Importance of membrane fusion mediated by human immunodeficiency virus envelope glycoproteins for lysis of primary CD4-positive T cells

In established T-cell lines, the membrane-fusing capacity of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins mediates cytopathic effects, both syncytium formation and single-cell lysis. Furthermore, changes in the HIV-1 envelope glycoproteins are responsible for the increased CD4(+) T-cell-depleting ability observed in infected monkeys upon in vivo passage of simian-human immunodeficiency virus (SHIV) chimeras. In this study, a panel of SHIV envelope glycoproteins and their mutant counterparts defective in membrane-fusing capacity were expressed in primary human CD4(+) T cells. Compared with controls, all of the functional HIV-1 envelope glycoproteins induced cell death in primary CD4(+) T-cell cultures, whereas the membrane fusion-defective mutants did not. Death occurred almost exclusively in envelope glycoprotein-expressing cells and not in bystander cells. Under standard culture conditions, most dying cells underwent lysis as single cells. When the cells were cultured at high density to promote syncytium formation, the envelope glycoproteins of the passaged, pathogenic SHIVs induced more syncytia than those of the respective parental SHIV. These results demonstrate that the HIV-1 envelope glycoproteins induce the death of primary CD4(+) T lymphocytes by membrane fusion-dependent processes.     

Attenuation of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the transmembrane envelope glycoprotein.

The cytopathic effects of human immunodeficiency virus type 1 (HIV-1) infection are specific for cells that express the CD4 viral receptor and consist of syncytium formation and single-cell lysis. Here we report that a mutation (517A) affecting the amino terminus of the HIV-1 gp41 transmembrane envelope glycoprotein resulted in a virus that was markedly less cytopathic than was wild-type HIV-1. In systems in which cell-to-cell transmission of HIV-1 occurred, the replication ability of the 517A virus was comparable with that of the wild-type virus. Even though the levels of viral protein expression, virion production, and interaction of the envelope glycoproteins with CD4 were similar for the 517A and wild-type viruses, both syncytium formation and single-cell lysis were attenuated for the 517A mutant virus. These results demonstrate that an envelope glycoprotein region important for mediating post-receptor binding events in cell membrane fusion is important for the induction of cytopathic effects by HIV-1. These results also indicate that levels of HIV-1 viral proteins or viral particles produced in infected cells are in themselves not sufficient to induce cytopathic effects.     

Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.     

Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.     

Dissociation of unintegrated viral DNA accumulation from single-cell lysis induced by human immunodeficiency virus type 1.

Acute cytopathic retroviral infections are accompanied by the accumulation, due to superinfection, of large amounts of unintegrated viral DNA in the cells. The cytopathic effects of human immunodeficiency virus type 1 (HIV-1) infection are specific for cells that express the CD4 viral receptor and consist of syncytium formation and single-cell lysis. Here we investigated the relationship between superinfection and single-cell lysis by HIV-1. Antiviral agents were added to C8166 or Jurkat lymphocytes after HIV-1 infection had occurred. Treatment with azidothymidine or a neutralizing anti-gp120 monoclonal antibody reduced or eliminated, respectively, the formation of unintegrated viral DNA but did not inhibit single-cell killing. Furthermore, in the infected Jurkat cells, the levels of unintegrated viral DNA peaked several days before significant single-cell lysis was observed. Essentially complete superinfection resistance was established before the occurrence of single-cell killing. These results demonstrate that single-cell lysis by HIV-1 can be dissociated from superinfection and unintegrated viral DNA accumulation. These results also indicate that single-cell killing may involve envelope glycoprotein-receptor interactions not accessible to the exterior of the cell.     

Changes in the cytopathic effects of human immunodeficiency virus type 1 associated with a single amino acid alteration in the ectodomain of the gp41 transmembrane glycoprotein.

A human immunodeficiency virus type 1 mutant with a single amino acid change (designated 596 W/M) in the ectodomain of the gp41 transmembrane envelope glycoprotein replicated in T-cell lines and in CD4-positive peripheral blood mononuclear cells identically to the wild-type virus. However, the cytopathic effects associated with infection by the mutant virus were altered, with a marked attenuation of syncytium formation and a significant delay in single-cell lysis relative to those of the wild-type virus-infected culture. The 596 W/M mutant is apparently defective in a function that is dispensable for virus entry but that contributes to the efficiency of induction of viral cytopathic effects.     

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.     

trans-Dominant Interference with Human Immunodeficiency Virus Type 1 Replication and Transmission in CD4+ Cells by an Envelope Double Mutant

We previously reported that a human immunodeficiency virus type 1 (HIV-1) envelope (Env) mutant with the whole cytoplasmic domain deleted, denoted mutant TC, is able to dominantly interfere with wild-type (wt) virus infectivity. In the present study, the feasibility of developing a dominant negative mutant-based genetic anti-HIV strategy targeting the gp41 cytoplasmic domain was investigated. Mutants TC and 427,TC, a TC derivative with a Trp-to-Ser substitution introduced into residue 427 in the CD4-binding site, and a series of mutants with deletions in the cytoplasmic domain, effectively trans-dominantly interfered with wt Env-mediated viral infectivity, as demonstrated by an env trans-complementation assay. The syncytium formation-defective 427, TC double mutant not only inhibited heterologous LAV and ELI Env-mediated viral infectivity but also interfered with syncytium formation and infectivity mediated by the Env proteins of the two primary isolates 92BR and 92US. Stable HeLa-CD4-LTR-beta-gal clones that harbored Tat-controlled expression cassettes encoding the control DeltaKS, which had a deletion in the env gene, wt, or mutant env gene were generated. Viral transmission mediated by laboratory-adapted T-cell-tropic HXB2 and NL4-3 viruses was greatly reduced in the TC and 427,TC transfectants compared to that observed in the control DeltaKS and wt transfectants. Viral replication caused by HXB2 and NL4-3 viruses and by macrophage-tropic ConB and ADA-GG viruses was delayed or reduced in human CD4(+) T cells transfected with the 427,TC env construct compared to that observed in cells transfected with the control DeltaKS or TC env construct. The lack of significant interference by TC mutant was due neither to the lack of TC env gene integration into host DNA nor to the lack of TC Env expression upon Tat induction. These results indicate that this 427,TC Env double mutant has a role in the development of trans-dominant mutant-based genetic anti-HIV strategies.     

The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses.     

Syncytium-inducing capacity of human immunodeficiency virus (HIV): analysis by the use of cloned viruses

The marked cytopathic effects of human immunodeficiency virus HIV for susceptible cells are caused mainly by fusion between cells expressing viral envelope glycoproteins and cells expressing CD4 molecule. In this study, we tested the ability of different clones of HIV to induce syncytia in CD4-positive cells. We have reported marked difference in syncytium-inducing capacity of 2 clones of human T lymphotropic virus type III (HTLV-IIIB) isolate despite no detectable difference in expression of viral glycoprotein (gp120). This difference in syncytium induction could be explained by the difference detected in their infectivity and binding activities to CD4-positive cells. Meanwhile we reported difference in syncytium-inducing capacity of 2 clones of lymphadenopathy associated virus (LAV1) isolate parallel to the different amounts of gp120 and other viral proteins expressed by these 2 clones. These results suggest that viral factors like infectivity and binding affinity of the virus to the susceptible cells and the amount of viral gp120 expressed by the infected cells may interact in a complex manner affecting fusion activity and syncytium induction in CD4-positive cells.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.     

Role of the fusogenic peptide sequence in syncytium induction and infectivity of human immunodeficiency virus type 2.

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.     

Enhancement of human immunodeficiency virus type 1 infectivity by replacing the region including Env derived from defective particles with an ability to form particle-mediated syncytia in CD4+T cells

The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.     

Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120.

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.     

Both the V2 and V3 regions of the human immunodeficiency virus type 1 surface glycoprotein functionally interact with other envelope regions in syncytium formation.

To map the regions of the external envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) involved in the process of membrane fusion, we determined the syncytium-inducing capacity of a panel of transiently expressed chimeric envelope genes. This panel was generated by exchanging gene fragments between four previously studied envelope genes that exhibited a high degree of sequence homology yet displayed marked differences in syncytium-inducing capacity when expressed by recombinant vaccinia virus. The results demonstrate that multiple regions of the HIV-1 envelope glycoproteins are involved in syncytium formation. Some fragments, most notably those containing the V2 or V3 region, can transfer syncytium-inducing capacity to envelope proteins previously not capable of inducing syncytia. Moreover, it is shown that such regions functionally interact with other envelope regions, especially one encompassing the V4 and V5 regions of gp120 or a region encompassing part of gp41, to exert their function in membrane fusion.     

Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity.

The role of human immunodeficiency virus type 1 (HIV-1) accessory genes in pathogenesis has remained unclear because of the lack of a suitable in vivo model. The most controversial of these genes is nef. We investigated the requirement for Nef for in vivo replication and pathogenicity of two isolates of HIV-1 (HIV-1JR-CSF and HIV-1NL4-3) in human fetal thymus and liver implants in severe combined immunodeficient mice. HIV-1JR-CSF and HIV-1NL4-3 differ in their in vitro phenotypes in that HIV-1JR-CSF does not induce syncytia and is relatively noncytopathic, while HIV-1NL4-3 is highly cytopathic and readily induces syncytia. The nef mutants of both isolates grew with kinetics similar to those of parental virus strains in stimulated peripheral blood lymphocytes but demonstrated attenuated growth properties in vivo. HIV-1NL4-3 induced severe depletion of human thymocytes within 6 weeks of infection, whereas its nef mutant did not. Thus, HIV-1 Nef is required for efficient in vivo viral replication and pathogenicity.     

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.     

The highly conserved glycan at asparagine 260 of HIV-1 gp120 is indispensable for viral entry

Carbohydrate-binding agents bind to the N-glycans of HIV-1 envelope gp120 and prevent viral entry. Carbohydrate-binding agents can select for mutant viruses with deleted envelope glycans. Not all glycosylation motifs are mutated to the same extent. Site-directed mutagenesis revealed that deletions destroying the highly conserved (260)NGS(262) glycosylation motif resulted in non-infectious virus particles. We observed a significant lower CD4 binding in the case of the N260Q mutant gp120 virus strains, caused by a strikingly lower expression of gp120 and gp41 in the virus particle. In addition, the mutant N260Q HIV-1 envelope expressed in 293T cells was unable to form syncytia in co-cultures with U87.CD4.CXCR4.CCR5 cells, due to the lower expression of envelope protein on the surface of the transfected 293T cells. The detrimental consequence of this N-glycan deletion on virus infectivity could not be compensated for by the creation of novel glycosylation sites near this amino acid, leaving this uncovered envelope epitope susceptible to neutralizing antibody binding. Thus, the Asn-260 glycan in the gp120 envelope of HIV-1 represents a hot spot for targeting suicidal drugs or antibodies in a therapeutic effort to efficiently neutralize a broad array of virus strains.     

The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells

mAb 60.3 and IB4 to CD18, the common beta-subunit of the human leukocytic cell adhesion molecule family, efficiently inhibit syncytium formation induced by the interaction of HIV type 1 (HIV-1)-infected monocytoid cells and CD4+ T cells. The antibodies also interfere with cellfree HIV-1 infection of U-937 clone 16 cells. Virus-induced aggregation of these cells and the subsequent syncytia formation leading to massive cell death are efficiently blocked, and the number of infected cells remains at a very low level, 2 to 5%, for the entire culture period. However, anti-CD18 mAb do not inhibit binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4. The results indicate participation of CD18, or of the protein complex CD11a-c/CD18, in addition to CD4, in the infection and cytopathic effect of HIV-1. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.     

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.