Recognition of native hepatitis C virus E1E2 heterodimers by a human monoclonal antibody

The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.     

Role of disulfide bond formation in the folding and assembly of the envelope glycoproteins of a pestivirus

Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, closely related to, and used as a surrogate model for the hepatitis C virus. Its envelope contains the E1 and E2 glycoproteins, disulfide linked into homo- and heterodimers. In this study, we investigate the role of disulfide bond formation in the folding, assembly, and stability of BVDV glycoproteins. We provide molecular evidence that intact disulfide bonds are critical for the acquirement of a stable conformation of E2 monomers. Forcing the E2 glycoproteins to adopt a reduced conformation either co- or post-translationally before assembly into dimers, determines their misfolding and degradation by proteasome. In contrast, dimerization of E2 glycoproteins results in a conformation resistant to reducing agents and degradation. Furthermore, inhibition of the ER-alpha-mannosidase activity leads to impairment of misfolded E2 degradation, demonstrating the involvement of this enzyme in targeting viral proteins towards proteasomal degradation.     

Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.     

Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) are released from the polyprotein by signal peptidase-mediated cleavage and interact to form a heterodimer. Since properly folded subunits are usually required for specific recognition and stable oligomer formation, the rate of stable E1E2 complex formation, which is low, may be limited by the rate of HCV E1 and/or E2 folding. In this study, the folding of the HCV E1 and E2 glycoproteins was monitored by observing the kinetics of intramolecular disulfide bond formation. The association/dissociation of E1 and E2 with calnexin was also examined, since this molecular chaperone appears to play a major role in quality control via retention of incompletely folded or misfolded proteins in the endoplasmic reticulum. Our results indicate that the disulfide-dependent folding of E2 occurs rapidly and appears to be complete upon cleavage of the precursor E2-NS2. In contrast, folding of E1 is slow (> 1 h), suggesting that this step may be rate limiting for E1E2 oligomerization. Both HCV glycoproteins associated rapidly with calnexin, but dissociation was slow, consistent with the slow folding and assembly of E1E2 glycoprotein complexes. These results suggest a role for prolonged association with calnexin in the folding and assembly of HCV glycoprotein heterodimer complexes.     

Role of N-glycan trimming in the folding and secretion of the pestivirus protein E(rns)

N-glycosylation inhibitors have antiviral effect against bovine viral diarrhea virus. This effect is associated with inhibition of the productive folding pathway of E1 and E2 envelope glycoproteins. E(rns) is the third pestivirus envelope protein, essential for virus infectivity. The protein is heavily glycosylated, its N-linked glycans counting for half of the apparent molecular weight. In this report we address the importance of N-glycan trimming in the biosynthesis, folding, and intracellular trafficking of E(rns). We show that E(rns) folding is not assisted by calnexin and calreticulin; however, the protein strongly interacts with BiP. Consistently, the N-glycan trimming is not a prerequisite for either the acquirement of the E(rns) native conformation, as it retains the RNase enzymatic activity in the presence of alpha-glucosidase inhibitors, or for dimerization. However, E(rns) secretion into the medium is severely impaired suggesting a role for N-glycosylation in the transport of the glycoprotein through the secretory pathway.     

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.     

Folding of hepatitis C virus E1 glycoprotein in a cell-free system

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.     

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.     

CD81-dependent binding of hepatitis C virus E1E2 heterodimers

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.     

Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum

Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.     

Interactions between PE2, E1, and 6K required for assembly of alphaviruses studied with chimeric viruses.

During the assembly of alphaviruses, a preassembled nucleocapsid buds through the cell plasma membrane to acquire an envelope containing two virally encoded glycoproteins, E2 and E1. Using two chimeric viruses, we have studied interactions between E1, E2, and a viral peptide called 6K, which are required for budding. A chimeric Sindbis virus (SIN) in which the 6K gene had been replaced with that from Ross River virus (RR) produced wild-type levels of nucleocapsids and abundant PE2/E1 heterodimers that were processed and transported to the cell surface. However, only about 10% as much chimeric virus as wild-type virus was assembled, demonstrating that there is a sequence-specific interaction between 6K and the glycoproteins required for efficient virus assembly. In addition, the conformation of E1 in the E2/E1 heterodimer on the cell surface was different for the chimeric virus from that for the wild type, suggesting that one function of 6K is to promote proper folding of E1 in the heterodimer. A second chimeric SIN, in which both the 6K and E1 genes, as well as the 3' nontranslated region, were replaced with the corresponding regions of RR also resulted in the production of large numbers of intracellular nucleocapsids and of PE2/E1 heterodimers that were cleaved and transported to the cell surface. Budding of this chimera was severely impaired, however, and the yield of the chimera was only approximately 10(-7) of the SIN yield in a parallel infection. The conformation of the SIN E2/RR E1 heterodimer on the cell surface was different from that of the SIN E2/SIN E1 heterodimer, and no interaction between viral glycoproteins and nucleocapsids at the cell plasma membrane could be detected in the electron microscope. We suggest that proper folding of the E2/E1 heterodimer must occur before the E2 tail is positioned properly in the cytoplasm for budding and before heterodimer trimerization can occur to drive virus budding.     

Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.

Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.     

Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin.

Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N-linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP-glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP-glucose enhanced the amount of folded transferrin and reduced the disulfide-linked aggregates. Analysis of transferrin folding in briefly heat-treated microsomes revealed that UDP-glucose was also effective in elimination of heat-induced misfolding. Incubation of the microsomes with an alpha-glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.     

Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.

Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.     

Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

E1, along with E(rns) and E2, is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini, and E(rns) loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1 mediated by disulfide bridges between cysteine residues. The E1 protein of CSFV strain Brescia contains six cysteine residues at positions 5, 20, 24, 94, 123, and 171. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Here we observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein still have formation of E1-E2 heterodimers which are functional in terms of allowing efficient virus progeny yields in infected primary swine cells. Additionally, these single cysteine mutant viruses were virulent in infected swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1ΔCys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1ΔCys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days postinfection.     

The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins

We have identified the membrane-active regions of the full sequences of the HCV E1 and E2 envelope glycoproteins by performing an exhaustive study of membrane leakage, hemifusion, and fusion induced by 18-mer peptide libraries on model membranes having different phospholipid compositions. The data and their comparison have led us to identify different E1 and E2 membrane-active segments which might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. Moreover, it has permitted us to suggest that the fusion peptide might be located in the E1 glycoprotein and, more specifically, the segment comprised by amino acid residues 265-296. The identification of these membrane-active segments from the E1 and E2 envelope glycoproteins, as well as their membranotropic propensity, supports their direct role in HCV-mediated membrane fusion, sustains the notion that different segments provide the driving force for the merging of the viral and target cell membranes, and defines those segments as attractive targets for further development of new antiviral compounds.     

Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum

The lumen of the endoplasmic reticulum (ER) provides a unique folding environment that is distinct from other organelles supporting protein folding. The relatively oxidizing milieu allows the formation of disulfide bonds. N-linked oligosaccharides that are attached during synthesis play multiple roles in the folding process of glycoproteins. They stabilize folded domains and increase protein solubility, which prevents aggregation of folding intermediates. Glycans mediate the interaction of newly synthesized glycoproteins with some resident ER folding factors, such as calnexin and calreticulin. Here we present an overview of the present knowledge on the folding process of the heavily glycosylated human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein in the ER.     

Identification of interactions in the E1E2 heterodimer of hepatitis C virus important for cell entry

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.     

Hepatitis C virus envelope glycoprotein E2 glycans modulate entry, CD81 binding, and neutralization

Hepatitis C virus (HCV) is a major human pathogen that causes serious liver disease, including cirrhosis and hepatocellular carcinoma. The primary target cells of HCV are hepatocytes, and entry is restricted by interactions of the envelope glycoproteins, E1 and E2, with cellular receptors. E1 and E2 form noncovalently linked heterodimers and are heavily glycosylated. Glycans contribute to protein folding and transport as well as protein function. In addition, glycans associated with viral envelopes mask important functional domains from the immune system and attenuate viral immunogenicity. Here, we explored the role of N- and O-linked glycans on E2, which is the receptor binding subunit of the HCV envelope. We identified a number of glycans that are critical for viral entry. Importantly, we showed that the removal of several glycans significantly increased the inhibition of entry by sera from HCV-positive individuals. Only some of the glycans that affected entry and neutralization were also important for CD81 binding. Our results show that HCV envelope-associated glycans play a crucial role in masking functionally important regions of E2 and suggest a new strategy for eliciting highly neutralizing antibodies against this virus.