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.     

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.     

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.     

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.     

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.     

Enhancement of human immunodeficiency virus type 1 envelope-mediated fusion by a CD4-gp120 complex-specific monoclonal antibody.

The entry of human immunodeficiency virus type 1 (HIV-1) into cells is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. The gp120-CD4 complex formed at the cell surface undergoes conformational changes that may allow its association with an additional membrane component(s) and the eventual formation of the fusion complex. These conformational rearrangements are accompanied by immunological changes manifested by altered reactivity with monoclonal antibodies specific for the individual components and presentation of new epitopes unique to the postbinding complex. In order to analyze the structure and function of the gp120-CD4 complex, monoclonal antibodies were generated from splenocytes of BALB/c mice immunized with soluble CD4-gp120 (IIIB) molecules (J. M. Gershoni, G. Denisova, D. Raviv, N. I. Smorodinsky, and D. Buyaner, FASEB J. 7:1185-1187 1993). One of those monoclonal antibodies, CG10, was found to be strictly complex specific. Here we demonstrate that this monoclonal antibody can significantly enhance the fusion of CD4+ cells with effector cells expressing multiple HIV-1 envelopes. Both T-cell-line-tropic and macrophage-tropic envelope-mediated cell fusion were enhanced, albeit at different optimal doses. Furthermore, infection of HeLa CD4+ (MAGI) cells by HIV-1 LAI, ELI1, and ELI2 strains was increased two- to fourfold in the presence of CG10 monoclonal antibodies, suggesting an effect on viral entry. These findings indicate the existence of a novel, conserved CD4-gp120 intermediate structure that plays an important role in HIV-1 cell fusion.     

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.     

Spontaneous reversion of human immunodeficiency virus type 1 neutralization-resistant variant HXB2thr582: in vitro selection against cytopathicity highlights gp120-gp41 interactive regions.

Spontaneous revertants of the immune-selected variant HXB2thr582, which resists neutralization by certain conformationally dependent antibodies specific for the CD4-binding site on gp120 (such as F105), appeared after long-term culture in the absence of immune-selecting serum. Molecular analysis showed some of the viruses in the revertant stock contained a simple back mutation, whereas others retained the Thr-582 codon but contained a substitution of serine for phenylalanine in gp41 at position 673. Neutralization sensitivity to the selecting serum and to F105 of infectious clones containing either the back mutation or the compensatory mutation, HXB2thr582ser673, was confirmed. HXB2thr582-infected cells have a greater propensity for syncytium formation and single cell killing than do either the parental HXB2 or the revertant HXB2thr582ser673. This suggests that the revertant arose by selection in vitro for a less cytopathic virus. Our results link three envelope regions shown to influence virus-cell fusion as well as neutralization by antibody: the CD4-binding region, the leucine zipper domain, and a region hidden to antipeptide antibodies upon envelope oligomerization. Taken together they illustrate the functional importance of the gp120-gp41 interaction and emphasize the impact of the interplay between envelope regions on overall conformation and function and on recognition by neutralizing antibodies.     

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.     

Mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41.

The mechanism of heparan sulfate (HS)-mediated human immunodeficiency virus type 1 (HIV-1) binding to and infection of T cells was investigated with a clone (H9h) of the T-cell line H9 selected on the basis of its high level of cell surface CD4 expression. Semiquantitative PCR analysis revealed that enzymatic removal of cell surface HS by heparitinase resulted in a reduction of the amount of HIV-1 DNA present in H9h cells 4 h after exposure to virus. Assays of the binding of recombinant envelope proteins to H9h cells demonstrated a structural requirement for an oligomeric form of gp120/gp41 for HS-dependent binding to the cell surface. The ability of the HIV-1 envelope to bind simultaneously to HS and CD4 was shown by immunoprecipitation of HS with either antienvelope or anti-CD4 antibodies from 35SO4(2-)-labeled H9h cells that had been incubated with soluble gp140. Soluble HS blocked the binding of monoclonal antibodies that recognize the V3 and C4 domains of the envelope protein to the surface of H9 cells chronically infected with HIV-1IIIB. The V3 domain was shown to be the major site of envelope-HS interaction by examining the effects of both antienvelope monoclonal antibodies and heparitinase on the binding of soluble gp140 to H9h cells.     

Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion.     

Structure and dynamics of micelle-associated human immunodeficiency virus gp41 fusion domain

The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.     

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.     

Role of hydrophobic residues in the central ectodomain of gp41 in maintaining the association between human immunodeficiency virus type 1 envelope glycoprotein subunits gp120 and gp41

The interaction between the gp120 and gp41 subunits of the human immunodeficiency virus envelope glycoprotein serves to stabilize the virion form of the complex and to transmit receptor-induced conformational changes in gp120 to trigger the membrane fusion activity of gp41. In this study, we used site-directed mutagenesis to identify amino acid residues in the central ectodomain of gp41 that contribute to the stability of the gp120-gp41 association. We identified alanine mutations at six positions, including four tryptophan residues, which result in mutant envelope glycoprotein complexes that fail to retain gp120 on the cell surface. These envelope glycoproteins readily shed their gp120 and are unable to mediate cell-cell fusion. These findings suggest an important role for the conserved bulky hydrophobic residues in stabilizing the gp120-gp41 complex.     

Oligomeric organization of gp120 on infectious human immunodeficiency virus type 1 particles.

The oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein (gp120) was examined by treating infectious virions with chemical cross-linking agents and subjecting the protein to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and velocity centrifugation. Immunoblots of cross-linked samples revealed three gp120 bands and an approximately threefold shift in gp120 sedimentation. Our finding of cross-linking solely between gp120 suggests that the gp120 subunits are closely associated in the native envelope structure.     

Amphotericin B derivative blocks human immunodeficiency virus type 1 entry after CD4 binding: effect on virus-cell fusion but not on cell-cell fusion.

The antiviral effect of MS8209, an amphotericin B derivative, was studied in CD4+ cells transfected with a lacZ gene inducible upon human immunodeficiency virus type 1 (HIV-1) infection. MS8209 was shown to block virus entry after receptor binding and probably before virus-cell membrane fusion, but it had no effect on syncytium formation, although both processes are mediated by HIV-1 envelope proteins and CD4.     

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.     

Model for intracellular folding of the human immunodeficiency virus type 1 gp120.

The intracellular folding of the human immunodeficiency virus type 1 gp120 has been assessed by analyzing the ability of the glycoprotein to bind to the viral receptor CD4. Pulse-chase experiments revealed that the glycoprotein was initially produced in a conformation that was unable to bind to CD4 and that the protein attained the appropriate tertiary structure for binding with a half-life of approximately 30 min. The protein appears to fold within the rough endoplasmic reticulum, since blocking of transport to the Golgi apparatus by the oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone did not appear to perturb the folding kinetics of the molecule. The relatively lengthy folding time was not due to modification of the large number of N-linked glycosylation sites on gp120, since inhibition of the first steps in oligosaccharide modification by the inhibitors deoxynojirimycin or deoxymannojirimycin did not impair the CD4-binding activity of the glycoprotein. However, production of the glycoprotein in the presence of tunicamycin and removal of the N-linked sugars by endoglycosidase H treatment both resulted in deglycosylated proteins that were unable to bind to CD4, suggesting in agreement with previous results, that glycosylation contributes to the ability of gp120 to bind to CD4. Interestingly, incomplete endoglycosidase H treatment revealed that a partially glycosylated glycoprotein could bind to the receptor, implying that a subset of glycosylation sites, perhaps some of those conserved in different isolates of human immunodeficiency virus type 1, might be important for binding of the viral glycoprotein to the CD4 receptor.     

Covalently linked human immunodeficiency virus type 1 gp120/gp41 is stably anchored in rhabdovirus particles and exposes critical neutralizing epitopes

Rabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Because the gp120 and gp41 portions are covalently attached in the gp140 SOS molecule, the protein is maintained on the surface of the RV virion throughout purification. Surface immunostaining and fluorescence-activated cell sorting analysis with anti-envelope antibodies show that the gp140 SOS protein is stably expressed on the surface of infected cells and maintains CD4 binding capabilities. Furthermore, Western blot and immunoprecipitation experiments with infected-cell lysates and purified virions show that a panel of neutralizing anti-envelope antibodies efficiently recognize the gp140 SOS protein. The antigenic properties of this recombinant RV particle containing covalently attached Env, as well as the ability to present Env in a membrane-bound form, suggest that this approach could be a useful component of a HIV-1 vaccine strategy.     

Determinants of human immunodeficiency virus type 1 resistance to membrane-anchored gp41-derived peptides

The expression of a membrane-anchored gp41-derived peptide (M87) has been shown to confer protection from infection through human immunodeficiency virus type 1 (HIV-1) (Hildinger et al., J. Virol. 75:3038-3042, 2001). In an effort to characterize the mechanism of action of this membrane-anchored peptide in comparison to the soluble peptide T-20, we selected resistant variants of HIV-1(NL4-3) and HIV-1(BaL) by serial virus passage using PM1 cells stably expressing peptide M87. Sequence analysis of the resistant isolates showed different patterns of selected point mutations in heptad repeat regions 1 and 2 (HR1 and HR2, respectively) for the two viruses analyzed. For HIV-1(NL4-3) a single amino acid change at position 33 in HR1 (L33S) was selected, whereas for HIV-1(BaL) the majority of the sequences obtained showed two amino acid changes, one in HR1 and one in HR2 (I48V/N126K). In both selections the most important contiguous 3-amino-acid sequence, GIV, within HR1, associated with resistance to soluble T-20, was not changed. Site-directed mutagenesis studies confirmed the importance of the characterized point mutations to confer resistance to M87 as well as to soluble T-20 and T-649. Replication capacity and dual-color competition assays revealed that the double mutation I48V/N126K in HIV-1(BaL) results in a strong reduction of viral fitness, whereas the L33S mutation in HIV-1(NL4-3) did enhance viral fitness compared to the respective parental viruses. However, the selected point mutations did not confer resistance to the more recently described optimized membrane-anchored fusion inhibitor M87o (Egelhofer et al., J. Virol. 78:568-575, 2004), strengthening the importance of this novel antiviral concept for gene therapy approaches.