INTRODUCTION

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Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, which also comprises the genera Flavivirus and Hepacivirus (24). In the absence of an efficient cell culture system able to support hepatitis C virus (HCV) replication, BVDV has been adopted as a model and surrogate for HCV (1), as both viruses share molecular and virological features. HCV and BVDV are small, enveloped viruses with positive single-stranded RNA genomes of approximately 9,600 and 12,600 nucleotides, respectively. The polypeptide precursor is transcribed from a single large open reading frame and subsequently co- and posttranslationally processed into structural and nonstructural proteins. Most BVDV and HCV proteins are functionally homologous. The envelope glycoproteins E1 and E2 interact either noncovalently (HCV) (7) or through disulfide bonds (BVDV) (26, 28) to form a dimer, which has been proposed as the functional complex present on the surfaces of mature virions.

We have previously shown that endoplasmic reticulum (ER) -glucosidase inhibitors containing the glucose analogue deoxynojirimycin (DNJ) as the head group have a strong antiviral effect on BVDV (32). Castanospermine (CST), another -glucosidase inhibitor, was shown to cause misfolding of recombinant HCV E1 and E2 glycoproteins expressed in BHK-21 cells and reduce their association into native dimers (5). Similarly, CST affected the morphogenesis and assembly of Dengue virus (DENV), another member of the Flaviviridae family (6).

The ER -glucosidases perform the stepwise removal of the three glucose residues on N-linked glycans attached to nascent polypeptides. This removal enables folding intermediates to associate with the lectin-like ER chaperones calnexin and calreticulin, which interact with monoglucosylated glycoproteins and which retain incompletely folded polypeptides and oligomers in the ER (2, 23). However, not all cellular proteins are dependent on the -glucosidase-mediated folding pathway, and certain cell lines lacking -glucosidase expression are viable (22). Importantly, the folding of certain viral glycoproteins has been shown to be calnexin dependent (19, 20, 29). Thus, targeting the ER -glucosidases may potentially be of therapeutic use in treating viral infections, without affecting host cell viability. In addition, since the enzymes are host cell and not virus encoded, emergence of drug-resistant viruses is less likely to occur.

While the folding of recombinant HCV envelope proteins has been thoroughly investigated and its dependence upon the calnexin-mediated pathway has been clearly established (9), nothing is known about the folding of BVDV-encoded glycoproteins. Characterization of the folding pathway of the BVDV glycoproteins is important if we want to use BVDV as a model system for HCV to study drugs which interfere with the assembly and secretion of the envelope proteins. BVDV (National Animal Disease Laboratory [NADL] strain) encodes three envelope glycoproteins, E^rns, E1, and E2, which carry eight, two, and four potential N-glycosylation sites, respectively (24). Unlike E1 and E2, E^rns lacks a membrane anchor and is secreted from infected cells. In this paper we investigate the folding of the BVDV envelope glycoproteins E1 and E2 and the role played by the ER chaperones calnexin and calreticulin in this process. To this end, we have monitored the kinetics of intra- and intermolecular disulfide bond formation and the association of E1 and E2 glycoproteins into heterodimers. The mechanism of the sensitivity of BVDV to -glucosidase inhibition was also examined. The usefulness of BVDV as a model system for screening anti-HCV drugs is discussed and compared to that of the HCV system available.

	MATERIALS AND METHODS

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Cell culture, virus, inhibitors, and enzymes. Noncytopatic BVDV-free MDBK cells (European Collection of Animal Cell Cultures, Porton Down, United Kingdom) and cytopathic BVDV virus (NADL strain; American Type Culture Collection, Manassas, Va.) were used in this study. MDBK cells were grown in RPMI 1640 medium (GIBCO/BRL) supplemented with 10% BVDV-free fetal calf serum (PAA Laboratories, Teddington, United Kingdom). N-Butyl-DNJ (NB-DNJ) was a gift from Searle/Monsanto. N-Butyldeoxygalactojirimycin (NB-DGJ) was purchased from Boehringer Mannheim. The inhibitors were made up as 200 mM stock solutions in water and filtered before use. Partially purified ER -glucosidases I and II were kindly provided by T. Butters (Oxford Glycobiology Institute).

Antibodies. Monoclonal antibodies (MAbs) 158 and 214 raised against BVDV E2 glycoprotein were purchased from the Veterinary Laboratories Agency, Weybridge, United Kingdom. The anticalnexin and anticalreticulin polyclonal antibodies were purchased from Bioquote Limited, York, United Kingdom. The antirabbit and antimouse horseradish peroxidase-conjugated secondary antibodies were from Sigma.

BVDV plaque reduction assay. MDBK cells were grown to subconfluent monolayers in six-well plates and infected with cytopathic BVDV at a multiplicity of infection (MOI) of 1 PFU/cell for 1 h at 37°C. After the inoculum was removed, the cells were washed with phosphate-buffered saline (PBS) and incubated for 3 days in the presence or absence of inhibitors. The medium containing secreted virus was then removed from the wells, centrifuged at low speed to remove cellular debris, and used to infect fresh monolayers of MDBK cells grown in six-well plates. The resulting plaques were counted after 2 days.

Western blotting. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to nitrocellulose membranes using a semidry electroblotter (Millipore) and detected with anti-BVDV E2 antibodies (dilution, 1/1,000) or anticalnexin antibodies (dilution, 1/4,000) followed by antimouse (dilution, 1/2,000) or antirabbit (dilution, 1/10,000) antibodies conjugated to horseradish peroxidase. The proteins were detected using an enhanced-chemiluminescence detection system (Amersham) by following the manufacturer's instructions.

-Glucosidase digestion. MDBK cells were infected with BVDV at an MOI of 1 and treated or not treated with 2 mM NB-DNJ. Twenty milligrams of total cellular extract was incubated overnight at 37°C using 5 U (each) of ER -glucosidases I and II. Samples were analyzed by SDS-10% PAGE and stained for Western blotting with anti-E2 antibodies.

Pulse-labeling and chase. Subconfluent MDBK cell monolayers grown in 25-cm² flasks were infected with BVDV at an MOI of 1. After 1 h of incubation at 37°C, the viral inoculum was replaced with medium containing 10% fetal calf serum. Eighteen hours postinfection (p.i.), the monolayers were washed once with PBS and incubated in methionine- and cysteine-free RPMI 1640 medium (ICN Flow, Thame, Oxfordshire, United Kingdom). After 1 h, the cells were pulse-labeled with 100 µCi of [³⁵S]methionine-[³⁵S]cysteine (Tran ³⁵S-label, 1,100 Ci/mmol; ICN Flow) per ml at 37°C for the times indicated in the figures. Following labeling, the isotope-supplemented medium was removed and the cells were washed once with PBS and chased for various times in RPMI 1640 medium containing 10 mM unlabeled methionine. At the time points indicated in the figures, the chase media were discarded and the cells were harvested. For experiments in which the association of BVDV envelope proteins with calnexin or calreticulin was analyzed, the cells were treated with actinomycin D (4 µg/ml) before being labeled. When the effect of the inhibition of ER -glucosidases I and II was investigated, NB-DNJ was added to the cells 2 h before the pulse at the concentrations indicated in Results and was present throughout the chase period. Cells were then lysed for 1 h on ice in a buffer containing 0.5% Triton X-100, 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 2 mM EDTA (Triton-TSE buffer) and a mixture of protease inhibitors (Sigma). In experiments in which the formation of disulfide bonds was monitored, 20 mM iodoacetamide was included in the lysis buffer to alkylate free sulfhydryl groups and avoid unspecific aggregation. When the interaction with calnexin or calreticulin was studied, cell lysis was performed under mild conditions using a CHAPS-HSE buffer (2% CHAPS {3-[(3-chloramidopropyl)-dimethylammonio]-1-propanesulfonate} in 50 mM HEPES [pH 7.5]-200 mM NaCl-2 mM EDTA).

Immunoprecipitation and SDS-PAGE. Labeled cell lysates were clarified by centrifugation at 12,000 × g for 15 min and precleared with 20 µl of either protein A-Sepharose (when polyclonal antibodies were used) or protein G-Sepharose (when MAbs were used) for 1 h at 4°C. The lysates were then briefly centrifuged, and the supernatants were incubated with either anti-BVDV E2 or antichaperone antibodies (diluted 1:50 or 1:200, respectively) overnight at 4°C. Protein A- or G-Sepharose (30 µl) was then added to the supernatants, and the incubation continued for 1 h at 4°C. The slurry was washed six times with 0.2% Triton X-100 in TSE buffer. The washing buffer was replaced by 0.5% CHAPS in HSE buffer for immunoprecipitation with anticalnexin or -calreticulin antibodies. For coimmunoprecipitation experiments, the lysates were first immunoprecipitated with anticalnexin or -calreticulin antibodies (diluted 1:200); the slurry was then washed twice in 0.5% CHAPS-HSE buffer, and bound proteins were eluted by boiling the samples in 1% SDS. The eluates were diluted 10 times with washing buffer and reprecipitated with anti-E2 antibodies (diluted 1:50). The immunoprecipitated complexes were eluted by boiling the samples for 10 min in SDS-PAGE sample buffer, in the presence (reducing conditions) or absence (nonreducing conditions) of 5% 2-mercaptoethanol. Samples were either quantified by liquid scintillation counting or separated by SDS-PAGE. After electrophoresis, the gels were treated with Amplyfier (Amersham), dried, and exposed at 70°C to Hyperfilm-MP (Amersham). The intensities of the bands on the resulting autoradiograms were measured by scan densitometry.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Folding and processing of BVDV envelope proteins in MDBK cells. The presence of intermolecular disulfide bridges between structural proteins encoded by pestiviruses has been previously shown for classical swine fever virus (CSFV) and the Osloss strain of BVDV (26, 28). Both CSFV gp55 and BVDV gp53 (E2) form homo- and heterodimers, which are also present in mature virions (26). However, the folding of the BVDV envelope glycoproteins involving the formation of intramolecular disulfide bonds, as well as the kinetics of the subsequent association into heterodimers, has not been investigated.

View larger version (129K): [in this window] [in a new window]   FIG. 1.   Analysis of BVDV envelope glycoproteins expressed in MBDK cells. (A) MBDK cells were infected with BVDV (NADL strain) at an MOI of 1. Eighteen hours p.i. the cells were lysed and analyzed by SDS-10% PAGE under nonreducing (lane N) and reducing (lane R) conditions, followed by Western blot analysis with MAb 214. (B) MBDK cells were infected with BVDV at an MOI of 1. Eighteen hours p.i. the cells were pulse-labeled with [35S]methionine and [35S]cysteine for 15 min, chased for the times indicated, lysed, and immunoprecipitated with MAb 214. Immunoprecipitated proteins were separated by SDS-10% PAGE under nonreducing conditions and analyzed by autoradiography. (C) Conditions were the same as described for panel B, except that the sample at min 90 of chase was analyzed by SDS-12% PAGE, under both nonreducing and reducing conditions. Mock-infected cells (mock inf.) were included as controls.

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Analysis of intra- and intermolecular disulfide bond formation of BVDV envelope glycoproteins. MBDK cells were infected with BVDV at an MOI of 1. Eighteen hours p.i., the cells were either pulse-labeled with [35S]methionine and [35S]cysteine for 15 min and chased for the times indicated (A) or pulse-labeled for 2.5 min and harvested immediately (B). Cell lysates were immunoprecipitated with MAb 214, and proteins bound were analyzed by SDS-10% PAGE under nonreducing conditions (A) and both nonreducing (lane N) and reducing (lane R) conditions (B). Mock-infected cells (mock) were included as controls.

Association of E1 and E2 glycoproteins with calnexin and calreticulin. Calnexin and calreticulin assist in the folding of various glycoproteins, including viral proteins, with incompletely or incorrectly folded ones being retained in the ER (23). To determine whether BVDV-encoded envelope glycoproteins make use of this folding pathway, a pulse-chase experiment was performed, followed by the sequential immunoprecipitation with anticalnexin and anti-E2 antibodies. Approximately 40% of the total amount of E2 or E2-p7 precipitated by MAb 214 was coprecipitated by anticalnexin antibodies immediately after a 2.5-min pulse (Fig. 3A). Neither E2 nor E2-p7 was found in association with calnexin at min 10 of chase, suggesting that their interaction with calnexin is rapid and that dissociation occurs shortly after translation.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Interaction of E1 and E2 BVDV glycoproteins with calnexin. MDBK cells were either infected with BVDV at an MOI of 1 (inf.) or mock infected (mock). (A and C) Eighteen hours p.i., cells were pulse-labeled for 2.5 min with [35S]methionine and [35S]cysteine, chased for the times indicated, and either immunoprecipitated with MAb 214 and polyclonal anticalnexin antibodies ( cal) alone or coimmunoprecipitated with both MAb 214 and  cal, as indicated. The immunoprecipitated proteins were analyzed by SDS-10% (A) or SDS-12% PAGE under reducing conditions and visualized by autoradiography (C, graph). Calnexin-bound E1 was quantified by densitometric analysis (C, graph), with the amount of E1 associated with calnexin at time point 0 equaling 100%. (B) Fractions of the cell lysates were analyzed either before immunoprecipitation (lanes 3 and 4) or following immunoprecipitation with MAb 214 and elution of bound proteins under reducing conditions. The proteins were separated by SDS-10% PAGE and analyzed with a Western blot stained with  cal.

Folding of BVDV envelope glycoproteins in the presence of NB-DNJ. Having established that the association of BVDV envelope proteins with calnexin was inhibited in the presence of NB-DNJ, we wanted to determine the effect of this inhibition on the folding of the viral proteins.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Biosynthesis and processing of BVDV envelope glycoproteins in the presence of NB-DNJ. MDBK cells were infected with BVDV at an MOI of 1. (A) Eighteen hours p.i., the cells were treated (+) or not treated () with 2 mM NB-DNJ. Two hours later, the cells were pulse-labeled with [35S]methionine and [35S]cysteine for 15 min, chased for the times indicated in the continuous presence of the drug, and immunoprecipitated with MAb 214. The proteins were analyzed by SDS-10% PAGE under reducing conditions and visualized by autoradiography. (B) Infected cells were grown for 18 h in the absence () or presence (+) of 2 mM NB-DNJ. Cell lysates were analyzed for protein content, and the equivalent of 20 µg of protein was digested (+) or not digested () with a mixture of -glucosidases I and II. The proteins were separated by SDS-10% PAGE under reducing conditions and analyzed with a Western blot stained with MAb 214. (C) Infected cells were grown for 18 h in the absence () or presence (+) of 2 mM NB-DNJ. Cell lysates were boiled for 5 min in the absence () or presence (+) of 5% -mercaptoethanol prior to separation by SDS-10% PAGE. The proteins were analyzed with a Western blot stained with MAb 158. Calnexin was detected with an anticalnexin antibody and used as loading control. (D) Conditions were the same as described for panel A, except that infected cells were lysed immediately after the 15 min of pulse and immunoprecipitated with MAb 158.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Folding and assembly of E1 and E2 glycoproteins in NB-DNJ-treated cells. (A and B) MDBK cells were infected with BVDV at an MOI of 1. Eighteen hours p.i., the cells were treated (+) or not treated () with 2 mM NB-DNJ for 2 h before being pulse-labeled with [35S]methionine and [35S]cysteine for 15 min. At the chase times indicated, the cells were lysed and immunoprecipitated with MAb 158. Proteins bound were separated by SDS-10% PAGE under nonreducing conditions (A) or by SDS-12% PAGE under reducing conditions (B) and visualized by autoradiography. (C) Conditions were the same as those described for panel B, except that the cell lysates were immunoprecipitated with MAb 214.

The antiviral effect of NB-DNJ correlates with misfolding of the E2 glycoprotein. NB-DNJ has previously been shown to have an impact on the yield of secreted infectious BVDV at 3 days p.i. at a low MOI (0.01 PFU/cell) (32). However, to be able to investigate the impact of -glucosidase inhibition on the folding of the viral envelope proteins, we needed to increase the MOI in order to achieve a higher level of viral protein expression. We therefore reanalyzed the effect of NB-DNJ on BVDV plaque formation, this time with MDBK cells which were infected at an MOI of 1 (Fig. 6A). NB-DGJ, an iminosugar derivative which does not inhibit the -glucosidases (18), was included as a control. While NB-DNJ strongly inhibited the cytopathic effect of BVDV on MBDK cells in a dose-dependent manner, NB-DGJ had no impact on plaque formation.

View larger version (33K): [in this window] [in a new window]   FIG. 6.   BVDV protein folding and infectivity in NB-DNJ-treated cells. (A) MDBK cells were grown to subconfluence in six-well plates and infected with BVDV at an MOI of 1. After 1 h the inoculum was removed and the cells were grown for 24 h in medium containing either NB-DNJ or NB-DGJ at the indicated concentrations (plaque assay). The supernatants containing secreted virus were then used to infect fresh MDBK monolayers, and the plaques were counted after 24 h (yield assay). Results are the percentages of the number of plaques resulting from infection with the inhibitor-free plaque assay supernatant (considered as 100%). (B) MDBK cells were infected with BVDV at an MOI of 1 and grown in the absence or presence of increasing concentrations of NB-DNJ. Eighteen hours p.i., the cells were labeled for 15 min with [35S]methionine and [35S]cysteine and lysed. The amount of radiolabeled protein in the cell lysates was adjusted to 3 × 106 cpm/ml, before immunoprecipitation with MAb 158 and antiactin antibodies (loading control). The amount of immunoprecipitated protein was quantified by liquid scintillation counting and expressed as the percentage of counts per minute determined for the drug-free samples. (C) MDBK cells were infected with BVDV at an MOI of 1 and grown in the absence or presence of NB-DNJ at the concentrations indicated. Eighteen hours p.i., the cells were lysed and the assembly of the viral proteins was analyzed by SDS-10% PAGE under nonreducing conditions followed by Western blotting using MAb 214. Calnexin and actin were detected on the same membrane (loading control) (gels). The band intensities of E2 monomers as well as of E1-E2 and E2-E2 dimers were quantified by densitometric analysis (graph).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We used the conformation-dependent and -independent anti-BVDV E2 MAbs available to investigate the disulfide bond-associated folding of BVDV envelope glycoproteins in infected MDBK cells. Our results suggest that, while E2 and E2-p7 acquire a compact configuration stabilized by intramolecular disulfide bonds, either cotranslationally or immediately after translation has been completed, the folding of E1 glycoprotein is rather slow. Both E2 and E2-p7 can be detected as stable polypeptides as early as 2.5 min after pulse-labeling of infected cells, and no further processing at the E2-p7 site occurred within the 4 h of chase. The same unusual lack of a precursor-product relationship between E2-p7 and E2 was reported for HCV (16) and, more recently, for the BVDV CP7 strain (13). Cleavage between E2 and p7 has been reported to be mediated by cellular signal peptidases (10), which cleave the polypeptides at specific sequences, most often immediately after their translocation into the ER. The ability of E2-p7 to acquire very rapidly a conformation stabilized by disulfide bonds in the absence of peptidase peptidase processing indicates that folding and cleavage of this polypeptide may be competing processes. In this hypothetical scenario, a change in conformation may occur during folding of E2-p7, which may lead to the obstruction of the consensus sequence recognized by the host peptidase, preventing subsequent processing.

Interaction of E2 or E2-p7 with calnexin is short, and the kinetics correlate with the formation of intramolecular disulfide bonds. In contrast to E2, E1 remains posttranslationally associated with calnexin for up to 30 min. The release of E1 from calnexin coincides with the beginning of association of E1 and E2 into native, disulfide-linked heterodimers. Since protein assembly into quaternary complexes requires correct folding of the monomers, the folding of E1 appears to be the rate-limiting step for the formation of E1-E2 dimers. The longer association of E1 with calnexin may well play a role in the assembly of the virus, allowing proper interactions between the envelope proteins to occur and promoting formation of functional E1-E2 heterodimers. Although both envelope proteins were found at the same time in calnexin coimmunoprecipitates, no association between E1 and E2 could be detected earlier than 30 min postpulse, suggesting that each monomer interacted independently with calnexin. However, we cannot entirely rule out the possibility that weaker, noncovalent interactions between the two polypeptides may occur before the E1-E2 heterodimer is stabilized by intermolecular disulfide bonds.

In the presence of the -glucosidase inhibitor NB-DNJ, the association of both BVDV envelope glycoproteins with calnexin was inhibited, indicating that N-glycan trimming is necessary for the interaction to occur. This conclusion is consistent with previously published reports in which calnexin is shown to act solely as a lectin (25, 31). In the absence of the interaction with calnexin, the folding associated with the formation of intramolecular disulfide bonds of E2 is inefficient.

The amount of E2 recognized by a conformation-dependent MAb decreased with increasing concentrations of NB-DNJ and was reduced to approximately 50% of that in controls for the highest concentration used. Since the antibody used is able to recognize a disulfide-dependent epitope, the simplest explanation for this result is that, in the presence of the inhibitor, E2 glycoproteins exist as a mixture of native and misfolded polypeptides rather than as a uniform, partially misfolded population.

E1 was also shown to interact with calnexin, and its folding is therefore expected to be impaired by NB-DNJ treatment. Unfortunately, the lack of anti-E1 antibodies makes a more detailed analysis of the folding state of the polypeptide very difficult. Similarly, potentially heavily N-glycosylated E^rns may be a target for the drug, and alterations in E^rns folding may interfere with viral infectivity. Misfolding of the BVDV envelope glycoproteins impairs the association of E1 and E2 into heterodimers. A similar result was obtained for the HCV envelope proteins expressed from a vaccinia virus vector in the presence of CST (5). CST has also been shown to cause the misfolding of DENV prM and E envelope proteins and the formation of unstable prME heterodimers (6). In the HCV system, a second constitutive, nonproductive pathway leading to aggregation of misfolded envelope proteins is activated in the presence of -glucosidase inhibitors. In the DENV system, treatment with -glucosidase inhibitors leads to a delay of envelope protein assembly. In the recombinant HCV system, calreticulin interacts preferentially with these misfolded aggregates, whereas calnexin preferentially associates with either the monomeric form or the noncovalent complexes (5). In BVDV, neither monomers nor dimers are found to be associated with calreticulin and no high-molecular-weight aggregates can be detected with a panel of four MAbs against E2 (data not shown), indicating that, in a natural pestivirus infection, this nonproductive pathway does not occur.

NB-DNJ treatment of BVDV-infected MDBK cells results in a dose-dependent reduction of the amount of infectious virus. The inhibitor concentration which reduces the number of plaques to 50% compared to the number in untreated controls is about 125 µM, when the cells are infected with an MOI of 1. The antiviral effect correlates with the misfolding of E2 and the inefficient association of E1 and E2 into heterodimers. The E1-E2 dimer is the major component of mature virions and is thought to play a central role during infection. Therefore, any conformational changes in the subunits of the complex may interfere with virus binding to host cell receptors or with other postbinding events, leading to reduced viral infectivity.

BVDV has been suggested as a model for HCV since both viruses share molecular and virological features, such as similar genome organizations, replication strategies, and protein functions (24). In this study we show for the first time that BVDV and HCV have a common dependence on calnexin for the folding of their envelope glycoproteins. BVDV E1 and E2 associate rapidly with calnexin and dissociate at different rates. The disulfide-dependent folding of E2 is fast, while E1 folds rather slowly, as judged by the longer interaction with calnexin. The inhibition of calnexin binding to the envelope proteins by -glucosidase inhibitors leads to E2 (and probably E1) misfolding and a decrease in the formation of E1-E2 heterodimers.

A difference between the two viruses may reside in the nature of the interaction between E1 and E2 within the dimers: while the BVDV E1-E2 complex is clearly disulfide bonded, both inside the cells and in secreted mature virions (reference 26 and our own observations), the situation for HCV is less clear. Generally, the noncovalently linked E1-E2 heterodimer found in the recombinant HCV system is thought to be the native complex which is subsequently incorporated into mature secreted virions (8, 12). Interestingly, for Newcastle disease virus, it has been reported that the virus produces two forms of the hemagglutinin-neuraminidase dimer, one linked by disulfide bonds and the other one not, and that both forms are transported to the cell surface with identical kinetics. However, only the form containing intermolecular disulfide bonds is incorporated into virions; the noncovalently linked form is not incorporated and presumably degraded (21). For HCV, so far it has not been possible to identify the functional envelope glycoprotein complex in virions.

Treatment with -glucosidase inhibitors affect the life cycles of other enveloped viruses by inducing misfolding of viral structural proteins. For example, the V1-V2 loop of human immunodeficiency virus gp120, which is involved in virus binding to host cells, has an altered conformation in the presence of NB-DNJ, and this correlates with inhibition of virus entry into the target cells (11). Similarly, recent studies have shown that correct N-glycan trimming is necessary for the secretion of HBV virions and that proper folding and transport of the HBV M and L proteins depend upon the interaction with calnexin (3, 29, 30). The generality of these effects to other viruses remains to be established, for it crucially depends on their envelope glycoproteins using the calnexin-mediated folding pathway.