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编号:11202109
Basic Residues in Hypervariable Region 1 of Hepati
     CNRS-UPR2511, Institut de Biologie de Lille-Institut Pasteur de Lille, Lille

    Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, INSERM U412, IFR74, Ecole Normale Supérieure de Lyon, Lyon

    INSERM-U635, Hopital Henri Mondor, Université Paris XII, Paris

    UMR5086 CNRS-Université Lyon 1, IFR128 BioSciences Lyon-Gerland, Institut de Biologie et de Chimie des Protéines, 69367 Lyon, France

    ABSTRACT

    The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.

    INTRODUCTION

    Hepatitis C virus (HCV) is a small enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (32). Its genome encodes a single polyprotein precursor of 3,010 amino acid residues. HCV polyprotein is synthesized on endoplasmic reticulum-associated ribosomes and is cleaved co- and posttranslationally by cellular and viral proteases to yield at least 10 mature products (reviewed in reference 32). The two envelope glycoproteins E1 and E2 are released from the polyprotein by signal peptidase cleavages (19). HCV glycoproteins are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (38).

    After their synthesis, HCV glycoproteins E1 and E2 assemble as a noncovalent heterodimer (16). The transmembrane domains of HCV envelope glycoproteins play a major role in the assembly of E1E2 heterodimer (39) as well as its subcellular localization (9, 11). The envelope glycoprotein complex E1E2 is proposed to be essential for HCV entry (3, 30). For a long time, HCV entry studies have remained limited because of the lack of a robust cell culture system to amplify HCV. Recently, infectious pseudotyped particles (HCVpp) that consist of unmodified HCV envelope glycoproteins assembled onto retroviral core particles have successfully been generated, and they are very useful to study HCV entry (3, 17, 30).

    The E1E2 heterodimer is the functional unit present in the envelope of HCVpp (40), and E2 is the subunit involved in interactions with the candidate receptors. The E2 glycoprotein has indeed been shown to interact with the CD81 tetraspanin molecule (45), the scavenger receptor class B type I (SR-BI), a high-density lipoprotein (HDL)-binding molecule (49), the mannose binding lectins DC-SIGN and L-SIGN (27, 34, 46), and glycosaminoglycans (2). Experimental data indicate that at least CD81 and SR-BI play a role in HCVpp entry (3, 5, 30, 31, 58). Studies of E2-CD81 interactions and identification of epitopes recognized by antibodies that inhibit these interactions suggest that the CD81-binding region consists of discrete segments of E2 that are organized within the same domain during E2 folding (8, 23, 24, 41, 57). Besides this putative binding region, the hypervariable region 1 (HVR1) (56), a 27-amino-acid-long segment found at the N terminus of E2, has also been suggested to play a role in cell attachment (44). HVR1 has been proposed to modulate accessibility to either CD81 or SR-BI, since deletion of HVR1 increased binding to CD81 (47) but abrogated binding to SR-BI (49). In addition, HVR1 has also been reported as a target for anti-HCV neutralizing antibodies (22).

    The envelope glycoproteins E1 and E2 of HCV exhibit the highest degree of genetic heterogeneity, especially in HVR1 (56). It evolves rapidly in infected individuals, suggesting that it is under strong immune pressure (reviewed in reference 36). Although an HCV clone lacking HVR1 was shown to be infectious in chimpanzees, this mutant virus was attenuated, suggesting that HVR1 plays a facilitating role in HCV infectivity (25). Despite strong amino acid sequence variability related to strong host pressure towards change, the chemicophysical properties and conformation of HVR1 are highly conserved, and HVR1 is a globally basic stretch, with basic residues located at specific sequence positions (44). Here, we used HCVpp harboring mutated envelope glycoproteins to determine the role of basic residues of HVR1 in HCV entry. Our results show that positively charged residues in HVR1 modulate the entry functions of HCV envelope glycoproteins, indicating that HVR1 is a region involved in interaction with a host molecule involved in HCV entry.

    MATERIALS AND METHODS

    Collection and analyses of HVR1 sequences. Patient M was selected for extensive HVR1 quasispecies analyses in a cohort of 113 patients with chronic hepatitis C enrolled in a clinical trial and treated with 3 million units of alpa interferon 2a (Roferon-A; Roche Laboratories, Basel, Switzerland) subcutaneously three times a week for 6 months (43). This patient, infected with a genotype 1a strain, did not clear HCV RNA and was classified as a nonresponder, since viremia was detectable during treatment and throughout the 6-month follow-up period. Blood samples were taken from this patient at months 0 (prior to therapy), 3 (during therapy), 6 (end of therapy), and 12 (end of follow-up). HVR1 quasispecies sequences were determined and analyzed as previously described (42). A total of 80 HVR1 clones (20 clones per time point) were generated.

    All sequence analyses were made using the Institut de Biologie et Chimie des Proteines (IBCP) euHCVdb database website facilities (http://euhcvdb.ibcp.fr) (13). HVR1 sequences within the 30,000 deposited HCV sequences were searched with the sequence query tool using the "site" keyword "HVR1" in the protein features panel. From this search, 11,050 HVR1 sequences were retrieved, but for most of them, no genotype has been deposited or no provisional genotype could be assigned according to the consensus recommendations of Simmonds and coauthors (50). The genotyped HVR1 sequences were retrieved by selecting the genotypes in the corresponding menu of the sequence query tool. Among the 3,500 retrieved sequences, a final set of 1,489 nonredundant sequences were selected and aligned with the CLUSTAL W program (52). For amino acid HVR1 repertoire construction, the residue types and their respective frequencies at each sequence position were obtained using the "AA repertoire" button in the CLUSTAL W result window.

    Cell culture. Huh-7 human hepatocarcinoma cells (37) and 293T human embryo kidney cells (293tsA1609neo) obtained from the American Type Culture Collection (Manassas, VA) were grown in Dulbecco's modified essential medium (Invitrogen) supplemented with 10% fetal bovine serum.

    Plasmid constructs. The DNA sequences used to construct HVR1 mutants were derived from H strain (genotype 1a) (26). In order to facilitate plasmid constructions containing HVR1 mutants, mutations were introduced in the context of a plasmid expressing E2 with its signal peptide (phCMV-E2) (3). In addition, the ATC codon at amino acid position 411 (position on the polyprotein) was changed into a CTG codon to create a unique PstI restriction site immediately downstream of HVR1. This leads to an Ile-to-Leu change at position 411, a residue which is very frequently observed at this position in natural variants and which does not affect the physicochemical properties of this position. To study the role of basic residues in HVR1, two series of HVR1 mutants were produced. We created a first series of mutants by replacing the HVR1 sequence in E2 of H strain by HVR1 sequences of several quasispecies isolated from a patient infected by a genotype 1a isolate (isolate M) (Fig. 1). The second series of mutants was created by introducing or replacing basic residues, in the HVR1 sequence of H strain, at positions where such residues are naturally observed in isolates of genotype 1a. For all the HVR1 mutants, DNA fragments covering the signal peptide and N terminus of E2 glycoprotein and containing wild-type or mutant sequences were supplied by Genaert (Regensburg, Germany) and were subcloned into the phCMV-E2 plasmid (3). A similar approach was used to introduce a deletion in HVR1 (phCMV-HVR1). For this construct, a deletion removing amino acids 384 to 411 was introduced into phCMV-E2 as described previously (54).

    Antibodies. Monoclonal antibodies (MAbs) A4 (anti-E1) (18), H53 (anti-E2) (11), H47 (12), and R187 (anticapsid of MLV) (ATCC CRL1912) were produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer.

    Production of HCVpp and infection assays. Production of HCVpp and infection assays have been described previously (3, 40). In this work, we used the Gag-Pol murine leukemia virus (MLV) packaging construct, containing the MLV gag and pol genes, and the MLV-Luc plasmid, encoding an MLV-based transfer vector containing a CMV-Luc internal transcriptional unit. In addition, to facilitate the mutagenesis in HVR1, HCV envelope glycoproteins were expressed on two different plasmids, phCMV-E1 and phCMV-E2, as described previously (3). Analyses of the enhancement of HCVpp infectivity in the presence of HDL was performed as described previously (54). To analyze the incorporation of HCV envelope glycoproteins into pseudotyped particles, HCVpp were pelleted by centrifugation through a 30% sucrose cushion and analyzed by Western blotting. Within a given preparation of virions, similar amounts of virion-associated MLV capsid proteins were detected for HCVpp generated with the different mutants. However, as previously observed, important differences in the absolute quantities of virion-associated capsid can be noticed when two independent preparations of HCVpp are compared (29, 31). Thus, to minimize variations due to differences in the quality of preparations, each evaluation experiment was conducted using HCVpp generated concurrently.

    Western blotting. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein preparations were transferred to nitrocellulose membranes (Hybond-ECL; Amersham) by using a Trans-Blot apparatus (Bio-Rad) and revealed with a specific MAb followed by rabbit anti-mouse immunoglobulin conjugated to peroxidase (Dako; dilution, 1/1,000). The proteins of interest were revealed by enhanced chemiluminescence detection (ECL; Amersham) as recommended by the manufacturer.

    Immunoprecipitation. Monolayers of 293T cells grown in 6-well plates were transfected with phCMV plasmids expressing E1 and E2 using a PEI Exgen 500-based protocol (Euromedex). At 16 h posttransfection, cells were washed and incubated in Dulbecco's modified medium without L-methionine and L-cysteine (Invitrogen) for 20 min. Transfected cells were pulse labeled for 15 min with 100 μCi per ml of Promix ([35S]methionine-[35S]cysteine; Amersham) as described previously (18). Cells were washed twice with medium containing a 10-fold excess of methionine and cysteine, followed by a 4-h chase. Cells were lysed with 1 ml of 0.5% Igepal CA-630 in phosphate-buffered saline in the presence of 20 mM iodoacetamide, and immunoprecipitations were carried out as previously described (20). Immunoprecipitates were eluted from protein A-Sepharose beads in 25 μl of 4x nonreducing Laemmli sample buffer by heating for 10 min at 70°C and run on SDS-15% PAGE. After electrophoresis, the gels were treated with sodium salicylate, dried, and exposed at –70°C to preflashed Hyperfilm-MP (Amersham).

    CD81 pull-down assay. Recombinant fusion proteins containing the large extracellular loop (LEL) of human or murine CD81 fused to glutathione S-transferase (GST) were preadsorbed onto glutathione-Sepharose 4B beads according to the manufacturer's recommendations (Pharmacia Biotech). Pull-down experiments were performed as previously described (10). The precipitates were separated by SDS-PAGE (12% polyacrylamide) followed by Western blotting with anti-E1 (A4) or anti-E2 (H47) MAb.

    Heparin-Sepharose binding assay. To analyze the interactions between heparin and HCV envelope glycoproteins, 293T cells expressing E1 and E2 or HCVpp were lysed with 0.5% Igepal CA-630 in TBS (50 mM Tris-HCl, pH 7.5; 150 mM NaCl) and incubated for 2 h at 4°C with heparin-Sepharose beads (Pharmacia Biotech). The beads were washed five times with 0.2% Igepal CA-630 in TBS, resuspended in Laemmli sample buffer, and heated at 70°C for 10 min. Precipitated proteins were separated by SDS-PAGE (12% polyacrylamide) followed by Western blotting with anti-E1 (A4) or anti-E2 (H47) MAb.

    RESULTS

    Analyses of basic residues in natural variants of HVR1. We reported previously from the analysis of 1,382 HCV sequences that HVR1 is intrinsically a basic segment, with the basic residues located at specific positions (44). The present availability in HCV databases of nearly 11,050 HVR1 sequences, including 1,489 nonredundant sequences among the 3,841 sequences of known genotypes, allowed us to update and to tailor this analysis to each of the main genotypes. Whatever the genotype, about 40% of nonredundant HVR1 sequences contained 3 basic residues (H, K, and R) and 90% contained 3 ± 1 basic residues. In contrast, 93% of the sequences exhibited no or only one acidic residue, generally at HVR1 position 1. Figure 1A shows that most basic residues are observed at positions 3, 11, 14, 25, and 27. Besides these main positions, basic residues could also be observed at positions 1, 8, 15, 18, 21, and 22. However, a detailed analysis revealed that the frequency of basic residues at each position is genotype dependent. For instance, basic residues are less frequent at positions 14 and 27 in genotype 1a (Fig. 1B). Basic residues are also less frequent at position 25 in genotype 1b (Fig. 1C). This genotype-dependent variability is clearly highlighted when examining the most frequent basic patterns observed in HVR1 sequences (Table 1). The most frequent basic pattern (pattern a) is largely observed in any genotype except genotype 1b. In contrast, pattern c is most frequently observed in genotypes 1b, 2, and 3 but not in genotypes 1a and 4. In addition, some patterns appeared to be specific for only one genotype (e.g., f in genotype 3, g in genotype 4, i in genotype 2, and j and k in genotype 3).

    The nature of basic residues observed at the various HVR1 positions is also reported in Fig. 1. Globally, the three residues H, K, and R were observed in various proportions at any position except position 3, where K was never observed. However, some genotype specificities were observed; for instance, no H residue was observed at position 25 in genotype 1a or at position 27 in genotype 1b. Therefore, it seems that, besides the genotype-dependent specificity of basic patterns, there is likely an additional relative specificity in the presence of a specific basic residue at a specific position.

    Natural variants of HVR1 modulate HCVpp infectivity. As a first step to study the role of basic residues of HVR1 in HCVpp entry, we analyzed the quasispecies isolated from HCV-infected patients. The objective was to select natural variants of HVR1 showing variations in the numbers and positions of basic residues but with limited variability at other HVR1 positions. To facilitate our functional studies, we cloned HVR1 sequences into the backbone of E2 protein of H strain. Indeed, the envelope proteins of this genotype 1a strain are well characterized, and a large panel of MAbs is available to analyze them (40). Since we cannot exclude interactions between HVR1 and the core of E2 glycoprotein, we focused our analyses on genotype 1a isolates. One can indeed expect a good conservation of intramolecular interactions within the same subtype.

    We focused our functional studies on HVR1 sequences of several quasispecies isolated from a selected patient infected by a genotype 1a isolate (denoted M; Table 2). This isolate was selected because its HVR1 consensus sequence was rather close to that of the H strain (48% identity and 85% similarity), and it exhibited numerous variants (26 different sequences over 80 clones analyzed; see Materials and Methods). Based on the number of basic residues, we selected class 1 to 5 sequences among the nine classes of variants identified for the M isolate. To facilitate our functional studies, we cloned the HVR1 sequences of the M isolate into the backbone of E2 protein of H strain. Because HCV envelope glycoproteins can be expressed in trans without affecting their entry functions (3), and to facilitate plasmid constructions HVR1 cassettes were introduced in the context of a plasmid expressing E2 with its signal peptide. Infectivity of the variants was compared to HCVpp generated with wild-type envelope glycoproteins. Particles produced in the absence of envelope proteins were used as a negative control of infectivity.

    As shown in Fig. 2A, compared to HCVpp produced with wild-type proteins, a strong reduction in HCVpp infectivity was observed for all the M variants. Indeed, infectivity of the M variants was reduced by at least 60%. The differences of infectivity were not due to variation in the incorporation of HCV envelope glycoproteins into HCVpp. Indeed, as shown in Fig. 2B, similar amounts of HCV envelope glycoproteins were incorporated into HCVpp. The level of infectivity displayed by the various HVR1 variants appeared to be globally related to the percentage of sequence similarities with HVR1 of the H strain (Table 2). Typically, M1, M2, and M3, which exhibit 81 to 85% sequence similarities with the H strain, showed the highest infectivities (39, 40, and 29%, respectively). In contrast, M4 and M5 exhibiting 78% sequence similarities with the H strain had infectivities reduced to 24 and 9%, respectively. In other words, the higher the sequence similarities to the H strain, the higher the infectivity was. Hence, although HVR1 sequences from the M isolate are from the same genotype as the H strain, this result suggests that the reduction in infectivity is at least partially due to alteration in interactions between HVR1 sequences of the M isolate and the backbone of E2 glycoprotein, which is from another strain. It has also to be noted that the positions of the basic residues are different in the M isolate compared to the H strain. Indeed, in the H strain, basic residues are observed at major positions (3, 11, and 25), whereas in the M isolate, except for position 27, basic residues occupy minor positions (Table 2). One therefore cannot exclude that the absence of basic residues at some major positions might also be responsible for the decrease in HCVpp infectivity observed in Fig. 2A. Interestingly, when we compared the infectivity of the M variants with their content or position of basic residues, differences in the levels of infectivity were observed (Fig. 2A). The strongest reduction was observed for M5, which exhibited only 9% infectivity, while its sequence is close to that of M4, which exhibited 24% infectivity. It is worth noting that a basic residue is present at position 10 in the M5 variant, and among the 1,489 genotyped HVR1 sequences analyzed, this position was never occupied by a basic residue. The presence of this basic residue might therefore potentially reduce HCVpp infectivity. However, other mutations are also present in HVR1 of M5 compared to M4, and we cannot exclude a modulating effect of these residues on HCVpp infectivity. The presence of a basic residue at the minor position 8 in M4 and M5 variants might also potentially affect HCVpp infectivity compared to M1, M2, and M3 variants. The M1 and M2 variants had identical sequences except for the presence of an additional basic residue at position 18 in M2. However, the presence of a basic residue at this minor position had no effect on HCVpp infectivity (compare M2 to M1 sequences, 40 and 39% infectivity, respectively). In contrast, a basic residue at minor position 11 had a decreasing effect on infectivity (compare M3 to M1, 29 and 39% infectivity, respectively). Although no clear conclusion can be drawn on the role of basic residues, these data clearly indicate that some amino acid changes in HVR1 sequence within the quasispecies of a patient modulate HCVpp infectivity. This finding is in keeping with a role of HVR1 in HCV entry.

    Charged residues in HVR1 modulate HCVpp infectivity. To investigate the effect of HVR1 basic residues on HCVpp infectivity, we chose to directly modify HVR1 sequence of strain H by site-directed mutagenesis. This strain has one of the most frequent patterns of basic residues in genotype 1a (16.6%, pattern b; Table 1) with positions 3, 11, and 25 occupied by basic residues. Inaddition, the basic residues occupying these positions are also among the most frequently observed (i.e., H in position 3s and 11 and K in position 25; Fig. 1B). We therefore chose to remove or add basic residues, either at major or minor positions (Table 3). For some mutations, we have also changed the type of amino acid at canonical positions.

    Basic positions identified by sequence analysis of 469 nonredundant sequences of genotype 1a in addition to the 36 nonredundant sequences of patient M were selected for mutagenesis (Table 3). For each group, mutants were constructed and cloned into the phCMVE2 plasmid. For some mutants basic residues have been replaced by a Tyr, a Gln, and an Asn residue at positions 3, 11, and 25, respectively. These residues have been chosen because they are frequently observed at these positions, and they are therefore unlikely to affect the structure of HVR1. Although the absence of basic residues in HVR1 was seldom observed (Table 3), a mutant containing no basic residue in this region was constructed to analyze its infectivity in the context of HCVpp. A mutant E2 protein with a deletion of HVR1 was also used to analyze the level of HCVpp infectivity in the absence of HVR1.

    In the absence of basic residues in HVR1, the infectivity of HCVpp was reduced to 28% (Fig. 3A), and a similar level of infectivity was observed with HCVpp containing an E2 protein deleted of HVR1. These data confirm the role of HVR1 in HCV entry and suggest that basic residues within this region might play a role in this function. Interestingly, mutations that increased the number of basic residues in HVR1 had a tendency to increase HCVpp infectivity. Indeed, mutants containing 0 (0B), 1 (1B), 2 (2Ba and 2Bc), 3 (3Ba), or 4 (4Ba and 4Bc) basic residues had an infectivity of 28% (0B), 48% (1B), 60% (2Ba), 82% (2Bc), 92% (3Ba), 109% (4Ba), and 141% (4Bc) (Fig. 3A).

    Importantly, the differences of infectivity observed between the mutants were not due to variations in the incorporation of HCV envelope glycoproteins into HCVpp. Indeed, as shown in Fig. 3B, similar amounts of HCV envelope glycoproteins were incorporated into HCVpp. Surprisingly, the intensity of the Gag precursor was always lower in the absence of HCV glycoproteins (Fig. 3B). One explanation for this observation is that assembly of HCVpp and pseudotyped particles that contain no envelope protein might take place at different subcellular compartments, which might in turn affect Gag maturation.

    We also analyzed the effect of the mutations on the formation of noncovalent E1E2 heterodimer by immunoprecipitation with a conformation-sensitive E2-specific MAb (H53) that has been shown to specifically precipitate the native E1E2 complex (21). No alteration in the assembly of E1E2 heterodimer was observed (data not shown), indicating that mutations in HVR1 did not alter E1-E2 interactions.

    Although increasing the number of basic residues in HVR1 had a tendency to improve HCVpp infectivity, the positions of these charged residues also had a modulating effect on infectivity. Indeed, differences in the levels of infectivity were observed among the mutants containing 2 or 3 basic residues (Fig. 3A and Table 3). For instance, mutants 2Ba, 2Bb, 2Bc, and 2Bd had an infectivity of 60%, 54%, 82%, and 65%, respectively. Mutants 4Ba, 4Bb, and 4Bc had an infectivity of 109%, 65%, and 141%, respectively. These data indicate that an additional basic residue at position 21 and to a lesser extent at position 22 has a positive effect on HCVpp infectivity compared to the wild-type HVR1 (Fig. 3A and Table 3). On the contrary, the presence of a Lys at position 1 of HVR1 had a negative effect on HCVpp infectivity. Indeed, as shown for mutant 4Bb, it decreased HCVpp infectivity by 35% compared to wild-type E2, while the only difference is the Glu-to-Lys change at position 1 of HVR1 in 4Bb. However, the level of incorporation of HCV envelope glycoproteins into HCVpp was slightly lower for this mutant, and we cannot exclude that this might slightly affect HCVpp infectivity. Interestingly, a shift of an Arg residue from position 22 to position 21 in mutants containing 4 basic residues improved HCVpp infectivity at a higher level than wild-type HVR1 (Fig. 3A, compare mutants 4Ba and 4Bc). Similarly, the presence of an Arg residue at position 15, 18, or 21 of HVR1 modulated HCVpp infectivity in mutants containing 2 basic residues (Fig. 3A, compare mutants 2Bb, 2Bc, and 2Bd). Although the presence of an Arg residue at position 21 of HVR1 improved HCVpp infectivity in the context of 4 basic residues, it had a limited effect in the context of 2 basic residues (Fig. 3A, compare mutants 2Bd and 4Bc). These data suggest that modulation of HCVpp infectivity by changing the position of a basic residue depends on the global context of the basic motif.

    Finally, as H, K, or R residues could be observed at most basic positions (see above), we investigated, for a given position, how the type of basic residue itself can also modulate HCVpp infectivity. Basic amino acids were therefore changed into another basic residue in the context of a motif containing 3 basic residues (Table 3, compare strains H, 3Ba, and 3Bb). The mutations were again chosen on the basis of H, K, and R motifs observed in natural variants. Although we did not construct all the potential mutants, we observed that changing the HHK motif (wild type) into a RRK motif (3Bb) reduced HCVpp infectivity to 69%, whereas its change into an HRR motif (3Ba) did not affect HCVpp infectivity (Fig. 3). These results indicate that replacing the basic residues with other positively charged residues can affect HCVpp infectivity, suggesting a fine tuning in the selection of the basic residue at each position. Interestingly, the less frequently observed motif RRK (7.7%, 3Bd; Table 3) is the less infectious variant, suggesting that the most frequently observed motifs in quasispecies are better adapted for infectivity.

    Altogether, these data indicate that (i) basic residues in HVR1 affect HCVpp infectivity, (ii) HCVpp infectivity globally increases with the number of basic residues in HVR1, and (iii) the positions of some charged residues modulate the level of HCVpp infectivity.

    Basic residues within HVR1 do not affect the binding of E2 to heparin. It has been suggested that the presence of positively charged residues within HVR1 might affect the interaction of E2 with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). Interestingly, recent data indicate that E2 interacts with heparin and liver-derived highly sulfated heparan sulfate and that HVR1 might be involved in this interaction (2, 6). We therefore analyzed the effect of our mutants on E2 binding to heparin. Heparin is not a constituent of cell membranes but is a close structural homologue of highly sulfated heparan sulfate, which is expressed in various forms on the surface of defined cells and extracellular matrices, including hepatocytes (7). After treatment with a nonionic detergent, cell- and HCVpp-associated envelope glycoproteins were incubated with heparin-Sepharose beads and the interaction between E2 and heparin was revealed by pull-down followed by Western blotting with an anti-E2 MAb.

    As previously shown for a truncated form of E2 (2), the intracellular form of wild-type E2 interacted with heparin in our assay (Fig. 4). Some differences were observed between mutant M and wild-type E2 (Fig. 4) for the binding of E2 on heparin. However, changing the number of basic residues within the HVR1 region of E2 of H strain did not affect the binding of E2 to heparin (data not shown), suggesting that HVR1 is not directly involved in this interaction. In addition, in the context of HCVpp, no interaction was observed between E2 and heparin, suggesting that the heparin-binding domain of E2 is not accessible in the mature form of E2 associated with HCVpp. Similar results were observed for the M mutants as well as for the mutants of HVR1 basic motifs (Fig. 4 and data not shown). Together, these data suggest that molecules other than heparin-like proteoglycans are involved in interactions with the basic residues of HVR1.

    Basic residues in HVR1 do not affect the binding of E2 to CD81. HVR1 has been proposed to modulate accessibility to CD81, since deletion of HVR1 has been shown to increase E2 binding to CD81 (47). We therefore analyzed the effect of our mutants on E2 binding to CD81 in a pull-down assay. After treatment with a nonionic detergent, HCVpp-associated envelope glycoproteins were incubated with GST-CD81-LEL and interaction between E2 and CD81 was revealed by pull-down with glutathione-Sepharose beads followed by Western blotting with an anti-E2 MAb.

    As previously shown (40), wild-type E2 associated with HCVpp interacted with human CD81-LEL but not with the murine molecule (Fig. 5). However, contrary to what has been previously reported (47), deletion of HVR1 reduced the recognition of E2 by CD81-LEL. Indeed, in comparison to wild-type E2, human CD81-LEL brought down only 15% of E2 deleted of HVR1 (Fig. 5). These discrepancies are likely due to differences in the forms of E2 used to analyze the interactions with CD81. Indeed, in their study, Roccasecca and coworkers (47) used a truncated form of E2, whereas in our case E1E2 complexes were used, and recent data indicate that analyzing E2-CD81 interactions by using a truncated form of E2 does not necessarily reflect the reality of protein-protein interaction (10) or the effect on infectivity (58). Our data suggest that some residues within HVR1 might be involved in binding to CD81 in the context of E1E2. Alternatively, the presence of E1 might alter the recognition of the CD81 binding domain when HVR1 is deleted. Alteration in CD81-LEL recognition of the mutant lacking HVR1 was not due to the lack of basic residues, because CD81-LEL recognized very well an E2 glycoprotein that does not contain basic residues in HVR1 (Fig. 5, compare WT and 0B). In addition, all the other mutants were well recognized by CD81-LEL (Fig. 5). Together, these data indicate that there is no correlation between E2-CD81 interactions and modulation of HCVpp infectivity by changing the content of basic residues in HVR1.

    Effect of HVR1 basic residues on HDL enhancement of infectivity. Deletion of HVR1 has been shown to abrogate E2 binding to SR-BI (49), and HVR1 deletion mutants are no longer subject to neutralization with anti-SRBI sera, indicating that HVR1 is directly or indirectly involved in interactions with SR-BI. The basic residues in HVR1 might therefore play a role in the interaction between E2 and SR-BI. In the absence of a reliable assay to measure the interactions between HCV envelope glycoproteins and SR-BI in native conditions, an alternative approach is to measure the effect of the natural ligand of SR-BI, the high-density lipoproteins (HDL). Indeed, it has recently been shown that HDL markedly enhance HCVpp entry in an SR-BI-dependent manner (4, 54). We therefore analyzed whether mutations of charged residues within HVR1 might affect HDL-mediated enhancement of HCVpp infectivity.

    As previously shown (54), deletion of HVR1 led to an alteration in HDL enhancement of infectivity (Fig. 6, compare the wild type and HVR). In the presence of HDL, most mutants showed an approximately twofold increase of infectivity (Fig. 6). However, a reduction in HDL facilitation of infectivity was observed for the mutant that contained no basic residue (0B) as well as some other mutants (3Bb, 4Ba, and 4Bb) (Fig. 6). Compared to the 223% increase of infectivity for wild-type HCVpp in the presence of HDL, the enhancement of infectivity was 153%, 136%, 147%, and 155% for mutants 0B, 3Bb, 4Ba, and 4Bb, respectively. Since these mutants contain 0, 3, or 4 basic residues, no correlation can be established between the number of basic residues and the HDL enhancement effect. If one compares the infectivities within the groups of mutants containing 3 or 4 basic residues, it seems, however, that the position as well as the specific basic residue occupying the position can modulate the HDL enhancement effect (Fig. 6, compare 3Ba and 3Bb as well as 4Ba, 4Bb, and 4Bc). Together, these data indicate that there is no obvious correlation between the number of basic residues in HVR1 and HDL enhancement of infectivity. However, positions of some basic residues can modulate the HDL enhancement effect.

    DISCUSSION

    HVR1 has been proposed to modulate HCV entry. Indeed, deletion of HVR1 has been shown to abrogate E2 binding to SR-BI (49) and to reduce HCVpp infectivity in cell culture (5) and HCV infectivity in chimpanzee (25). In addition, HDL enhancement of HCVpp infectivity is suppressed when HVR1 is deleted or mutated at specific positions (4, 54). Interestingly, despite the high degree of variability, the conformation of HVR1 is highly conserved, and this sequence forms a globally basic stretch, with basic residues located at specific sequence positions, suggesting that the presence of positively charged residues within HVR1 might affect the interaction of E2 with molecules involved in HCV entry (44). Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position as well as the basic patterns are genotype dependent. In addition, our data with HCVpp containing mutations in HVR1 sequence indicate that basic residues within this region play a role in HCV entry. Indeed, in the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. In addition, HCVpp infectivity increased with the number of basic residues in HVR1, and the presence or absence of basic residues at specific positions modulated HCVpp infectivity.

    The role of basic residues of HVR1 in HCV entry indicates that this region is involved in interactions with the host molecule(s) involved in HCV entry. Several candidate receptors for HCV have recently been proposed. They include the low-density lipoprotein (LDL) receptor (1, 35), CD81 (45), SR-BI (49), the mannose binding lectins DC-SIGN and L-SIGN (27, 34, 46), glycosaminoglycans (2, 28), and the asialoglycoprotein receptor (48). Recently, HCVpp have been used to investigate the role of most of these candidate receptors. Experimental data indicate that CD81 and SR-BI play a role in HCVpp entry (3, 5, 30, 31, 58), whereas there is no evidence for a direct involvement of DC-SIGN, L-SIGN, and the LDL receptor in HCVpp entry (3, 14, 30, 33).

    Our analyses indicate that changing the content of basic residues in HVR1 does not affect the recognition of CD81, indicating that basic residues in HVR1 are not involved in E2-CD81 interactions. However, deletion of HVR1 reduced the recognition of E2 by CD81-LEL, suggesting that some residues within HVR1 might be involved in binding to CD81 in the context of E1E2. Alternatively, the presence of E1 might alter the recognition of the CD81 binding domain when HVR1 is deleted. Interestingly, there was no correlation between reduction in E2-CD81 interaction and HCVpp infectivity. Indeed, the mutant lacking HVR1 was as infectious as the mutant containing no basic residues, despite differences in E2-CD81 interactions. Similar discrepancies have recently been reported with mutants of CD81 (58).

    Due to the role of SR-BI in HDL-mediated enhancement of HCVpp entry (4, 54), we analyzed whether mutations of charged residues within HVR1 might affect HDL-mediated enhancement of HCVpp infectivity. Our data indicate that there is no correlation between the number of basic residues in HVR1 and HDL enhancement of infectivity, indicating that there is no simple relationship between HVR1, SR-BI, and HDL in HCV entry. Since HDL facilitate HCVpp entry through SR-BI, one would have expected that an increase in HDL enhancement of infectivity would correlate with improvement of HCVpp entry by increasing binding to SR-BI. Our assumption was based on the hypothesis that SR-BI interacts directly with HVR1, because deletion of HVR1 alters E2 binding to SR-BI (49). However, we cannot exclude that deletion of HVR1 indirectly affects the SR-BI binding domain. If this is the case, basic residues in HVR1 might therefore not be involved in interaction with SR-BI but instead with another cellular molecule involved in HCV entry, and this interaction would be affected by mutation of the charged residues. Interestingly, all the cells permissive to HCVpp coexpress CD81 and SR-BI and are of liver origin (5). However, there are some other cell lines coexpressing CD81 and SR-BI that are nonpermissive to HCVpp infection and which are of nonhepatic origin (5, 30). These results suggest that an additional molecule(s), expressed in hepatic cells only, is necessary for HCV entry. Whether this unidentified molecule interacts with HVR1 will need to be investigated.

    Based on a study of interactions between HCV envelope glycoprotein E2 and heparin, it has been postulated that highly sulfated heparan sulfate may serve as the initial docking site for HCV attachment (2). In addition, HVR1 was proposed to play a major role in this interaction. Due to the existence of other candidate receptors, the authors of this study have also proposed that, after binding to heparan sulfate, HCV may be transferred to a second high-affinity receptor, triggering entry. GAG chains on cell surface proteoglycans indeed provide primary docking sites for the binding of various viruses to host cells (53). Although, as described by Barth and coworkers (2), we observed an interaction between the intracellular form of E2 and heparin, E2 glycoprotein associated with HCVpp was not recognized by heparin. Similar results were obtained when wild-type E1 and E2 were coexpressed from the same polyprotein (data not shown), indicating that the lack of interaction with heparin in the context of E2 associated with pseudotyped particles is not an artifact of the coexpression of E1 and E2 in trans. These data suggest that the heparin-binding domain of E2 is not accessible in the mature form of E2 associated with HCVpp. Since HCV envelope glycoproteins associated with HCVpp are modified by Golgi enzymes (40), one can speculate that some modifications of N-linked glycans might induce a masking of the heparin-binding domain. Alternatively, only a fraction of E2 interacts with heparin, and, potentially due to misfolding, this fraction is retained within the endoplasmic reticulum and is therefore not incorporated into HCVpp. Our data suggest that, at least in the context of HCVpp, heparin-like proteoglycans are not involved in HCV entry.

    Although we clearly show the involvement of HVR1 basic residues for HCVpp infectivity, their precise role remains elusive, especially in the absence of reliable three-dimensional structure models for HCV E2 and E1 glycoproteins. Indeed, the genotype-dependent basic patterns and frequency of basic residues at specific positions is possibly associated with specific interactions between HVR1 and the rest of E2 protein and/or with E1. Alternatively, HVR1 might potentially be involved in the binding of undetermined (co)receptors exhibiting some genotype-dependent specificity. For some viruses, the initial steps of entry can be very complex and involve the successive use of multiple attachment factors and receptors (51). In the case of HCV, several putative receptors have been proposed (55), suggesting that HCV entry might also potentially involve successive interactions. CD81 has been proposed as a coreceptor (15), suggesting that initial contact with another cellular molecule might be necessary for HCV entry. It is tempting to speculate that interaction with SR-BI precedes the contact with CD81; however, this remains to be determined. In addition, an as-yet unidentified molecule seems to be necessary for HCV entry (5, 30). Due to its potential accessibility at the surface of the particle, HVR1 likely plays a major role in the initial contact between HCV particle and the cell surface by potentially interacting with SR-BI or an as-yet unidentified molecule. Further studies will be necessary to decipher the coordinated interactions between regions of HCV envelope glycoproteins and cellular molecules involved in HCV entry.

    In conclusion, the modulation of infectivity by the presence or absence of basic residues at specific positions in HVR1 likely reflects the fitness of HCV quasispecies to both overcome the host immune pressures and allow efficient infection of target cells.

    ACKNOWLEDGMENTS

    We thank André Pillez and Sophana Ung for their technical assistance. We are grateful to M. Andrawiss for providing us with reagents.

    This work was supported by EU grant QLRT-2000-01120 and QLRT-2001-01329 and grants from the "Agence Nationale de Recherche sur le Sida et les Hépatites virales" (ANRS), INSERM "ATC-Hépatite C," and the ARC. J.D. is an international scholar of the Howard Hughes Medical Institute.

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