A metastable form of the large envelope protein of duck hepatitis B virus: low-pH release results in a transition to a hydrophobic, potentially fusogenic conformation

We have examined the structure and fusion potential of the duck hepatitis B virus (DHBV) envelope proteins by treating subviral particles with deforming agents known to release envelope proteins of viruses from a metastable to a fusion-active state. Exposure of DHBV particles to low pH triggered a major structural change in the large envelope protein (L), resulting in exposure of trypsin sites within its S domain but without affecting the same region in the small surface protein (S) subunits. This conformational change was associated with increased hydrophobicity of the particle surface, most likely arising from surface exposure of the hydrophobic first transmembrane domain (TM1). In the hydrophobic conformation, DHBV particles were able to bind to liposomes and intact cells, while in their absence these particles aggregated, resulting in viral inactivation. These results suggests that some L molecules are in a spring-loaded metastable state which, when released, exposes a previously hidden hydrophobic domain, a transition potentially representing the fusion-active state of the envelope.     

Topology of the large envelope protein of duck hepatitis B virus suggests a mechanism for membrane translocation during particle morphogenesis.

We have investigated the membrane topology of the large envelope protein of duck hepatitis B virus (DHBV) by protease protection and Western blot analysis, using monoclonal antibodies specific for the pre-S and S regions of the DHBV envelope to characterize protease-resistant polypeptides. These studies showed that DHBV L protein exhibits a mixed membrane topology similar to that of human hepatitis B virus L, with approximately half of the L molecules displaying pre-S on the surface of virus particles and the remainder with pre-S sequestered inside the virus envelope. The C-terminal region of DHBV pre-S was susceptible to protease digestion on all DHBV particle L protein, indicating that this region was externally disposed. DHBV L protein pre-S was entirely cytosolic immediately after synthesis. Our data, therefore, suggested that an intermediate form of the DHBV L molecule exists in mature envelope particles in which L is partially translocated or exists in a translocation-ready conformation. Incubation of virus particles at low pH and 37 degrees C triggered conversion of this intermediate into a fully translocated form. We have proposed a model for pre-S translocation based on our results that invokes the presence of an aqueous pore in the virus envelope, most likely created by oligomerization of transmembrane domains in the S region. The model predicts that pre-S is transported through this pore and that a loop structure is formed because the N terminus remains anchored to the inner face of the membrane. This translocation process occurs during particle morphogenesis and may also be a prerequisite to virus uncoating during infection.     

A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.     

Hepadnavirus envelope topology: insertion of a loop region in the membrane and role of S in L protein translocation

A unique feature of the large hepadnavirus envelope protein (L) is its mixed transmembrane topology, resulting from partial posttranslational translocation of the pre-S domain. Using protease protection analysis, we demonstrate for duck hepatitis B virus an essential role for the small envelope protein (S) in this process, providing the first experimental evidence for an S translocation channel. Further analysis revealed that the presumed cytoplasmic loop between TM1 and TM2 in the C-terminal S domain is membrane embedded and protrudes to the particle surface. These data suggest that some L molecules form a highly folded, potentially spring-loaded topology with five membrane-spanning regions and a membrane-traversing pre-S chain.     

Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation.

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.     

A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-terminal additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.     

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.     

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM alpha-helical bundle. We have shown previously that the topology of the alpha-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM alpha-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric alpha-helical bundle model.     

Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation

The large (L) envelope protein of the hepatitis B virus (HBV) has the peculiar capacity to form two transmembrane topologies via an as yet uncharacterized process of partial post-translational translocation of its pre-S domain across membranes. In view of a current model that predicts an HBV-specific channel generated during virion envelope assembly to enable pre-S translocation, we have examined parameters influencing L topogenesis by using protease protection analysis of wild-type and mutant L proteins synthesized in transfected cells. We demonstrate that contrary to expectation, all determinants, thought to be responsible for channel formation, are dispensable for pre-S reorientation. In particular, we observed that this process does not require (i) the helper function of the HBV S (small) and M (middle) envelope proteins, (ii) covalent dimer formation of envelope chains, or (iii) either of the three amphipathic transmembrane segments of L. Rather, the most hydrophobic transmembrane segment 2 of L was identified as a vital topogenic determinant, essential and sufficient for post-translational pre-S translocation. Cell fractionation studies revealed that pre-S refolding and thus the dual topology of L is established at the endoplasmic reticulum (ER) membrane rather than at a post-ER compartment as originally supposed. Together our data provide evidence to suggest that the topological reorientation of L is facilitated by a host cell transmembrane transport machinery such as the ER translocon.     

Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion.

The hepatitis delta virus (HDV) is coated with large (L), middle (M), and small (S) envelope proteins encoded by coinfecting hepatitis B virus (HBV). To study the role of the HBV envelope proteins in the assembly and infectivity of HDV, we produced three types of recombinant particles in Huh7 cells by transfection with HBV DNA and HDV cDNA: (i) particles with an envelope containing the S HBV envelope protein only, (ii) particles with an envelope containing S and M proteins, and (iii) particles with an envelope containing S, M, and L proteins. Although the resulting S-, SM-, and SML-HDV particles contained both hepatitis delta antigen and HDV RNA, only particles coated with all three envelope proteins (SML) showed evidence of infectivity in an in vitro culture system susceptible to HDV infection. We concluded that the L HBV envelope protein, and more specifically the pre-S1 domain, is important for infectivity of HDV particles and that the M protein, which has been reported to bear a site for binding to polymerized albumin in the pre-S2 domain, is not sufficient for infectivity. Our data also show that the helper HBV is not required for initiation of HDV infection. The mechanism by which the L protein may affect HDV infectivity is discussed herein.     

Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity.     

The critical N-linked glycan of murine leukemia virus envelope protein promotes both folding of the C-terminal domains of the precursor polyprotein and stability of the postcleavage envelope complex.

The infectivity of Friend ecotropic murine leukemia virus was previously shown to be highly sensitive to modification in its envelope protein (Env) at only one of the eight signals for N-linked glycan attachment, the fourth from the N terminus (gs4). In the present study, a set of six single-amino-acid substitutions in or near gs4 was used to determine the function of this region of Env and the role played by the glycan itself. One mutant that lacked the gs4 glycan was fully infectious, while one that retained this glycan was completely noninfectious, indicating that the gs4 glycan per se is not required for Env function. Infectivity correlated with the level of mature Env complex incorporated into virus particles, which was determined by the severity of defects in transport of the envelope precursor protein (gPrEnv) from the endoplasmic reticulum into the Golgi apparatus, in cleavage of gPrEnv into the two envelope subunits (the surface protein [SU] and the transmembrane protein [TM]), and in the association of SU with cellular membranes. All of the mutants induced the wild-type level of superinfection interference, indicating that the gs4 region mutations did not interfere with proper folding of the N-terminal domain of SU. These results suggest that the gs4 region mediates folding of the C-terminal domains of gPrEnv and stability of the interaction between SU and TM. Although the gs4 glycan was not essential for infectivity, processing of all mutant Envs lacking this glycan was significantly impaired, suggesting that efficient folding of gPrEnv requires a glycan at this position. The conservation of a glycosylation site homologous to gs4 across a broad range of retroviruses suggests that this sequence may play a similar role in many retroviral Envs.     

Novel transmembrane topology of the hepatitis B virus envelope proteins.

The small (S), middle (M) and large (L) envelope proteins of the hepatitis B virus (HBV) are initially synthesized as multispanning membrane proteins of the endoplasmic reticulum membrane. We now demonstrate that all envelope proteins synthesized in transfected cells or in a cell-free system adopt more than one transmembrane orientation. The L protein disposes its N-terminal preS domain both to the cytoplasmic and the luminal side of the membrane. This unusual topology does not depend on interaction with the viral nucleocapsid, but is preserved in secreted empty envelope particles. Pulse-chase analysis suggests a novel process of post-translational translocation leading to the non-uniform topology. Analysis of L deletion mutants indicates that the block to co-translational translocation can be attributed to a specific sequence within preS, suggesting that translocation of L may be regulated. Additional topological heterogeneity is displayed in the S region of the envelope proteins and in the S protein itself, as assayed in a cell-free system. S proteins integrated into microsomal membranes exhibit both a luminal and a cytoplasmic orientation of the internal hydrophilic region carrying the major antigenic determinants. This may explain the unusual partial glycosylation of the HBV envelope proteins.     

Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.     

Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.

The preS domain at the N-terminus of the large envelope protein (LHBs) of the hepatitis B virus is involved in (i) envelopment of viral nucleocapsids and (ii) binding to the host cell. While the first function suggests a cytosolic location of the preS domain during virion assembly, the function as an attachment site requires its translocation across the lipid bilayer and final exposure on the virion surface. We compared the transmembrane topology of newly synthesized LHBs in the endoplasmic reticulum (ER) membrane with its topology in the envelope of secreted virions. Protease sensitivity and the absence of glycosylation suggest that the entire preS domain of newly synthesized LHBs remains at the cytosolic side of ER vesicles. However, virions secreted from transfected cell cultures or isolated from the blood of persistent virus carriers expose antibody binding sites and proteolytic cleavage sites of the preS domain at their surface in approximately half of the LHBs molecules. Thus, preS domains appear to be transported across the viral lipid barrier by a novel post-translational translocation mechanism to fulfil a dual function in virion assembly and attachment to the host cell.     

Chaperones involved in hepatitis B virus morphogenesis

Little is known about host cell factors necessary for hepatitis B virus (HBV) assembly which involves envelopment of cytosolic nucleocapsids by the S, M and L transmembrane viral envelope proteins and subsequent budding into intraluminal cisternae. Central to virogenesis is the L protein that mediates hepatocyte receptor binding and envelopment of capsids. To serve these topologically conflicting roles, L protein exhibits an unusual dual membrane topology, disposing its N-terminal preS domain inside and outside of the virion lipid envelope. The mixed topology is achieved by posttranslational preS translocation of about half of the L protein molecules across a post-endoplasmic reticulum membrane. Here we identify and characterize a preS-specific sequence that confers the suppression of cotranslational translocation even of a model reporter. This cytosolic anchorage sequence specifically binds the cognate heat shock protein Hsc70, thus indicating chaperone participitation in HBV morphogenesis. Conversely, the M envelope protein needs the assistance of the chaperone calnexin for proper folding and trafficking. Calnexin selectively binds to the N-glycan, specific for M, rather than to the N-glycan, common to all three envelope proteins. As inhibition of the calnexin-M interaction blocks the secretion of viral envelopes, we propose an essential role for calnexin, as well as for Hsc70, in chaperoning HBV assembly.     

Respiratory syncytial virus F envelope protein associates with lipid rafts without a requirement for other virus proteins

Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.     

Phenotypic mixing of rodent but not avian hepadnavirus surface proteins into human hepatitis B virus particles.

The virus family Hepadnaviridae comprises two genera: orthohepadnaviruses isolated from humans (hepatitis B virus [HBV]) and rodents (e.g., woodchuck hepatitis virus [WHV]) and avihepadnaviruses isolated from birds (e.g., duck hepatitis B virus [DHBV]). They carry in their envelopes two (DHBV) or three (HBV and WHV) coterminal proteins referred to as small (S), middle (M), or large (L) surface protein. These proteins are also secreted from infected cells as subviral particles consisting of surface protein and lipid (e.g., 20-nm hepatitis B surface antigen for HBV). To investigate the assembly of these proteins, we asked whether surface proteins from different hepadnaviruses are able to mix phenotypically with each other. By coexpression and coimmunoprecipitation with species-specific antibodies, we could show the formation of mixed subviral particles and disulfide-linked heterodimers between the WHV S and HBV M proteins whereas the DHBV and HBV surface proteins did not coassemble. Complementation of HBV genomes defective in expressing the S or L protein and therefore incompetent to form virions was possible with the closely related WHV S protein or a WHV pre-S-HBV S chimera, respectively, but not with the less related DHBV S or L protein or with a DHBV L-HBV S chimera. The results suggest that the assembly of HBV subviral particles and virion envelopes requires relatively precise molecular interactions of their surface proteins, which are not conserved between the two hepadnavirus genera. This contrasts with the ability of, e.g., rhabdoviruses or retroviruses, to incorporate envelope proteins even from unrelated viruses.     

Interaction between duck hepatitis B virus and a 170-kilodalton cellular protein is mediated through a neutralizing epitope of the pre-S region and occurs during viral infection.

Identification of cell surface viral binding proteins is important for understanding viral attachment and internalization. We have fused the pre-S domain of the duck hepatitis B virus (DHBV) large envelope protein to glutathione S-transferase and demonstrated a 170-kDa binding protein (p170) in [35S]methionine-labeled duck hepatocyte lysates. This glycoprotein was found abundantly in all extrahepatic tissues infectible with DHBV and in some noninfectible tissues, though it is not secreted into the blood. The interaction of pre-S fusion protein with p170 was competitively inhibited by wild-type DHBV in a dose-dependent manner. In addition, infection of hepatocytes with DHBV blocked the binding of pre-S fusion protein to p170, which suggests a biological role for p170 during natural infection. The p170 binding site was mapped to a conserved sequence of 16 amino acid residues (positions 87 to 102) by using 24 pre-S deletion mutants; this binding domain coincides with a major virus-neutralizing antibody epitope. Furthermore, site-directed mutagenesis revealed that an arginine residue at position 97 is critical for p170 binding. p170 was purified by a combination of ion-exchange and affinity chromatographies, and four peptide sequences were obtained. Two peptides showed significant similarities to human and animal carboxypeptides H, M, and N. Taken together, these results raise the possibility that the p170 binding protein is important during the replication cycle of DHBV.     

Early assembly step of a retroviral envelope glycoprotein: analysis using a dominant negative assay

As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.