HCV envelope glycoproteins in their mature form, as they are present at the surface of the particle, have never been characterized. Because HCVpp contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, are supposed to be in a mature conformation similar to that present on native HCV particles. HCVpp therefore represent the best tool currently available to characterize functional HCV envelope glycoproteins. Here, we characterized HCVpp-associated envelope proteins with conformation-dependent MAbs, some of which had been shown to have neutralizing activity against HCVpp. We showed that these HCV envelope glycoproteins associated with HCVpp formed a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. In contrast to what has been observed for most viral envelope proteins, we did not detect any cleavage by a cellular endoprotease during their transport through the secretory pathway. In addition, HCV envelope glycoproteins were recognized by a large panel of MAbs and were shown to interact with CD81 and to be sensitive to low-pH treatment.
Noncovalent E1E2 heterodimers are associated with HCVpp. From sedimentation analyses, there is currently no evidence that larger oligomers of E1E2 are formed (A. Op De Beeck, unpublished data). Interestingly, some of the MAbs used to characterize the E1E2 complex show neutralizing activity (4
; this work), indicating that they recognize fully functional envelope glycoproteins. Earlier studies of HCV envelope glycoproteins analyzed in transient expression systems have shown that these proteins can form noncovalent E1E2 heterodimers as well as heterogeneous disulfide-linked aggregates (19
). Extensive characterization of the noncovalent heterodimer with conformation-dependent MAbs has strongly suggested that this oligomer is most likely the prebudding form of the functional complex (15
). We now show that the mature HCV glycoproteins form a complex showing some similarities to the prebudding E1E2 heterodimer. Very recently, a MAb produced by immunizing mice with an HCV antigen obtained from a chronically infected plasma has been shown to recognize disulfide-bound complexes that might potentially be formed of E1 and E2 (49
). However, there is no evidence that the HCV envelope glycoproteins recognized by this MAb are associated with infectious particles, because there was no purification of viral particles either to generate the antibody or to analyze its reactivity. There is, therefore, no clear evidence that this MAb recognizes envelope glycoproteins that are functional in HCV entry.
HCV envelope glycoproteins E1 and E2 possess up to 6 and 11 potential glycosylation sites, respectively (27
). It has been confirmed experimentally by site-directed mutagenesis that four of the five putative glycosylation sites of E1 of genotype 1a can be occupied by N-glycans (42
). The glycosylation sites occupied in E2 have not been determined yet. However, expression of HCV glycoprotein E2 followed by deglycosylation indicates that a large number of glycosylation sites are occupied. Analyses of the glycans bound to the intracellular HCV envelope glycoprotein heterodimer have indicated that high-mannose type oligosaccharides are associated with these proteins (15
). A fraction of HCV envelope glycoproteins overexpressed in 293T cells has been shown to accumulate at the plasma membrane, where they are supposed to be incorporated into HCVpp (4
). However, our results suggest that expression of E1 and E2 on the cell surface is likely due to the accumulation of small amounts of E1E2 proteins escaping the ER retention machinery. The envelope glycoproteins associated with HCVpp contain complex type glycans. These proteins have therefore been transported through the secretory pathway, and some of the N-linked glycans associated with them have been modified by Golgi enzymes. This is in agreement with lectin binding analyses of HCV particles isolated from infected patients which suggest that the envelope glycoproteins of HCV might contain complex type glycans (56
). Although the glycans associated with E2 proteins incorporated into HCVpp are modified by Golgi enzymes, most E1 glycans remain high-mannose type glycans. Indeed, endo H treatment experiments suggest that E1 might have a single glycan of the complex type. This conclusion contrasts with the previous observation that a C-terminally truncated form of E1 had all its glycans resistant to endo H treatment after secretion (44
). These conflicting observations suggest that the presence of E2 masks the access of the Golgi enzymes to most E1 glycans. Alternatively, the truncated form of E1 might be more accessible to Golgi enzymes due to its misfolding. It has also been shown previously that different glycoforms of intracellular E1 can be observed (18
). Here, we show that only the fully glycosylated form of E1 is incorporated into HCVpp.
The surface proteins of many enveloped viruses are initially synthesized as inactive precursors, and proteolytic cleavage is often required for maturation and full functional activity. In several virus families, this processing step is carried out by cellular proprotein convertases (32
). In the case of the flaviviruses, the envelope contains two proteins, E and M. The latter is synthesized as a precursor called prM (29
). Newly synthesized E and prM proteins associate to form heterodimers (2
) that are incorporated into immature virions by budding into the ER lumen (38
). The particles are then transported through the secretory pathway, and shortly before release from the cell, they are converted to the active form by cleavage of prM by a cellular furin protease in the trans
-Golgi network (58
). Heterodimeric interactions between prM and E are important for proper folding of E (3
) and probably also for protection of the immature virion against acid inactivation during transport through acidic vesicles (29
). Such a maturation of the envelope glycoprotein complex is not observed for HCV. Whether modification of the glycans associated with HCV envelope proteins is responsible for the maturation of the fusion-competent E1E2 complex remains to be determined.
HCV envelope glycoproteins associated with HCVpp are sensitive to low-pH treatment. This finding is in agreement with the observation that HCVpp cell entry is pH dependent (5
). Conformational changes in viral fusion proteins are necessary for exposure of the fusion peptide, which interacts with the target membrane and thus initiates fusion. Interestingly, low-pH treatment induces dissociation of the E1E2 heterodimer. This is likely necessary to induce homo-oligomerization of the active form of the fusion protein, as shown for alphavirus envelope glycoproteins (61
). There is, however, some controversy about the identity of the HCV fusion protein. It was first proposed that E1 might be a good candidate, because sequence analyses suggest that the ectodomain of E1 might contain a putative fusion peptide (26
). However, potential structural homology with other fusion proteins from the same family, as well as with other type II fusion proteins, suggests that E2 should be the fusion protein (35
). Mutagenesis studies of the putative fusion peptides of the envelope glycoproteins associated with HCVpp, as described for the flavivirus envelope protein E (1
), will be helpful for further characterization of the HCV fusion protein.