Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.     

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.     

Role of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein in cell-cell fusion and virus infection

The membrane-spanning domain (MSD) of the human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein is critical for its biological activity. Previous C-terminal truncation studies have predicted an almost invariant core structure of 12 amino acid residues flanked by basic amino acids in the HIV-1 MSD that function to anchor the glycoprotein in the lipid bilayer. To further understand the role of specific amino acids within the MSD core, we initially replaced the core region with 12 leucine residues and then constructed recovery-of-function mutants in which specific amino acid residues (including a GGXXG motif) were reintroduced. We show here that conservation of the MSD core sequence is not required for normal expression, processing, intracellular transport, and incorporation into virions of the envelope glycoprotein (Env). However, the amino acid composition of the MSD core does influence the ability of Env to mediate cell-cell fusion and plays a critical role in the infectivity of HIV-1. Replacement of conserved amino acid residues with leucine blocked virus-to-cell fusion and subsequent viral entry into target cells. This restriction could not be released by C-terminal truncation of the gp41 glycoprotein. These studies imply that the highly conserved core residues of the HIV Env MSD, in addition to serving as a membrane anchor, play an important role in mediating membrane fusion during viral entry.     

Lateral mobility of reconstituted Sendai virus envelope glycoproteins on human erythrocytes: correlation with cell-cell fusion

We have recently employed fluorescence photobleaching recovery (FPR) to demonstrate that the envelope glycoproteins of Sendai virions become laterally mobile on the surface of human erythrocytes following fusion [Henis, Y. I., Gutman, O., & Loyter, A. (1985) Exp. Cell Res. 160, 514-526]. In order to investigate whether this lateral mobilization is involved in the mechanism of virally mediated cell-cell fusion, or is merely a result of viral envelope-cell fusion, we have now performed FPR studies on erythrocytes fused with reconstituted Sendai virus envelopes (RSVE). These RSVE, which were prepared by solubilization of Sendai virions with Triton X-100 followed by removal of the detergent through adsorption to SM-2 Bio-beads, fused with human erythrocytes as efficiently as native virions but induced cell-cell fusion to a much lower degree. The fraction of the viral envelope glycoproteins that became laterally mobile in the erythrocyte membrane following fusion was markedly lower in the case of RSVE than in the case of native virions. The lower cell-cell fusion activity of the RSVE does not appear to be due to inactivation of the viral fusion protein, since the envelope-cell fusion and hemolytic activities of the RSVE were similar to those of native virions. Moreover, fusion with RSVE or with native virions resulted in the incorporation of rather similar amounts of viral glycoproteins into the cell membrane. Since the reduced fraction of laterally mobile viral glycoproteins correlates with the lower cell-cell fusion activity of the RSVE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate binding properties of the envelope glycoproteins of human immunodeficiency virus type 1

Here, we confirm and extend our previous findings on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteinN-acetylglucosaminyl binding properties. We show the occurrence of saturable, temperature, pH, and calcium dependent carbohydrate-specific interactions between recombinant precursor gp160 (rgp160) and two affinity matrices:d-mannose-divinylsulfone-agarose, and natural glycoprotein, fetuin, also coupled to agarose. Binding of rgp160 to the matrices was inhibited by soluble mannosyl derivatives, -d-Man₁₇-BSA and mannan, by -d-GlcNAc₄₇-BSA and by glycopeptides from Pronase-treated porcine thyroglobulin, which produces oligomannose and complex N-linked glycans. Glycopeptides from Endoglycosidase H-treated thyroglobulin partially inhibited rgp160 binding, as did the asialo-agalacto-tetraantennary precursor oligosaccharide of human ₁-acid glycoprotein for binding to fetuin-agarose. -d-Glucan and -d-Gal₁₇-BSA had no or only limited effect. Also, surface unit rgp120 specifically interacted with fetuin-agarose and soluble fetuin, but in the latter case with a twofold reduced affinity relative to rgp160. After affinity chromatography, rgp160 was specifically retained by the two matrices and eluted by mannan in both cases, while rgp120 was not retained by fetuin-agarose but only eluted as a significantly retarded peak, which confirms its specific but weak interaction. Thus, rgp160 interacts with both oligomannose type, and the mannosyl core of complex type N-linked glycans, and its gp120 region plays a role in this interaction. Because fetuin and asialofetuin inhibit to nearly the same extent, the binding of rgp160 or rgp120 to fetuin-agarose, interaction with sialic acid or -d-galactosyl structures of complex N- or O-linked glycans can be ruled out. Specific rgp160 and rgp120 binding to ap-aminophenyl--d-GlcNAc-agarose matrix, which was inhibited by -d-GlcNAc₄₇-BSA and by fetuin, confirms that HIV-1 envelope glycoproteins can also specifically interact with theN-acetylglucosaminyl core of oligosaccharide structures.     

Peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites

Human immunodeficiency virus type 1 (HIV-1) entry into target cells requires folding of two heptad-repeat regions (N-HR and C-HR) of gp41 into a trimer of N-HR and C-HR hairpins, which brings viral and target cell membranes together to facilitate membrane fusion. Peptides corresponding to the N-HR and C-HR of gp41 are potent inhibitors of HIV infection. Here we report new findings on the mechanism of inhibition of a N-HR peptide and compare these data with inhibition by a C-HR peptide. Using intact envelope glycoprotein (Env) under fusogenic conditions, we show that the N-HR peptide preferentially binds receptor-activated Env and that CD4 binding is sufficient for triggering conformational changes that allow the peptide to bind Env, results similar to those seen with the C-HR peptide. However, activation by both CD4 and chemokine receptors further enhances Env binding by both peptides. We also show that a nonconservative mutation in the N-HR of gp41 abolishes C-HR peptide but not N-HR peptide binding to gp41. These results indicate that there are two distinct sites in receptor-activated Env that are potential targets for drug or vaccine development.     

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.     

Membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion

We have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (HIV-1) gp41, different from the fusion peptide, that interacts strongly with membranes. This conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the Wimley-White interfacial hydrophobicity scale. We have investigated here the membrane effects induced by NH(2)-DKWASLWNWFNITNWLWYIK-CONH(2) (HIV(c)), the membrane interface-partitioning region at the C terminus of the gp41 ectodomain, in comparison to those caused by NH(2)-AVGIGALFLGFLGAAGSTMGARS-CONH(2) (HIV(n)), the fusion peptide at the N terminus of the subunit. Both HIV(c) and HIV(n) were seen to induce membrane fusion and permeabilization, although lower doses of HIV(c) were required for comparable effects to be detected. Experiments in which equimolar mixtures of HIV(c) and HIV(n) were used indicated that both peptides may act in a cooperative way. Peptide-membrane and peptide-peptide interactions underlying those effects were further confirmed by analyzing the changes in fluorescence of peptide Trp residues. Replacement of the first three Trp residues by Ala, known to render a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, also abrogated the HIV(c) ability to induce membrane fusion or form complexes with HIV(n) but not its ability to associate with vesicles. Hydropathy analysis indicated that the presence of two membrane-partitioning stretches separated by a collapsible intervening sequence is a common structural motif among other viral envelope proteins. Moreover, sequences with membrane surface-residing residues preceding the transmembrane anchor appeared to be a common feature in viral fusion proteins of several virus families. According to our experimental results, such a feature might be related to their fusogenic function.     

Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay

BACKGROUND: In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. METHODS: We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. RESULTS: Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). CONCLUSIONS: The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion.     

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.     

Mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity.

A chimeric protein consisting of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) ectodomain joined to the transmembrane and cytoplasmic-tail domains of vesicular stomatitis virus G protein lost the ability to fuse CD4+ HeLa cells yet was transported to the cell surface and cleaved normally. These results suggested some critical role of the HIV gp41 transmembrane or cytoplasmic domain in fusion. Subsequent mutagenic analysis of the HIV-1 Env transmembrane domain revealed that the sequence of amino acid residues from positions 696 to 707 of the transmembrane domain was important for fusion function but was not required for anchoring of the Env protein in the lipid bilayer or for transport to the cell surface. Further analysis indicated that the basic residues at positions 696 and 707 were critical for membrane fusion activity, as was the spacing between these residues. These results demonstrate that in addition to providing an anchoring function, the specific amino acid sequence in the transmembrane domain plays a crucial role in the membrane fusion process.     

Functional analysis of hepatitis C virus envelope proteins, using a cell-cell fusion assay

Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.     

Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160DeltaCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160DeltaCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160DeltaCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.     

Photoinactivation and kinetics of membrane fusion mediated by the human immunodeficiency virus type 1 envelope glycoprotein.

The fusion kinetics of cells expressing the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with CD4 target cells was continuously monitored by image-enhanced Nomarski differential interference contrast optics. The analysis of the videotape recordings showed that (i) cells made contact relatively rapidly (within minutes), in many cases by using microspikes to "touch" and adhere to adjoining cells; (ii) the adhered cells fused after a relatively long waiting period, which varied from 15 min to hours; (iii) the morphological changes after membrane fusion, which led to disappearance of the interface separating the two cells, were rapid (less than 1 min); and (iv) the process of syncytium formation involved subsequent fusion with other cells and not simultaneous fusion of many cells. To measure the kinetics of early stages of cell fusion, we used the recently developed very stable membrane-soluble dye, PKH26, which redistributes between labeled and unlabeled membranes after fusion but does not exchange spontaneously between membranes for prolonged periods. We found that photoactivation of this dye by illumination with green light inhibits fusion of cell membranes as indicated by the lack of dye transfer from the labeled HIV-1 envelope-expressing cells to unlabeled CD4 cells. The inhibitory effect was localized in space and time, which allowed us to develop a new assay for measuring the kinetics of membrane fusion by illuminating the cell mixture at different times after coculture. This assay has also been used to monitor the fusion kinetics of HIV-1 and recombinant vaccinia virus. The photoactivation of nonexchangeable membrane-soluble fluorescent dyes may be useful for development of new assays for measuring the kinetics of membrane fusion and could also be important in designing new antiviral approaches.     

Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.     

Synthesis of functional human immunodeficiency virus tat protein in baculovirus as determined by a cell-cell fusion assay.

The human immunodeficiency virus tat protein is a strong trans-activator of the expression of mRNAs originating from the viral long terminal repeat. We have expressed the first 72 amino acids (coding exon 1) of this protein in eucaryotic Spodoptera frugiperda SF9 cells by using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. We show that the baculovirus vector stably produced the 72-amino-acid form of the tat protein but was unable to stably synthesize a larger 101-amino-acid full-length version of the same polypeptide. The 72-amino-acid tat protein, when introduced into mammalian fibroblasts by using a cell-cell fusion technique, functionally trans-activated the expression of the human immunodeficiency virus long terminal repeat.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.     

Mannosyl/N-acetyl-beta-D-glucosaminyl binding properties of the envelope glycoprotein of human immunodeficiency virus type 2.

We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein precursor gp160 (rgp160) behaves as a mannosyl/N-acetylglucosaminyl (GlcNAc) binding protein. If such a carbohydrate-binding property were of biological relevance it should be shared by other related primate immunodeficiency viruses such as HIV-2. The present study confirms this hypothesis and extends these findings by showing that HIV-2 recombinant gp140 (rgp140) specifically interacts with three affinity matrices substituted by synthetic or natural carbohydrate structures: D-mannose-divinylsulphone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and the natural glycoprotein, bovine fetuin, also coupled to agarose. Binding of rpg140 to the matrices was inhibited by alpha-D-Man17-BSA (where BSA is bovine serum albumin), beta-D-GlcNAc47-BSA and fetuin, and by glycopeptides derived from pronase-treated porcine thyroglobulin. Glycopeptides obtained after endoglycosidase H treatment of thyroglobulin had a limited inhibitory effect, whereas beta-D-Gal17-BSA and beta-D-glucan had no effect. These results indicate that, like HIV-1 envelope glycoprotein, HIV-2 rgp140 interacts with high-mannose and with the mannosyl core of complex-type N-linked glycans, as well as with the N-acetylglucosaminyl core of oligosaccharidic structures.     

Human immunodeficiency virus envelope glycoprotein/CD4-mediated fusion of nonprimate cells with human cells.

Human immunodeficiency virus (HIV) infects human cells by binding to surface CD4 molecules and directly fusing with the cell membrane. Although mouse cells expressing human CD4 bind HIV, they do not become infected, apparently because of a block in membrane fusion. To study this problem, we constructed a recombinant vaccinia virus that can infect and promote transient expression of full-length CD4 in mammalian cells. This virus, together with another vaccinia recombinant encoding biologically active HIV envelope glycoprotein gp160, allowed us to study CD4/gp160-mediated cell-cell fusion in a wide variety of human and nonhuman cells in the absence of other HIV proteins. By using syncytium formation assays in which a single cell type expressed both CD4 and gp160, we demonstrated membrane fusion in lymphoid and nonlymphoid human cells but not in any of the 23 tested nonhuman cell types, derived from African green monkey, baboon, rabbit, hamster, rat, or mouse. However, in mixing experiments with one cell type expressing CD4 and the other cell type expressing gp160, all of these nonhuman cells could form CD4/gp160-mediated syncytia when mixed with human cells; in 20 of 23 cases, membrane fusion occurred only if the CD4 molecule was expressed on the human cells whereas in the other three cases, CD4 could be expressed on either one of the fusing partners. Interestingly, in one mouse cell line, CD4-dependent syncytia formed without a human partner, but only if a C-terminally truncated form of the HIV envelope glycoprotein was employed. Our results indicate that nonhuman cells are intrinsically capable of undergoing CD4/gp160-mediated membrane fusion, but this fusion is usually prevented by the lack of helper or the presence of inhibitory factors in the nonhuman cell membranes.     

Consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virus-infected macaques

We have analyzed changes to proviral Env gp120 sequences and the development of neutralizing antibodies (NAbs) during 1 year of simian/human immunodeficiency virus SHIV-89.6P infection in 11 Macaca nemestrina macaques. Seven macaques had significant env divergence from that of the inoculum, and macaques with greater divergence had higher titers of homologous NAbs. Substitutions in sequons encoding potential N-linked glycosylation sites (PNGs) were among the first to be established, although overall the total number of sequons did not increase significantly. The majority (19 of 23) of PNGs present in the inoculum were conserved in the sequences from all macaques. Statistically significant variations in PNGs occurred in multiple macaques within constrained regions we term "hot spots," resulting in the selection of sequences more similar to the B consensus. These included additions on V1, the N-terminal side of V4, and the outer region of C2. Complex mutational patterns resulted in convergent PNG shifts in V2 and V5. Charge changes in Env V1V2, resulting in a net acidic charge, and a proline addition in V5 occurred in several macaques. Molecular modeling of the 89.6P sequence showed that the conserved glycans lie on the silent face of Env and that many are proximal to disulfide bonds, while PNG additions and shifts are proximal to the CD4 binding site. Nonsynonymous-to-synonymous substitution ratios suggest that these changes result from selective pressure. This longitudinal and cross-sectional study of mutations in human immunodeficiency virus (HIV) env in the SHIV background provides evidence that there are more constraints on the configuration of the glycan shield than were previously appreciated.