in a L3 laboratory according to European safety regulations. 24 h before harvesting viral particle-containing supernatants, electroporated cells were incubated in Dulbecco's modified Eagle's medium containing 2% lipoprotein-deficient fetal bovine serum (Sigma). Viral particle-containing supernatants were used directly in infection assays using Huh7-Lunet target cells or after purification as described above for HCVpp.

Reagents and Antibodies—The HDL (Calbiochem) preparation (density 1.063-1.2 mg/ml) contained a mixture of HDL2 and HDL3. Purified apolipoproteins were purchased from Athens Research and Technology (Athens, GA). The BLT-4 SR-BI lipid transfer inhibitor (48) was obtained from Chembridge. The rabbit anti-apoC-I antibody was from Biodesign. The 9/27 (10), AP33 (49), H53 (8, 50), and the E2mAb-15 are E2-specific mAbs. A4 (51) is an E1-specific mAb. The 83A25 mAb (a kind gift of L. Evans), 2F5 mAb (NIH AIDS Research and Reference Reagent Program), and RD114 SU goat antiserum (ViroMed Biosafety Laboratories) are antibodies against MLV, HIV, and RD114 envelope glycoproteins, respectively. Lectin from Galanthus nivalis was obtained from Sigma. The recombinant CD81-LEL fragment (amino acids 112-202) and a truncated soluble form of E2 glycoprotein (sE2) (amino-acids 384-664) were fused to a His tag, produced in cells, and purified on nickel nitrilotriacetic acid resin (Qiagen). The protein A and G coupled to Sepharose beads were purchased from Amersham Biosciences. Phosphatidylcholine from egg yolk (99% pure), cholesterol (99% pure), and Triton X-100 were from Sigma. Phospholipid oxidation was routinely checked by spectrophotometry. Octadecylrhodamine B chloride (R18) was from Molecular Probes, and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) and N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (N-NBD-PE) were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids.

Infection Assays—For infection assays with HCVpp and HCVcc, Huh-7 and Huh-7-Lunet cells were, respectively, seeded 24 h before inoculation (6, 8). 2 h before infection target cells were preincubated in Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum. Medium was then removed, and dilutions of viral supernatants and various compounds were added to the cells as indicated. After 4 h, supernatants were removed, and the infected cells were kept in regular medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum) for 72 h before analysis.

The infectious titers were deduced from the percentage of green fluorescent protein-positive cells, as determined by fluorescence-activated cell sorter analysis (8). Infections with HCVpp were controlled by pseudo-particles devoid of E1E2 glycoproteins, which resulted in background titers below 5 x 102 infectious units/ml.

Binding Assays—Binding of HCVpp was performed as previously described for HCVpp or for other types of pseudo-particles (26, 52, 53). Briefly, 50 µl of virus particles purified on a 20% sucrose cushion were incubated with 106 CHO cells expressing SR-B1 and/or CD81 or Huh-7 cells in the presence of 0.1% sodium azide for 1 h. Cells were then washed twice with PBFA (PBS, 2% fetal bovine serum, and 0.1% sodium azide) and incubated with the H53 anti-HCV-E2 (40 µg/ml) or the A4 anti-HCV-E1 mAbs (40 µg/ml) for 1 h at 4 °C. After two washes, cells were incubated with a goat anti-mouse-allophycocyanine antibody (Jackson Immunoresearch) diluted in PBFA (5 µg/ml) for 45 min at 4 °C. Fluorescence of living 10,000 cells was determined by fluorescence-activated cell sorter analysis in the FL4-H channel.

Surface Plasmon Resonance (SPR) Binding Assays—Biomolecular interactions were studied using a BIAcore-3000 instrument (BIAcore AB, Uppsala, Sweden). Purified apoC-I (200 µg/ml in 10 mM acetate buffer, pH 4) was covalently immobilized via its primary amino groups to the dextran matrix of a CM4 sensor chip (amine coupling kit, BIAcore AB) at a flow rate of 5 µl/min. Activation and blocking steps were performed as described previously (26, 54). A control flow cell was prepared according to the same procedure by omitting apoC-I in the coupling buffer. It was used to assess nonspecific binding to the sensor chip surface. Purified HCVpp and control viral particles (CONTpp), HIVpp, MLVpp, or RD114pp were injected over immobilized apoC-I in PBS containing 0.005% P20 surfactant (BIAcore AB) at a flow rate of 5 µl/min at 25 °C. Sensorgrams obtained on the control flow cell were automatically subtracted from the sensorgrams obtained over immobilized apoC-I. The surface was then regenerated with a pulse of 2 M guanidinium chloride.

The AP33, 9/27, E2mAb-1, H53, 83A25, 2F5 antibodies (100 µg/ml in 10 mM acetate buffer, pH 4.5) and G. nivalis lectin (200 µg/ml in 10 mM acetate buffer, pH 4) were covalently immobilized to the dextran matrix of a CM3 sensor chip via their primary amino groups as described above. A control flow cell was prepared by immobilizing an irrelevant antibody (mouse anti-interleukin 2) according to the same procedure. Purified HCVpp, MLVpp, HIVpp, or sE2 were injected over the antibodies in PBS containing 0.005% P20 surfactant (BIAcore AB) at a flow rate of 5 µl/min at 25 °C, as described previously (26), resulting in capture levels ranging from 500 up to 1000 resonance units. Sensorgrams obtained on the control flow cell representing nonspecific binding to the sensor chip surface were automatically subtracted from the sensorgrams obtained with captured HCVpp. Binding assays of apoC-I and CD81-LEL were performed at 25 °C in PBS with 0.005% P20 surfactant at a flow rate of 5 µl/min. The surface was then regenerated with pulse of 0.025% SDS or 2 M guanidinium chloride.

Reverse Transcriptase (RT) Release Assays—The release assays were performed as described previously (55, 56) using an RT assay kit (RetroSysTM, Innovagen, Sweden) following the manufacturer's instructions but using a sample dilution buffer devoid of Triton X-100.

Fusion Assays—Fusion assays with rhodamine-labeled liposomes were performed as previously described (11, 53). Large unilamellar vesicles containing egg yolk phosphatidylcholine and cholesterol in a 7:3 ratio and 1 mol % each of -Rh-PE and N-NBD-PE were made by extrusion through a 100-nm-defined pore polycarbonate filters as described before (11) and stored at 4 °C. Liposomes (15 µM final lipid concentration) were added to a cuvette containing 40 µl of purified viral particles produced in cell culture media devoid of serum lipoproteins diluted in 400 µl PBS, pH 7.4, and preheated to 37 °C. Fluorescence dequenching of rhodamine was recorded ({lambda}xc = 560 nm, {lambda}em = 590 nm) as a function of time on an SLM Aminco 8000 spectrofluorimeter over a 15-min time period. For reasons of comparison with supplemental Fig. 3, we chose to show the dequenching of rhodamine instead of the increase in fluorescence of NBD due to fluorescence resonance energy transfer release. However, NBD fluorescence was recorded (data not shown) as well as the decrease of fluorescence resonance energy transfer efficiency on rhodamine ({lambda}exc (NBD) = 460 nm, {lambda}em (Rh) = 590 nm). After 100 s (time 0 of the fusion kinetics), pH was decreased to 5.0 by the addition of diluted HCl to the cuvette. Maximal dequenching was measured after the addition of 0.1% Triton X-100 (final concentration) to the mixture. The initial rates of fusion were taken as the value of the slope of the tangent determined at the initial part of the kinetics at time 0.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HDL-mediated Stimulation of HCV Entry Requires ApoC-I and HVR1—HCV particles, either the J6/JFH-1 cell culture grown authentic HCV (HCVcc) or the HCV pseudo-particles (HCVpp), were produced in vitro in media devoid of serum lipoproteins. As shown in Fig. 1A, the addition of purified HDL during infection of Huh-7 target cells increased the infectivity of HCVcc and HCVpp but not of control viral particles. One of the protein components of HDL that induced this enhancement of infection is apoC-I. Indeed, antibodies against apoC-I (Fig. 1A), but not antibodies against the other HDL apolipoproteins (i.e. apoA-I, apoA-II, apoC-II, and apoC-III; data not shown), abrogated the effect. Moreover, preincubation of HCV particles with purified, lipoprotein-free apoC-I increased cell infection of HCVpp and HCVcc in a manner similar to HDL (Fig. 1B and data not shown). This enhancement of infection was specific of HCV E1E2 glycoproteins since it was not observed with viral particles bearing the surface glycoproteins from alternative enveloped viruses, such as RD114 cat endogenous virus, vesiculovirus, influenza virus, or murine leukemia virus (MLV) (Fig. 1B), in keeping with the HCV-specific infection enhancement detected with HDL (Fig. 1A and Ref 33). Of note, no stimulation of infection was detected with purified apoC-II, another exchangeable apolipoprotein of HDL structurally related to apoC-I (Fig. 1B). Finally, the addition of apoC-I antibodies to HCVcc or HCVpp preincubated with purified apoC-I resulted in specific inhibition of their infectivity (Fig. 1C), presumably by neutralization of the HCV particles complexed with apoC-I, as shown below. These results, therefore, extended previous data obtained with HCVpp (33, 35, 36) and validated them using the HCVcc infection assay.


Figure 1
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  		FIGURE 1.
ApoC-I enhances the infectivity of HCV particles. Results of infection assays on Huh-7 cells using HCVcc (J6/JFH-1), HCVpp harboring HCV E1E2 proteins, or control viral particles (CONTpp) are as specified below. The viral particles were produced in cell culture media devoid of serum lipoproteins and were diluted in serum-free medium to compare viral preparations containing equivalent input of infectious particles, i.e. ~104 infectious units). A, infection assays using HCVcc, HCVpp, and CONTpp harboring the glycoproteins of RD114 in the presence of 6 µg/ml cholesterol-HDL and anti-apoC-I antibody, as indicated. The results show the increases of infection (mean ± S.D.; n = 3) determined by calculating the ratios between the average infectious titers in the presence of the indicated components relative to the average infectious titers in the absence of antibody and/or HDL as specified (dashes). B, HCVpp and CONTpp harboring the glycoproteins of RD114 (RD114pp), VSV (VSV-Gpp), influenza (HApp), and MLV (MLVpp) viruses were preincubated 45 min at room temperature with 1.4 µg/ml apoC-I or apoC-II as indicated. The results show the increases of infection (mean ± S.D.; n = 3) as determined by calculating the ratios between the average infectious titers in the presence of the indicated apolipoprotein relative to the average infectious titers in the absence of apolipoproteins (dashes). C, HCVcc, HCVpp, and CONTpp harboring the RD114 glycoproteins were preincubated 45 min at room temperature with 1.4 µg/ml apoC-I before adding anti-apoC-I antibody for 45 min at the indicated concentrations. The results are expressed as the percentage of neutralization of the infectious titers relative to incubation with medium devoid of antibody.

 
We then sought to unravel the viral determinants and the cellular factors through which HCV infection is enhanced by either lipoprotein-free apoC-I or HDL. HDL mediates HCV infection enhancement via interaction with their common receptor, SR-BI (26, 33, 36). This triple interplay is controlled by the HVR1 region of HCV E2 and, more particularly, via a framework of conserved residues within this region (33, 36). To better put into evidence the differences in enhancement of infection using various HCVpp mutants and reagents modulating stimulation, we standardized the 3-5-fold increase of infection of wild type HCVpp detected with HDL or apoC-I to 100% (first columns of the left and right panels of Fig. 2A, respectively). Note furthermore that to allow a direct comparison of the stimulation observed with HDL and apo-CI, experiments in Fig. 2 were performed with 1.4 µg/ml purified apoC-I which corresponds to the physiological apoC-I concentration in the HDL preparations used here (i.e. 6 µg/ml cholesterol-HDL), which is the optimal amount for maximal infection enhancement (33, 36). Compared with the standardized infection enhancement observed with wild type HCVpp, the infectivity of HCVpp harboring a deletion of HVR1 ({Delta}HVR1-HCVpp) was not or was barely enhanced by HDL as well as apoC-I (Fig. 2A). We then performed infection assays with HCVpp bearing changes in conserved amino acid positions of HVR1 (33) that are thought to be essential for its conformation (57). As compared with parental HCVpp, mutation of some of these conserved residues, i.e. residues Gly-389 and Leu-399, induced gain (G389R) or loss (L399R) of HDL-mediated infection enhancement (Fig. 2A) as reported before (33). Consistently, although HCVpp displaying the G389L point mutant were more intensively stimulated by apoC-I than wild type HCVpp, we found that HCVpp harboring the L399R mutation was not enhanced by apoC-I (Fig. 2A). Altogether, these findings assigned to HVR1 a critical role in modulating infection enhancement by both soluble apoC-I and HDL and pointed to key amino acid residues mediating this mechanism.


Figure 2
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  		FIGURE 2.
Parameters of apoC-I-induced modulation of HCV infectivity. Results of infection assays on Huh-7 cells using HCVpp harboring wild type or mutant E1E2 proteins or control viral particles (CONTpp) harboring the glycoproteins of RD114 as indicated. The viral particles were produced in cell culture media devoid of serum lipoproteins. The input of HCVpp or CONTpp, i.e. ~104 infectious units, was adjusted by dilutions of either viral particle in serum-free medium. A, effect of HDL or apoC-I on infectivity of HCVpp harboring HVR1-deletion ({Delta}HVR1-HCVpp) or G389L,L399R point mutations in HVR1. The results are expressed as the average percentages of the infection enhancements of these mutants relative to that detected for wild type (WT) HCVpp set to 100%, which corresponded to 4.5- and 3-fold increases of infection (see Fig. 1, A and B) when using HDL and apoC-I concentrations of 6 µg/ml (as cholesterol-HDL) and 1.4 µg/ml, respectively (mean ± S.D., n = 3). The infectious titers of parental and mutant HCVpp were within the range of 2 x 104-2 x 105 infectious units/ml. B, effect of HDL or apoC-I (CI) in HCVpp-infected Huh-7 cells pretreated with a 1/100-diluted polyclonal anti-SR-BI mouse serum (anti-SR-BI) or with 50 µM BLT-4 (BLT), an SR-BI lipid transfer inhibitor, or with a SR-BI small interfering RNA (siRNA) vector (down-regulation). The results are expressed relatively to the average percentages of the infection enhancement detected for wild type HCVpp in absence of SR-BI blocking antibody, BLT-4, or SR-BI small interfering RNA set to 100%, which corresponded to 5- and 2.8-fold increases of infection (see Fig. 1, A and B) when using HDL and apoC-I concentrations of 6 µg/ml (as cholesterol-HDL) and 1.4 µg/ml, respectively (mean ± S.D., n = 3). Down-regulation of SR-BI was verified by Western blotting of small interfering RNA vector-treated Huh-7 cells (inset). C, HCVpp were preincubated for 45 min at room temperature with varying concentrations of apoC-I (as µg/ml) as indicated. The results are expressed relatively to the average percentages of the infection enhancement detected for wild type HCVpp at the concentration of 1.4 µg/ml set to 100% (see Fig. 1B), which corresponded to a 3.5-fold increase of infection (mean ± S.D., n = 5).

 
Stimulation of infection induced by apoC-I was detected for all HCVpp-susceptible cell types, including Huh-7, Hep3B, HepG2-CD81, and PLC hepatocarcinoma cells (data not shown). As for HDL, the mechanism by which soluble apoC-I stimulates cell entry may involve SR-BI, a multiligand receptor that mediates lipid transfer from HDL and that is expressed in these different cell types (33). To address this possibility, we blocked SR-BI functions using either SR-BI antibodies (33), BLT-4, a compound that inhibits SR-BI-mediated lipid transfer (48), or via SR-BI down-regulation (16). Although either SR-BI-blocking method abrogated the infection enhancement of HCV particles by HDL, as reported previously (33), they did not inhibit infection enhancement by apoC-I (Fig. 2B). Thus, these results suggested that unlike HDL, infection enhancement by soluble, lipoprotein-free apoC-I does not require an interaction with SR-BI.

As for HDL (33), infection enhancement by lipoprotein-free apoC-I was dose-dependent and reached a maximal level at 0.7-1.4 µg/ml (Fig. 2C). However, the stimulating effect progressively disappeared at higher apoC-I concentrations (Fig. 2C), in contrast to the saturable infection enhancement at high HDL concentrations (33). Furthermore, apoC-I concentrations higher than 7 µg/ml induced a dose-dependent inhibition of infectivity for both HCVpp (Fig. 2C) and HCVcc (data not shown). Such an inhibition was specific to apoC-I and HCV particles since it was neither detected with apoC-II, whatever the concentration was, nor with control viral particles bearing alternative surface glycoproteins (data not shown).

ApoC-I Specifically Binds HCV Particles—Because lipoprotein-free apoC-I does not require SR-BI to modulate HCV infection, we reasoned that it could exert its activity through a direct interaction with the surface of HCV particles. Thus, to study HCV·apoC-I interactions, we performed SPR assays. This was achieved using HCVpp since, for trivial safety reasons, live HCVcc could not be investigated in our BIAcore plate form. ApoC-I was covalently immobilized to the dextran matrix of a CM4 BIAcore sensor chip through activation of its amine groups. We found that upon injection in the BIAcore system, HCVpp bound to immobilized apoC-I, as shown by the increase of SPR signal during HCVpp injection (Fig. 3A). Furthermore, this binding was stable since no decrease of SPR signal was detected after the injection of HCVpp was stopped and during buffer flows on the sensor chip surface (Fig. 3A). Finally, this interaction was specific since no significant binding to CM4-immobilized apoC-I could be detected upon injection of control pseudo-particles bearing the glycoproteins from HIV (Fig. 3A), MLV, influenza virus, or RD114 (data not shown). Note that identical inputs of HCVpp and control pseudo-particles were compared (Fig. 3A, inset).


Figure 3
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  		FIGURE 3.
ApoC-I interacts with HCVpp. Detection of HCVpp·apoC-I interactions by surface plasmon resonance analysis (BIAcore). The viral particles, HCVpp, or control particles (CONTpp), as specified below, were produced in cell culture media devoid of serum lipoproteins and purified through a sucrose-cushion. A, apoC-I was immobilized on the dextran matrix of a CM4 sensor chip. Purified HCVpp or control pseudo-particles displaying the glycoproteins of HIV (CONTpp) as indicated were injected over immobilized apoC-I (pp injection), and the binding of either viral particle type was determined by the number of resonance units (RU). Equivalent input of either viral particle, i.e. ~104 infectious units was checked by the detection of their capsid protein (CA) by Western blotting (inset). The spikes detected at the start and at the end of injection of the viral particles are due to change in buffer and/or to the on-line subtraction of the sensorgram recorded on the control flow cell. B, HCVpp were captured via the AP33 E2-mAb covalently immobilized to the dextran matrix of the CM3 sensor chip, and the binding was determined by the number of resonance units (RU). Purified apoC-I was injected over captured HCVpp at 100 µg/ml. C, injection of apoC-I at varying concentrations and of apoC-II at 100 µg/ml on HCVpp captured with the AP33 mAb, as in panel B. D, HCVpp and control pseudo-particles displaying the glycoproteins of HIV (CONTpp) were captured via the AP33 and 2F5 mAbs, respectively, covalently immobilized to the dextran matrix of the CM3 sensor chip. Equivalent capture levels of HCVpp and CONTpp (data not shown) were obtained by adjusting the input of viral particles. Purified apoC-I was injected over captured HCVpp and CONTpp at 100 µg/ml. E, disruption of viral membrane was determined by the release of an inner component of the viral particles (RT) using an enzymatic assay. HCVpp and CONTpp harboring the glycoproteins of HIV (HIVpp), RD114 (RD114pp), and MLV (MLVpp) were preincubated for 45 min with various concentrations of apoC-I (left panel) or apoC-II (right panel) in the absence of Triton X-100 in the sample dilution buffer. Equivalent input of either virus particle was checked by the detection of their capsid protein by Western blotting. The results show the ratio of the release of RT activity at each concentration of apoC-I or apoC-II relative to the release of RT in the absence of apoC-I and apoC-II. These data are representative of three independent experiments.

 
To confirm apoC-I binding to HCV particles, we preincubated HCVpp or control pseudo-particles with or without 1.4 µg/ml soluble apoC-I or HDL at equivalent apoC-I concentrations; i.e. 6 µg/ml cholesterol-HDL (see apoC-I bands in the input Western blot panel of supplemental Fig. 1A). These samples were then immunoprecipitated using apoC-I antibodies, and the pellets were analyzed by immunoblotting. We could detect the E1 and E2 proteins in samples from HCVpp preincubated with lipoprotein-free apoC-I but not with HDL (supplemental Fig. 1A). In contrast, control particles harboring RD114 glycoproteins preincubated with apoC-I were not immunoprecipitated with apoC-I antibodies, as shown by the lack of detection of RD114 surface glycoproteins, Env, in the pellets (supplemental Fig. 1B). These results indicated that soluble apoC-I, but not HDL-embedded apoC-I, could specifically associate to HCV particles.

Next, to investigate the properties of HCVpp·apoC-I complexes, we purified HCVpp on a sucrose cushion after incubation with soluble apoC-I or HDL. Again, we detected apoC-I on the viral particles preincubated with apoC-I but not with HDL (data not shown), indicating that soluble apoC-I, but not HDL-embedded apoC-I, could stably associate to the HCV particles. These HCV particles retained the apoC-I-mediated infection enhancement after purification (78.8 ± 4.15% of the infection enhancement detected before purification), suggesting a stable and functional HCVpp·apoC-I association. Furthermore, the infectivity of these purified HCV·apoC-I complexes could be neutralized by apoC-I antibodies (Table 1), which suggested a steric hindrance induced via binding of the antibodies onto the viral particles. Similar results were obtained when using HCVcc rather than HCVpp in such neutralization assays of purified HCV·apoC-I complexes (Table 1). Altogether, the results of infection inhibition, BIAcore, and immunoprecipitation analysis indicated that HDL-free apoC-I can associate to the surface of HCV particles and explained the results of HCVcc and HCVpp neutralization by apoC-I antibodies in Fig. 1C.


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  		TABLE 1
Neutralization of HCV.apoC-I complexes

 
ApoC-I Induces the Disruption of HCV Particles at High Concentrations—ApoC-I immobilized on a sensor chip may not have the flexibility necessary to mediate an effect on HCV particles beyond inducing their mere association (Fig. 3A). Thus, to further investigate the characteristics of HCVpp·apoC-I interaction, we performed alternative BIAcore studies by injecting lipoprotein-free apoC-I on captured pseudo-particles. HCVpp were captured via an E2-specific mAb covalently immobilized to a CM3 sensor chip (Fig. 3B). In those conditions, as reported before, HCVpp interacted with E1E2 ligands such as E1E2 antibodies or the large extracellular loop of CD81, CD81-LEL, but not with HDL (26). This indicated that although a part of HCVpp surface is occupied by the CM3-immobilized E2 mAb, a significant portion of the surface of the viral particles remains accessible for interacting with E1E2 partners. No or very poor binding of apoC-I to captured HCVpp could be detected upon its injection at low concentrations in the range of 0.1-3 µg/ml (data not shown). Strikingly, the injection over the captured HCVpp of apoC-I at higher concentrations, above 5 µg/ml, induced a rapid decrease of the SPR signal, suggesting a loss of mass from the chip surface via a partial removal of CM3-bound HCVpp (Fig. 3B). Such an effect was apoC-I dose-dependent (Fig. 3C), leading to up to 50-60% decrease of HCVpp-specific SPR signal (supplemental Fig. 2A) as deduced from the ratio of SPR decrease upon apoC-I injection relative to SPR signal after HCVpp capture (Fig. 3B). HCVpp loss was not detected either when apoC-II (Fig. 3C) or when HDL (26) was injected, even at high concentrations. Furthermore, whatever the concentration, apoC-I did not induce the loss of control particles displaying the surface glycoproteins of HIV (Fig. 3D) or MLV (data not shown) that were captured at levels similar to HCVpp via their respective CM3-immobilized Env antibodies, hence establishing the specificity of this particular HCVpp·apoC-I interaction. Then, to investigate whether the HVR1 determinant of HCV E2 glycoprotein plays a role in the disruption of CM3-bound HCVpp induced by apoC-I, we captured HVR1-deleted HCVpp ({Delta}HVR1-HCVpp) on the CM3 sensor chip, and we analyzed SPR signals upon apoC-I injection. As compared with wild type HCVpp, apoC-I only marginally reduced the SPR signal of the {Delta}HVR1-HCVpp (supplemental Fig. 2B). This confirmed that HVR1 is a critical determinant of HCVpp·apoC-I interplay, which can be visualized in both cell entry (Fig. 2A) and SPR assays.


Figure 4
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  		FIGURE 4.
ApoC-I does not modify cell surface binding of HCVpp. 50 µl of sucrose cushion-purified HCV particles produced in cell culture media devoid of serum lipoproteins were preincubated or not (solid line) with 1.4 µg/ml apoC-I or with 6 µg/ml cholesterol-HDL as indicated for 45 min at room temperature. The viral particles were then incubated for 1 h at 37 °C with Huh-7 (A) or with CHO-derived cells (B) expressing the human CD81 (CHO-CD81) and SR-BI (CHO-SR-BI) molecules. The cell-bound HCVpp were detected with either anti-E1 (A4) or anti-E2 (H53) mouse antibodies as indicated, then with allophycocyanin-conjugated anti-mouse antibodies. The fluorescence was analyzed with a fluorescence-activated cell sorter (FACSCalibur; BD Biosciences). The background fluorescence was provided using pseudo-particles without glycoproteins (A) or using parental CHO cells incubated with HCVpp only (B). The data are representative of three independent experiments. No difference in cell binding of VSV-Gpp control particles could be detected whether HDL or apoC-I was preincubated with such viral particles (data not shown).

 
The loss of SPR signal could be induced by partial release of the HCVpp from the immobilized E2 antibody via a competition of injected apoC-I for the same E2 binding site. However, other experimental setups to capture HCVpp on the CM3 sensor chip via different E2 antibodies that bind alternative epitopes or via G. nivalis lectins that bind E1E2 glycans also resulted in similar losses of HCVpp mass upon apoC-I injection (supplemental Fig. 2A). This indicated that apoC-I induced a disruption of HCVpp via interactions with the accessible portion (non-antibody-bound) of the E1E2 glycoproteins of the captured viral particles. Finally, apoC-I did not induce a modification of the SPR signal when soluble forms of the HCV glycoproteins, rather than whole HCVpp, were captured on the CM3 sensor chip via the same antibodies (supplemental Fig. 2C and data not shown). This indicated that the loss of CM3-bound HCVpp was specifically induced by apoC-I interaction with HCV glycoproteins as displayed in a native conformation on the membrane of viral particles.

That apoC-I induced the disruption of sensor chip-captured HCVpp (Fig. 3, A-D, and supplemental Fig. 2A) but not of captured sE2 (supplemental Fig. 2C) suggested that apoC-I exerts an effect on the HCV particle itself. To directly address this possibility, we incubated purified HCVpp with apoC-I and subsequently assessed the release of one of the inner components of these viral particles, the reverse-transcriptase, using an enzymatic assay that reflects disruption of the viral membrane (55). Little RT release was detected when HCVpp were incubated with apoC-II or when control viral particles were incubated with either apoC-I or apoC-II (Fig. 3E). In contrast, apoC-I could induce the disruption of HCV particle at concentrations above 10 µg/ml (Fig. 3E). Altogether, the loss of SPR signal and the release of RT upon apoC-I treatment of the HCVpp may explain the inhibition of infection at high apoC-I concentrations (Fig. 2C) by membrane disruption.

ApoC-I Stimulates HCV Membrane Fusion—Infection enhancement by low concentrations of apoC-I (Figs. 1B and 2C) may also be explained by less dramatic alterations of the HCV particle. Particularly, upon association with HCV particles, apoC-I may modify their binding properties to cell surface receptors. To address this possibility, we performed binding assays on Huh-7 cells or on CHO cells that individually expressed the CD81 or SR-BI HCV receptors. The binding of purified HCVpp was assessed by flow cytometry using either anti-E2 or anti-E1 antibodies, as described elsewhere (26, 53). No difference in cell binding of HCVpp (Fig. 4) or of control particles (data not shown) was observed whether the viral particles were incubated or not with HDL or apoC-I. Similar results were obtained in binding assays performed with sE2 (data not shown).

Because apoC-I does not modify HCVpp or sE2 binding to HCV receptors, an alternative possibility is that it stimulates the viral particles for membrane fusion processes. Because of its amphipathic {alpha}-helix structure, apoC-I may indeed interact with the viral surface in a manner-dependent on E1E2 presence and may predispose it for fusion with a target membrane via alterations of the viral membrane. To address this possibility, we investigated the membrane fusion process induced by HCVcc or HCVpp by using a recently developed HCVpp/liposome fusion assay (11, 53). This fusion assay is based upon direct measurement of lipid mixing between virion and liposome membranes.

Briefly, the assay relies on the relief of fluorescence self-quenching probes used to measure virus/cell fusion kinetics, i.e. either the pair of N-Rh-PE and N-NBD-PE probes (58), based on phospholipids stably inserted within the target membranes via their two long fatty acid chains or the R18 probe (59). Lipid dilution upon fusion between liposome and viral membranes at low pH leads to fluorescence dequenching of these probes. Thus, the fusion rates can be measured by the initial rates of fluorescence, as shown here for pH-dependent viruses such as HCV and influenza but not for pH-independent viruses such as MLV (Table 2; see Fig. 5A and supplemental Fig. 3, as representative curves for N-Rh-PE/N-NBD-PE and R18 probes, respectively). As reported before, fluorescence dequenching was observed only when the pH was decreased to 5.0, and no significant dequenching could be detected at neutral pH even after long incubation times (11).


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  		TABLE 2
Initial rates of lipid mixing in liposome fusion assays

The initial rates of lipid mixing (% max fluorescence·min–1) at pH 5 were determined as the tangents of the lipid mixing kinetics at t = 0 (see Fig. 5, as representative curves), which were averaged from three separate experiments. ND, not determined.

 
When apoC-I was preincubated with HCVpp (Table 2 and see Fig. 5B, as representative curves) at concentrations around 1 µg/ml, we found that lipid mixing with liposomes was stimulated, as shown by the increased initial rates of the fusion kinetics. The enhancement of fusogenicity remained acid pH-dependent since no fusion could be detected at neutral pH (data not shown), indicating that apoC-I promotes HCV fusogenicity in a specific manner. ApoC-I concentrations higher than 6 µg/ml resulted in inhibition of lipid mixing (Table 2 and Fig. 5B), which reflected the inhibition of infection at the same concentrations (Fig. 2C) and the disruption of HCVpp detected in SPR and RT release assays (Fig. 3). The stimulating effect of apoC-I on membrane fusion was highly specific of apoC-I interaction with HCV envelope since preincubation of apoC-I with control pseudo-particles harboring an influenza virus hemagglutinin (HApp) (Table 2 and Fig. 5C) and preincubation of apoC-II with either HCVpp or HApp (Table 2) did not change the fusion rates. Of note, the results obtained with the N-Rh-PE and N-NBD-PE probes were fully confirmed using the R18 probe (supplemental Fig. 3). Moreover, we also confirmed that low concentrations of apoC-I could also enhance membrane fusion by replicative HCVcc particles (supplemental Fig. 3B).

No effect of apoC-I could be detected on membrane fusion when apoC-I was preincubated with the liposomes rather than with the HCVpp (supplemental Fig. 3E). This suggested that the fusion-enhancing effect of apoC-I requires a prior interaction with the viral particles to promote fusion enhancement, most likely via its interplay with the HCV glycoprotein. Finally, we found that apoC-I could not stimulate liposome fusion of HCVpp harboring a deletion of HVR1 (Table 2 and supplemental Fig. 3F), congruent with the notion that HVR1 controls infection enhancement by apoC-I and HDL (Fig. 2A).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
HCV biology is strikingly linked to lipoproteins, which act at several steps of viral replication, including assembly, cell entry, and protection from the host immune response (26, 32, 33, 37, 60-71). Several groups have purified HCV from plasma to investigate virus/cell interactions in vitro. However, in addition to the inherent difficulties to measure viral infectivity of HCV purified from plasma, the current approaches to study viral particles isolated from patients do not allow the unequivocal molecular analysis of their interactions with serum components. Using novel infection assays that consist of infectious HCV particles produced in vitro in defined medium compositions our results reveal novel features of the interplay between HCV and lipoproteins. Our findings uncover an original mechanism by which HDL stimulates cell entry of HCV at a post-receptor binding stage. We demonstrate here that apoC-I, a protein subcomponent of HDL, is specifically recruited by HCV glycoproteins on the viral surface and intrinsically increases the fusogenicity of HCV particles. ApoC-I can be recruited either as an HDL-bound component through a triple interplay with SR-BI, a receptor for both HDL (29) and HCV (15, 31), or directly as lipoprotein-free protein in our experimental conditions. To our knowledge this is the first description of a host component that is specifically recruited to the surface of an enveloped virus to promote membrane fusion events. Interestingly, high apoC-I concentrations induced the disruption of the HCV particles and loss of infectivity, most likely through alteration of the viral membrane, indicating that a fine-tuning of apoC-I recruitment is required for optimizing infection enhancement.

The flexibility of the HCVpp assay, which facilitates the substitution or the modification of the viral surface glycoproteins, established the high specificity of the interaction between apoC-I and the HCV viral surface and the subsequent effect of this interaction on infectivity. Significantly, the most salient features of our findings could be confirmed with the J6/JFH-1-replicating HCV (HCVcc). Furthermore, we demonstrate that the HVR1 domain of HCV-E2 is a crucial viral component mediating the effect of apoC-I on virions. The importance of this HCV determinant is suggested in vivo by the attenuated phenotype of HVR1-deleted virus in chimpanzees and by abrogation of infectivity by HVR1 antibodies (72-74). The analysis of HVR1 sequences from different HCV strains indicated the involvement of this region during cell entry (57). Furthermore, the presence of basic residues in HVR1 was found to facilitate virus entry (75). Finally, HVR1 also modulates the interaction of HCV-E2 with SR-BI since deletion of HVR1 abrogates both SR-BI binding of soluble E2 (31) and HCV infection enhancement by HDL (33, 36). Our results indicate that the same HVR1-conserved residues are involved in infection enhancement by either HDL or lipoprotein-free apoC-I. This substantiates the notion that HVR1 sequences harbor a conserved function that modulates the effect of apoC-I on infectivity/fusogenicity (Fig. 2A and supplemental Figs. 2B and 3F) and/or its recruitment either directly, when apoC-I is provided at optimal concentrations as a lipoprotein-free protein to HCV particles, or alternatively, from HDL, most likely after HDL/SR-BI interaction, as discussed below.


Figure 5
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  		FIGURE 5.
ApoC-I increases membrane fusion. The fusion capacity of HCVpp harboring wild type E1E2 proteins and control pseudo-particles harboring influenza (HApp) glycoproteins, MLV glycoproteins (MLVpp), or no glycoprotein (noEnvpp) as indicated was tested using lipid mixing assays. 40 µl of purified viral particles produced in cell culture media devoid of serum lipoproteins were added to liposomes labeled with N-Rh-PE and N-NBD-PE lipids in PBS, pH 7.4. After a 2-min equilibration at 37 °C, fusion was initiated by decreasing the pH to 5 in the cuvette (time 0 of the fusion kinetics). No fluorescence dequenching was detected before acidification of the medium. The results are expressed as percentages of maximal fluorescence, obtained by the addition of Triton X-100 (final 0.1% vol:vol) to the particle/liposome suspensions. A, basic profiles of the fusion kinetics obtained with pH-dependent (HCVpp and HApp) and pH-independent (MLVpp) viral particles. Equivalent input of either virus particle was checked by the detection of their capsid protein (CA) by Western blotting (inset). B, apoC-I was preincubated for 45 min at low (0.7 µg/ml) versus high concentration (7 µg/ml) with HCVpp. C, apoC-I was preincubated for 45 min at low (0.7 µg/ml) versus high concentration (7 µg/ml) with HApp.

 
Because apoC-I in vivo is present in HDL mainly and in VLDL, but not as a lipoprotein-free form (40), it could be released from either lipoprotein type, in theory, to allow a subsequent recruitment on HCV particles. As expected, because of its association to LDL/VLDL as "lipo-viro-particles" (LVP) (61, 69, 71), we found that plasma-derived HCV contains apoC-I (data not shown). Although this suggests that in vivo HCV may also recruit apoC-I from VLDL, besides HDL, this possibility remains difficult to test experimentally. Indeed, because of the technical difficulties to unambiguously characterize LVPs in biochemical and functional assays, it is not possible to demonstrate if VLDL-bound apoC-I can be transferred to the viral membrane of LVP and if it regulates membrane fusion. Nevertheless, our data support the notion that apoC-I is most likely recruited from HDL because only the latter lipoprotein and not LDL/VLDL enhance the infection of HCVpp and HCVcc (26, 33, 35, 36, 82) and because HDL-mediated infection enhancement was fully blocked by apoC-I antibodies (Fig. 1A).

HDL itself does not directly interact with virions (26, 33, 36) and does not spontaneously exchange apoC-I (supplemental Fig. 1). We propose that in vivo apoC-I could be recruited on HCV particles after the interaction between HCV, HDL, and SR-BI. Indeed, recent studies indicate that HDL/SR-BI interaction results in consequent changes in the size, density, and lipid composition of HDL (76). Such changes modulate the affinity and stability of the exchangeable apolipoproteins on the lipoprotein and, hence, induce their dissociation, shedding and/or transfer to neighboring lipid surfaces (76-81). Because of the proximity of SR-BI-bound HDL with HCV particles, apoC-I desorbed from HDL could then be captured by the HCV glycoproteins and subsequently transferred to the viral lipid membrane. Such a model is compatible with our observation that the lipid transfer functions of SR-BI are required for HDL-mediated infection enhancement (26, 33, 36) but not for stimulation by lipoprotein-free apoC-I, which was fully maintained when SR-BI functions were inhibited by chemical compounds or blocking antibodies or by its down-regulation (Fig. 2B).

Our results indicate that apoC-I influences the infectivity of HCV particles at a post-binding stage by promoting their membrane fusion properties. On the one hand, apoC-I may modify the HCV glycoproteins conformation, thereby assisting a limiting transition stage in the refolding of the glycoproteins and, consequently, increasing the fusion rate. However, we did not find evidence for modification of the conformation of HCV glycoproteins after interaction of HCV particles with apoC-I (data not shown). Alternatively, our results indicate that apoC-I alters the viral membrane in a dose-dependent manner; it increases membrane fusion at low concentrations (Table 2, Fig. 5, and supplemental Fig. 3) but disrupts the viral particles at high concentrations (Fig. 3 and supplemental Fig. 2). These two effects may reflect the same property of apoC-I to interact with lipid membranes and are both reminiscent of a particular feature of membrane fusion processes.

The fusion between viral and cellular membranes involves a complex multistep conformational change of the viral glycoproteins (20). HCV entry is pH-dependent (10, 11, 15, 17), suggesting that the low pH induces the refolding of the E1E2 glycoprotein complex. The critical domains and the molecular events that mediate HCV membrane fusion remain poorly defined (53). For pH-dependent viruses, such as alphaviruses and flaviviruses, protonation of the ectodomain of the viral glycoproteins in the acidic endosomal environment triggers the first refolding event, which induces the dissociation of the Alphavirus E2-E1 heterodimers or of the Flavivirus E-E homodimers, insertion of the fusion peptide in the target cell membrane, apposition of the viral and cell membranes, and ultimate merging of the outer lipid leaflets (20).

Although the essential components of the fusion apparatus reside on the extra-virion side of the virion membrane (20), additional discrete segments or determinants of the viral glycoproteins modulate the extent of membrane fusion. For example, the transmembrane domains of several class I (83-85) and II (86) fusion proteins are essential for their activity at different stages of the fusion process, most likely during lipid mixing of the viral and cellular membranes. Additional segments located immediately before the transmembrane domains can also contribute in perturbation of the viral outer lipid leaflet and, hence, influence fusion with the cell membrane. Notably, a membrane-proximal tryptophan-rich region of HIV gp41 has been suggested to aid in the disruption of membrane during the gp41-mediated fusion process (87-89). This peptide forms a partially amphipathic helix (89), which by positioning in the outer membrane bilayer of the viral membrane (90), is believed to disrupt the water-phospholipid interface in a manner similar to the proposed mechanism of amphipathic structures present in antimicrobial peptides or exchangeable apolipoproteins (91-97). We propose that the recruitment of apoC-I on HCV particles similarly influences membrane fusion by facilitating the disruption of the outer phospholipid bilayer of the viral envelope. The {alpha}-helical amphipathic structure of apoC-I, its affinity to phospholipid surfaces, and its direct interaction with HCV particles are features shared with the aforementioned peptides. Indeed, as for the other exchangeable apolipoproteins, apoC-I contains a cluster of charged amino acids that interact with the polar head of negatively charged lipids and a cluster of hydrophobic amino acids that are embedded within the lipid aliphatic chains (96, 98-102). Furthermore, the strong interaction of exchangeable apolipoproteins with phospholipid bilayers is accompanied by microsolubilization of artificial as well as cell membranes through the modification of the lipid bilayer properties (93, 96) and, at high peptide/lipid ratios, disruption of the vesicles to form smaller discoidal complexes (103). Thus, binding of apoC-I to the surface of the HCV particle at a limited extent may induce small perturbations or deformations of the outer phospholipid layer, which may expose the hydrophobic interiors of bilayers and contribute to generate attractive forces between membranes that facilitate membrane fusion (Table 2, Fig. 5 and supplemental Fig. 3). At high apoC-I concentrations, however, extended disruption of the viral membrane would release the non lipid-linked viral components, e.g. the nucleo-capsid (Fig. 3E), only leaving patches of lipid-embedded E1E2 glycoproteins bound to the sensor chip (Fig. 3 and supplemental Fig. 2). These results, therefore, indicated the possibility that apoC-I-derived molecules could be used to develop HCV-specific inhibitors.

It is important to highlight the high specificity and concentration-dependent apoC-I effect on HCV particles. Indeed, neither membrane disruption nor fusion increase was detected with alternative viruses or with apoC-II, which has a amphipathic structure closely related to apoC-I. Moreover, infection enhancement versus disruption of HCV particles were induced at well defined concentrations, suggesting that HCV needs a regulatory mechanism to recruit the optimal dose of apoC-I on virions and favor its propagation. Such regulation could be achieved via HDL interaction with SR-BI, as above discussed, and/or via change of HVR1 residues involved in this function. Indeed, it is interesting that some mutations of HVR1-conserved residues resulted in either loss of function or gain of function (Fig. 2A) and that no infection inhibition was detected with HDL, even at saturating concentrations (33). This indicates that HCV interplay with HDL and SR-BI, which involves HVR1 conserved framework, results in release and subsequent recruitment of the appropriate quantity of apoC-I on viral particles. Thus, the unexpected role of a serum protein in promoting fusion enhancement is another remarkable feature of the ability of HCV to highjack blood components to facilitate its replication and highlights the intricate relationship between HCV and lipoproteins.


   FOOTNOTES
 
* This work was supported in part by INSERM, ENS Lyon, CNRS, and UCB Lyon-I and by grants from the Ligue Nationale Contre le Cancer, the European Union (LSHB-CT-2004-005246 "COMPUVAC"), and the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3. Back

1 Supported by a fellowship from the Région Rhône-Alpes. Back

2 These are equal contributions. Back

3 To whom correspondence should be addressed: ENS de Lyon, 46 Allée d'Italie, 69007 Lyon, France. Tel.: 33472728732; Fax: 33472728137; E-mail: flcosset{at}ens-lyon.fr.

4 The abbreviations used are: HCV, hepatitis C virus; SR-BI, scavenger receptor B-I; HDL, high density lipoprotein; VLDL, very low density lipoprotein; HVR1, hypervariable region-1; HA, hemagglutinin; R18, octadecylrhodamine B chloride; HIV, human immunodeficiency virus; mAb, monoclonal antibody; N-Rh-PE, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine; N-NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; SPR, surface plasmon resonance; RT, reverse transcription; MLV, murine leukemia virus; sE2, soluble E2. Back

5 C. Granier, G. Verneg, and F.-L. Cosset, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank V. Lotteau and P. André for providing HCV lipo-viro-particles purified from sera of HCV-infected patients. We thank J. Dubuisson, J. McKeating, A. H. Patel, and H. Greenberg for providing the H52 and H53, 9/27, AP33, and A4 monoclonal antibodies, respectively. We are grateful to T. Wakita and J. Bukh for gift of the JFH-1 and J6/CF isolates, respectively. We thank T. Pietschmann and R. Bartenschlager for providing the pFK-venus-Jc1 molecular clone and the Huh-7-Lunet cells. We are grateful to J. Fresquet and F. Alcaras for excellent technical assistance and to our co-workers and colleagues for encouragement and advice. We thank R. Barbaras and T. Huby for stimulating discussions and review of the manuscript. SPR measurements with the Biacore 3000 and fluorescence analysis with SLM Aminco 8000 were performed in the Protein Production and Analysis facility, IFR 128 Biosciences Lyon-Gerland (France).



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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