Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes.     

Intracellular processing of the gp160 HIV-1 envelope precursor. Endoproteolytic cleavage occurs in a cis or medial compartment of the Golgi complex

The intracellular processing of the gp160 HIV-1 envelope precursor was characterized in acutely infected CD4+ T cells. Our data show that gp160 undergoes endoproteolytic cleavage by a nonacid dependent protease(s) in the rough endoplasmic reticulum-Golgi complex, within cis or medial cisternae, and is not transported to the cell surface. Two-dimensional electrophoretic pulse-chase analysis indicates that it takes greater than 2 h for gp160 to be transported from the rough endoplasmic reticulum to the site of action of sialyltransferases in the trans Golgi. Evidence is presented that gp160 is subject to mannose trimming in the Golgi complex, which is inhibited by 1-deoxymannojirimycin (a specific Golgi alpha-mannosidase I inhibitor). Preliminary data also suggest that gp120 is post-translationally modified by sialylated O-linked oligosaccharides.     

Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4.

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.     

Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum.

The cell surface glycoprotein, CD4, is the receptor for human immunodeficiency virus (HIV) in T lymphocytes. Following HIV infection, there is reduced expression of CD4 on the cell surface, and this downregulation probably results, at least in part, from the formation of complexes containing the HIV type 1 (HIV-1) glycoprotein precursor (gp160) and CD4 that are not transported from the endoplasmic reticulum (ER). At the plasma membrane of T cells, CD4 is tightly associated with a cytoplasmic tyrosine kinase (p56lck) that is involved in T-cell activation. Using a transient expression system with HeLa cells, we show by pulse-labeling and immunoprecipitation that newly synthesized CD4 can associate with p56lck before CD4 is transported from the ER. In the presence of HIV-1 gp160, a ternary complex of gp160-CD4 and p56lck forms in the ER. Using confocal immunofluorescence microscopy, we observed complete retention of p56lck in the ER. Such mislocation of a tyrosine kinase to the cytoplasmic face of the ER could play a role in lymphocyte killing caused by HIV infection or expression of gp160 alone.     

Lateral diffusion of CD4 on the surface of a human neoplastic T-cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1)

The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein-labeled gp120 with CD4 was observed in a solid-phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV-1-infected cells on CEM cells was significantly inhibited in the presence of fluorescein-labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein-labeled gp120 was approximately 5 x 10(-10) cm2sec-1, with nearly 61% of the receptor molecules being mobile. Binding of anti-gp120 monoclonal antibody to the CD4-gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.     

The human immunodeficiency virus type 1 (HIV-1) CD4 receptor and its central role in promotion of HIV-1 infection.

Interactions between the viral envelope glycoprotein gp120 and the cell surface receptor CD4 are responsible for the entry of human immunodeficiency virus type 1 (HIV-1) into host cells in the vast majority of cases. HIV-1 replication is commonly followed by the disappearance or receptor downmodulation of cell surface CD4. This potentially renders cells nonsusceptible to subsequent infection by HIV-1, as well as by other viruses that use CD4 as a portal of entry. Disappearance of CD4 from the cell surface is mediated by several different viral proteins that act at various stages through the course of the viral life cycle, and it occurs in T-cell lines, peripheral blood CD4+ lymphocytes, and monocytes of both primary and cell line origin. At the cell surface, gp120 itself and in the form of antigen-antibody complexes can trigger cellular pathways leading to CD4 internalization. Intracellularly, the mechanisms leading to CD4 downmodulation by HIV-1 are multiple and complex; these include degradation of CD4 by Vpu, formation of intracellular complexes between CD4 and the envelope precursor gp160, and internalization by the Nef protein. Each of the above doubtless contributes to the ultimate depletion of cell surface CD4, although the relative contribution of each mechanism and the manner in which they interact remain to be definitively established.     

CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor.

We analyzed coexpression of the human immunodeficiency virus type 1 glycoprotein precursor, gp160, and its cellular receptor CD4 in HeLa cells to determine whether the two molecules can interact prior to transport to the cell surface. Results of studies employing coprecipitation, analysis of oligosaccharide processing, and immunocytochemistry showed that newly synthesized CD4 and gp160 form a complex prior to transport from the endoplasmic reticulum (ER). CD4 expressed by itself was transported efficiently from the ER to the cell surface, but the complex of CD4 and gp160 was retained in the ER. This retention of CD4 within the ER is probably a consequence of the very inefficient transport of gp160 itself (R. L. Willey, J. S. Bonifacino, B. J. Potts, M. A. Martin, and R. D. Klausner, Proc. Natl. Acad. Sci. USA 85:9580-9584, 1988). Retention of CD4 in the ER by gp160 may partially explain the down regulation of CD4 in human immunodeficiency virus type 1-infected T cells. Inhibition of CD4 transport appears to be a consequence of the interaction of two membrane-bound molecules, because a complex of CD4 and gp120 (the soluble extracellular domain of gp160) was transported rapidly and efficiently from the ER.     

Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein.

Various roles for the viral receptor, CD4, have been proposed in facilitating human immunodeficiency virus type 1 (HIV-1) entry, including virion binding to the target cell and the induction of conformational changes in the viral envelope glycoproteins required for the membrane fusion reaction. Here, we compare the structural requirements in the CDR2-like loop of CD4 domain 1, the major contact site of the gp120 envelope glycoprotein, for gp120 binding and virus entry. For every CD4 mutant examined, the level of cell surface expression and the gp120 binding affinity were sufficient to explain the relative ability to function as a viral receptor. The decrease in relative infectibility associated with decreased gp120 binding affinity was more pronounced at lower cell surface CD4 concentrations. These results imply that both receptor density and affinity determine the efficiency of HIV-1 entry and that specific structures in the CD4 residues examined are probably not required for HIV-1 entry functions other than gp120 binding.     

CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.     

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and CD4 binding

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.     

Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events.

The noncovalent association of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) is disrupted by soluble CD4 binding, resulting in shedding of the gp120 exterior envelope glycoprotein. This observation has led to the speculation that interaction of gp120 with the CD4 receptor triggers shedding of the exterior envelope glycoprotein, allowing exposure of gp41 domains necessary for membrane fusion steps involved in virus entry or syncytium formation. To test this hypothesis, a set of HIV-1 envelope glycoprotein mutants were used to examine the relationship of soluble CD4-induced shedding of the gp120 glycoprotein to envelope glycoprotein function in syncytium formation and virus entry. All mutants with a threefold or greater reduction in CD4-binding ability exhibited marked decreases in gp120 shedding in response to soluble CD4, even though several of these mutants exhibited significant levels of envelope glycoprotein function. Conversely, most fusion-defective mutants with wild-type gp120-CD4 binding affinity, including those with changes in the V3 loop, efficiently shed gp120 following soluble CD4 binding. Thus, soluble CD4-induced shedding of gp120 is not a generally useful marker for conformational changes in the HIV-1 envelope glycoproteins necessary for the virus entry or syncytium formation processes. Some gp120 mutants, despite being expressed on the cell surface and capable of efficiently binding soluble CD4, exhibited decreased gp120 shedding. These mutants were still sensitive to neutralization by soluble CD4, indicating that, for envelope glycoproteins exhibiting high affinity for soluble CD4, competitive inhibition may be more important than gp120 shedding for the antiviral effect.     

Human immunodeficiency virus type 1 Vpu has a CD4- and an envelope glycoprotein-independent function.

Vpu is a 16-kDa membrane-associated phosphoprotein that is expressed from the same, singly spliced message as the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor, gp160. Previous studies suggest that Vpu functions in the late stages of viral replication, possibly in virus egression from the cell. Recently, it has been demonstrated that Vpu functions to allow gp160 to be more efficiently processed by disrupting CD4-gp160 complexes generated by transfection of HeLa cells. We show here that the lack of expression of intact Vpu results in a 90% reduction in infectious virus produced over a single round of replication from HeLa cells in the absence of CD4 expression. This reduction persists when HIV-1 particles are pseudotyped with the HIV-2 or amphotropic murine leukemia virus envelope glycoprotein. Pulse-chase analysis of HIV-1 capsid protein (p24) in the absence of CD4 and envelope glycoprotein demonstrates that the rate of virus release is reduced when Vpu is not expressed. Our findings indicate that Vpu has a function involving particle release not dependent on CD4 or envelope glycoprotein expression.     

Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants

Naturally occurring human immunodeficiency virus (HIV-1) variants require the presence of CD4 and specific chemokine receptors to enter a cell. In the laboratory, HIV-1 variants that are capable of bypassing CD4 and utilizing only the CCR5 chemokine receptor for virus entry have been generated. Here we report that these CD4-independent viruses are significantly more sensitive to neutralization by soluble CD4 and a variety of antibodies. The same amino acid changes in the HIV-1 gp120 envelope glycoprotein determined CD4 independence and neutralization sensitivity. The CD4-independent envelope glycoproteins exhibited higher affinity for antibodies against CD4-induced gp120 epitopes but not other neutralizing ligands. The CD4-independent envelope glycoproteins did not exhibit increased lability relative to the wild-type envelope glycoproteins. The utilization of two receptors apparently allows HIV-1 to maintain a more neutralization-resistant state prior to engaging CD4 on the target cell, explaining the rarity of CD4 independence in wild-type HIV-1.     

Elicitation of neutralizing antibodies directed against CD4-induced epitope(s) using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.     

Apoptosis induced in CD4+ cells expressing gp160 of human immunodeficiency virus type 1.

In a previous study (Y. Koga, M. Sasaki, H. Yoshida, H. Wigzell, G. Kimura, and K. Nomoto, J. Immunol. 144:94-102, 1990), we demonstrated that the expression of gp160, a precursor form of envelope glycoprotein of human immunodeficiency virus type 1, in CD4+ cells causes the downregulation of surface CD4 and single-cell killing by forming intracellular gp160-CD4 complex. In the present study we investigated the events that lead to cell death in CD4+ cells expressing gp160. We found that apoptosis is induced in cells undergoing single-cell death. Moreover, even the cell clone, which expresses so little gp160 that it does not exhibit any apparent cytopathic effects, such as the inhibition of cell growth, was found to be highly susceptible to the apoptosis induction by the anti-Fas monoclonal antibody.     

Evidence by peptide mapping that the region CD4(81-92) is involved in gp120/CD4 interaction leading to HIV infection and HIV-induced syncytium formation.

Peptide fragments of the CD4 molecule were compared in their ability to 1) inhibit CD4-dependent HIV-induced cell fusion; 2) inhibit CD4-dependent HIV infection in vitro; and 3) block gp120 envelope glycoprotein binding to CD4. Peptides from the region CD4(81-92), although inactive when underivatized, were equipotent inhibitors of CD4-dependent virus infection, cell fusion, and CD4/gp120 binding when derivatized via benzylation and acetylation. Peptides of identical chemical composition, but altered sequence and derivatization pattern that blocked gp120 binding to either CD4-positive cells or solubilized CD4, also blocked infection and fusion with similar potencies. Those that did not block gp120/CD4 interaction were also inactive in HIV-1 infection and cell fusion assays. No other peptide fragments of the CD4 molecule inhibited fusion, infection, or CD4/gp120 interaction. The peptide CD4(23-56), derived from a region of CD4 implicated in binding of CD4 antibodies that neutralize HIV infection and cell fusion, had no effect on CD4-dependent cell fusion, HIV-1 infection, or CD4/gp120 binding, but did reverse OKT4A and anti-Leu 3a blockade of gp120 binding to CD4. These data provide evidence that the 81-92 region of CD4 is directly involved in gp120 binding leading to CD4-dependent HIV infection and syncytium formation. Previous observations with structural mutants of CD4 suggest that the CDR2-homologous region of CD4 is also involved, either directly or indirectly, in binding of gp120 to CD4. The CDR2- and CDR3-like domains of CD4 may both contribute to the binding of the HIV envelope necessary for HIV-1 infection and HIV-1-induced cell fusion.     

The CD4 molecule transmits biochemical information important in the regulation of T lymphocyte activity

The role of the CD4 molecule in the transmission and regulation of the biochemical signals involved in T cell activation was investigated using an anti-CD4 monoclonal antibody termed 6B10. 6B10 immunoprecipitated the 55-kDa CD4 molecule and detected an epitope of CD4 that overlapped with that detected by OKT4A, B, and D. 6B10, 6B10 Fab fragments and recombinant HIV envelope glycoprotein (gp120) induced calcium mobilization in PBMC. 6B10 stimulation also resulted in calcium mobilization in murine L cells expressing transfected CD4 gene products, indicating that CD4-mediated calcium mobilization occurred independently of the CD3/T cell receptor (TCR) complex. 6B10 induced a phosphatidylinositol response, but the response resulted in reduced inositol phosphate production compared to levels obtained using OKT3. Though 6B10 caused calcium mobilization and a phosphatidylinositol response, 6B10 did not induce DNA synthesis. The amount of inositol phosphates produced by 6B10 may be below the threshold necessary for cell cycle progression. We hypothesized that 6B10-mediated calcium mobilization is important in the regulation of T cell proliferation. 6B10, but not 6B10 Fab fragments, inhibited OKT3-induced DNA synthesis. Furthermore, 6B10 but not 6B10 Fab fragments inhibited OKT3-induced calcium mobilization, suggesting that crosslinking of CD4 may be an important factor determining whether signals result in both the up- and down-regulation of CD3/TCR complex function. The implication of this work is that signals generated via the CD4 molecule are important in the regulation of T cell function and that the signals generated as a result of HIV gp120 binding to CD4 can contribute to the mechanism by which HIV inhibits T cell function.     

Sulfation of the human immunodeficiency virus envelope glycoprotein.

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.