Mutations in human immunodeficiency virus type 1 gp41 affect sensitivity to neutralization by gp120 antibodies.

Three closely related molecular human immunodeficiency virus type 1 (HIV-1) clones, with differential neutralization phenotypes, were generated by cloning of an NcoI-BamHI envelope (env) gene fragment (HXB2R nucleotide positions 5221 to 8021) into the full-length HXB2 molecular clone of HIV-1 IIIB. These env gene fragments, containing the complete gp120 coding region and a major part of gp41, were obtained from three different biological clones derived from a chimpanzee-passaged HIV-1 IIIB isolate. Two of the viruses thus obtained (4.4 and 5.1) were strongly resistant to neutralization by infection-induced chimpanzee and human polyclonal antibodies and by HIV-1 IIIB V3-specific monoclonal antibodies and weakly resistant to soluble CD4 and a CD4-binding-site-specific monoclonal antibody. The third virus (6.8) was sensitive to neutralization by the same reagents. The V3 coding sequence and the gp120 amino acid residues important for the discontinuous neutralization epitope overlapping the CD4-binding site were completely conserved among the clones. However, the neutralization-resistant clones 4.4 and 5.1 differed from neutralization-sensitive clone 6.8 by two mutations in gp41. Exchange experiments confirmed that the 3' end of clone 6.8 (nucleotides 6806 to 8021; amino acids 346 to 752) conferred a neutralization-sensitive phenotype to both of the neutralization-resistant clones 4.4 and 5.1. From our study, we conclude that mutations in the extracellular portion of gp41 may affect neutralization sensitivity to gp120 antibodies.     

Resistance of a human serum-selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120.

We have selected an HXB2 variant which can replicate in the presence of a neutralizing human serum. Sequencing of the gp120 region of the env gene from the variant and parental viruses identified a single amino acid substitution in the third conserved region of gp120 at residue 375 (AGT-->AAT, Ser-->Asn; designated 375 S/N). The escape mutant was found to be resistant to neutralization by soluble CD4 (sCD4) and four monoclonal antibodies (MAbs), 39.13g, 1.5e, G13, and 448, binding to epitopes overlapping that of the CD4 binding site (CD4 b.s.). Introduction of the 375 S/N mutation into HXB2 by site-directed mutagenesis confirmed that this mutation is responsible for the neutralization-resistant phenotype. Both sCD4 and three of the CD4 b.s. MAbs (39.13g, 1.5e, and G13) demonstrated reduced binding to the native 375 S/N mutant gp120. The ability to select for an escape variant resistant to multiple independent CD4 b.s. MAbs by a human serum confirms the reports that antibodies to the discontinuous CD4 b.s. are a major component of the group-specific neutralizing activity in human sera.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody.

While one hypervariable, linear neutralizing determinant on the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein has been well characterized, little is known about the conserved, discontinuous gp120 epitopes recognized by neutralizing antibodies in infected individuals. Here, the epitope recognized by a broadly reactive neutralizing monoclonal antibody (F105) derived from an HIV-1-infected patient was characterized by examining the effects of changes in conserved gp120 amino acids on antibody reactivity. The F105 epitope was disrupted by changes in gp120 amino acids 256 and 257, 368 to 370, 421, and 470 to 484, which is consistent with the discontinuous nature of the epitope. Three of these regions are proximal to those previously shown to be important for CD4 binding, which is consistent with the ability of the F105 antibody to block gp120-CD4 interaction. Since F105 recognition was more sensitive to amino acid changes in each of the four identified gp120 regions than was envelope glycoprotein function, replication-competent mutant viruses that escaped neutralization by the F105 antibody were identified. These studies identify a conserved, functional HIV-1 gp120 epitope that is immunogenic in man and may serve as a target for therapeutic or prophylactic intervention.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.     

Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein.

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp41 glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp41 and the gp120 exterior glycoprotein. Amino acid changes in other parts of the gp41 glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp41 human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp41 ectodomain.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41.

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.     

Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120.

A panel of anti-gp120 human monoclonal antibodies (HuMAbs), CD4-IgG, and sera from people infected with human immunodeficiency virus type 1 (HIV-1) was tested for neutralization of nine primary HIV-1 isolates, one molecularly cloned primary strain (JR-CSF), and two strains (IIIB and MN) adapted for growth in transformed T-cell lines. All the viruses were grown in mitogen-stimulated peripheral blood mononuclear cells and were tested for their ability to infect these cells in the presence and absence of the reagents mentioned above. In general, the primary isolates were relatively resistant to neutralization by the MAbs tested, compared with the T-cell line-adapted strains. However, one HuMAb, IgG1b12, was able to neutralize most of the primary isolates at concentrations of < or = 1 microgram/ml. Usually, the inability of a HuMAb to neutralize a primary isolate was not due merely to the absence of the antibody epitope from the virus; the majority of the HuMAbs bound with high affinity to monomeric gp120 molecules derived from various strains but neutralized the viruses inefficiently. We infer therefore that the mechanism of resistance of primary isolates to most neutralizing antibodies is complex, and we suggest that it involves an inaccessibility of antibody binding sites in the context of the native glycoprotein complex on the virion. Such a mechanism would parallel that which was previously postulated for soluble CD4 resistance. We conclude that studies of HIV-1 neutralization that rely on strains adapted to growth in transformed T-cell lines yield the misleading impression that HIV-1 is readily neutralized. The more relevant primary HIV-1 isolates are relatively resistant to neutralization, although these isolates can be potently neutralized by a subset of human polyclonal or monoclonal antibodies.     

Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding.

Interaction with the CD4 receptor enhances the exposure on the human immunodeficiency type 1 gp120 exterior envelope glycoprotein of conserved, conformation-dependent epitopes recognized by the 17b and 48d neutralizing monoclonal antibodies. The 17b and 48d antibodies compete with anti-CD4 binding antibodies such as 15e or 21h, which recognize discontinuous gp120 sequences near the CD4 binding region. To characterize the 17b and 48d epitopes, a panel of human immunodeficiency virus type 1 gp120 mutants was tested for recognition by these antibodies in the absence or presence of soluble CD4. Single amino acid changes in five discontinuous, conserved, and generally hydrophobic regions of the gp120 glycoprotein resulted in decreased recognition and neutralization by the 17b and 48d antibodies. Some of these regions overlap those previously shown to be important for binding of the 15e and 21h antibodies or for CD4 binding. These results suggest that discontinuous, conserved epitopes proximal to the binding sites for both CD4 and anti-CD4 binding antibodies become better exposed upon CD4 binding and can serve as targets for neutralizing antibodies.     

The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site.

We have compared the expression of full-length gp160 envelope protein from human immunodeficiency virus type 1 with that of a deletion mutant lacking the N-terminal 31 amino acids of the mature protein (gp160 delta 32). The gp160 and gp160 delta 32 proteins are processed to yield gp41 and gp120 or gp120 delta 32, respectively. In contrast to full-length gp120, gp120 delta 32 failed to associate with gp41 at the cell surface, despite conformational integrity as judged by soluble CD4 binding. Thus, the N-terminal 31 amino acids of gp120, which contain hyperconserved sequences, are likely involved in forming a contact site for gp41.     

Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41.

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection. The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability. The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein. This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability. gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein. To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E. coli. Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain. Expression of the central region of gp41 comprising this domain was highly lytic for E. coli cells and increased membrane permeability to a number of compounds. These findings are discussed in the light of HIV-induced cytopathology and gp41 structure.     

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.

Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein.

Monoclonal antibodies have been isolated from human immunodeficiency virus type 1 (HIV-1)-infected patients that recognize discontinuous epitopes on the gp120 envelope glycoprotein, that block gp120 interaction with the CD4 receptor, and that neutralize a variety of HIV-1 isolates. Using a panel of HIV-1 gp120 mutants, we identified amino acids important for precipitation of the gp120 glycoprotein by three different monoclonal antibodies with these properties. These amino acids are located within seven discontinuous, conserved regions of the gp120 glycoprotein, four of which overlap those regions previously shown to be important for CD4 recognition. The pattern of sensitivity to amino acid change in these seven regions differed for each antibody and also differed from that of the CD4 glycoprotein. These results indicate that the CD4 receptor and this group of broadly neutralizing antibodies recognize distinct but overlapping gp120 determinants.     

Reevaluation of amino acid variability of the human immunodeficiency virus type 1 gp120 envelope glycoprotein and prediction of new discontinuous epitopes

To elucidate the evolutionary mechanisms of the human immunodeficiency virus type 1 gp120 envelope glycoprotein at the single-site level, the degree of amino acid variation and the numbers of synonymous and nonsynonymous substitutions were examined in 186 nucleotide sequences for gp120 (subtype B). Analyses of amino acid variabilities showed that the level of variability was very different from site to site in both conserved (C1 to C5) and variable (V1 to V5) regions previously assigned. To examine the relative importance of positive and negative selection for each amino acid position, the numbers of synonymous and nonsynonymous substitutions that occurred at each codon position were estimated by taking phylogenetic relationships into account. Among the 414 codon positions examined, we identified 33 positions where nonsynonymous substitutions were significantly predominant. These positions where positive selection may be operating, which we call putative positive selection (PS) sites, were found not only in the variable loops but also in the conserved regions (C1 to C4). In particular, we found seven PS sites at the surface positions of the alpha-helix (positions 335 to 347 in the C3 region) in the opposite face for CD4 binding. Furthermore, two PS sites in the C2 region and four PS sites in the C4 region were detected in the same face of the protein. The PS sites found in the C2, C3, and C4 regions were separated in the amino acid sequence but close together in the three-dimensional structure. This observation suggests the existence of discontinuous epitopes in the protein's surface including this alpha-helix, although the antigenicity of this area has not been reported yet.     

Target cell-specific determinants of membrane fusion within the human immunodeficiency virus type 1 gp120 third variable region and gp41 amino terminus.

The entry of human immunodeficiency virus type 1 (HIV-1) into target cells involves binding to the viral receptor (CD4) and membrane fusion events, the latter influenced by target cell factors other than CD4. The third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein and the amino terminus of the HIV-1 gp41 transmembrane envelope glycoprotein have been shown to be important for the membrane fusion process. Here we demonstrate that some HIV-1 envelope glycoproteins containing an altered V3 region or gp41 amino terminus exhibit qualitatively different abilities to mediate syncytium formation and virus entry when different target cells are used. These results demonstrate that the structure of these HIV-1 envelope glycoprotein regions determines the efficiency of membrane fusion in a target cell-specific manner and support a model in which the gp41 amino terminus interacts directly or indirectly with the target cell during virus entry.