Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited ≈1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.     

Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex.

To test whether antibodies that are neutralizing or nonneutralizing for human immunodeficiency virus type 1 (HIV-1) primary isolates can be distinguished by their affinities for the oligomeric envelope glycoproteins, we selected HIV-1(JR-FL) as a model primary virus and a panel of 13 human monoclonal antibodies (MAbs) and evaluated three parameters: (i) half-maximal binding to recombinant monomeric envelope, gp120(JR-FL); (ii) half-maximal binding to oligomeric envelope of HIV-1(JR-FL) expressed on the surface of transfected 293 cells; and (iii) neutralization of HIV-1(JR-FL) in a peripheral blood mononuclear cell-based neutralization assay. Two conclusions can be drawn from these experiments. First, we confirm that antibody interactions with monomeric gp120 do not predict primary virus neutralization. Second, we show that neutralization correlates qualitatively with the relative affinity of an antibody for the oligomeric envelope glycoproteins, at least for HIV-1(JR-FL).     

Varying effects of temperature, Ca(2+) and cytochalasin on fusion activity mediated by human immunodeficiency virus type 1 and type 2 glycoproteins

We examined fusion mediated by the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) envelope glycoproteins under various experimental conditions. Incubation of HeLa cells expressing HIV-2(ROD) and HIV-2(SBL/ISY) envelope glycoproteins with HeLa-CD4 target cells resulted in fusion at temperatures >/=25 degrees C whereas fusion with cells expressing HIV-1(Lai) occurred only at >/=31 degrees C. HIV-2 envelope glycoprotein-mediated fusion proceeded in the absence of Ca(2+) in the culture medium, whereas HIV-1 fusion required Ca(2+) ions for fusion. In contrast to HIV-2 envelope glycoprotein fusion, incubations in the presence of the 0.5 microM cytochalasin B completely inhibited HIV-1 envelope glycoprotein-mediated fusion. Our results suggest that in contrast to HIV-2, HIV-1 fusion is dependent on dynamic processes in the target membrane.     

Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins.     

Role of CD4 endocytosis in human immunodeficiency virus infection.

We have analyzed the role of CD4 endocytosis in human immunodeficiency virus (HIV) entry by measuring the infection of HeLa cells expressing various CD4 constructs with endocytosis rates of between 0.2 and 30%/min in a quantitative infectious focus assay. For a number of laboratory-adapted HIV-1 and HIV-2 strains, the highest levels of infection were found on cells with very limited CD4 endocytosis, while cells with efficient CD4 uptake were only poorly infectable, suggesting that CD4 internalization is not required for HIV entry. This was confirmed in a modified assay involving prebinding of HIV-1LAI to HeLa-CD4 cells at 4 degrees C, synchronized virus entry during warming to 37 degrees C, and neutralization of virions remaining at the cell surface with anti-V3 loop antibodies. Warming cells in hypertonic medium inhibited CD4 endocytosis but did not affect the rate or the extent of infection. These studies confirm that HIV infection does not require endocytosis and that laboratory-adapted virus strains can enter HeLa-CD4 cells by fusion at the plasma membrane.     

Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.     

Adaptation to persistent growth in the H9 cell line renders a primary isolate of human immunodeficiency virus type 1 sensitive to neutralization by vaccine sera.

Seven diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) were examined and found to be refractory to neutralization by antisera to recombinant gp120 (rgp120) protein from HIV-1 MN. This stands in marked contrast to the sensitivity exhibited by certain laboratory-adapted viruses. To understand the difference between primary and laboratory-adapted viruses, we adapted the primary virus ACH 168.10 to growth in the FDA/H9 cell line. ACH 168.10 was chosen because the V3 region of gp120 closely matches that of MN. After 4 weeks, infection became evident. The virus (168A) replicated in FDA/H9 cells with extensive cytopathic effect but was unchanged in sensitivity to antibody-mediated neutralization. Thus, growth in cell lines is not sufficient to render primary virus sensitive to neutralization. The 168A virus was, however, partially sensitive to CD4 immunoadhesin (CD4-Ig). Adaptation was continued to produce a persistently infected FDA/H9 culture that displayed minimal cytopathic effect. The virus (168C) was now sensitive to neutralization by MN rgp120 vaccine sera and by MN-specific monoclonal antibodies and showed increased sensitivity to HIVIG and CD4-Ig. 168C encoded three amino acid changes in gp120, including one within the V3 loop (I-166-->R, I-282-->N, G-318-->R). MN-specific monoclonal antibodies bound equally to the surface of cells infected with either neutralization-resistant or -sensitive virus. The coincidence of changes in neutralization sensitivity with changes in cell tropism and cytopathic effect suggests a common underlying mechanism(s) acting through the whole of the envelope protein complex.     

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

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

Functional role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization.

We have examined the influence of the V1/V2 region of the human immunodeficiency virus type 1 (HIV-1) gp120 on certain biologic properties of the virus. We observed that on the genomic background of the T-cell-line-tropic strain, HIV-1SF2mc, both the V1 and V2 domains of the macrophage-tropic strain, HIV-1SF162mc, in addition to the required V3 domain, are necessary to attain full macrophage tropism. Furthermore, the V2 domain modulates the sensitivity of HIV-1 to soluble CD4 neutralization. Structural studies of recombinant and mutant envelope glycoproteins suggest that the function of the V1/V2 region is to interact with the V3 domain and confer on the envelope gp120 of HIV-1SF2mc a conformation more similar to that of the macrophage-tropic strain HIV-1SF162mc. The conformation of the envelope gp120 appears to be strain specific and plays an important role in determining HIV-1 tissue tropism and sensitivity to soluble CD4 neutralization.     

Increased envelope spike density and stability are not required for the neutralization resistance of primary human immunodeficiency viruses.

Previous observations that the gp120 envelope glycoprotein contents of some primary, clade B human immunodeficiency virus type 1 (HIV-1) isolates were higher than those of laboratory-passaged HIV-1 isolates suggested the hypothesis that increased envelope glycoprotein spike density or stability contributes to the relative neutralization resistance of the primary viruses. To test this, the structural, replicative, and neutralization properties of a panel of recombinant viruses with HIV-1 envelope glycoproteins from divergent clades were examined in an env complementation assay. In this system, although the spike density and stability of envelope glycoproteins from primary HIV-1 isolates were not greater than those from a laboratory-adapted isolate, relative resistance to neutralizing antibodies and soluble CD4 was observed for the viruses with primary envelope glycoproteins. Thus, neither high envelope glycoprotein spike density nor stability is necessary for the relative neutralization resistance of primary HIV-1 viruses.     

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.     

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.     

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.     

Macrophage-tropic and T-cell line-adapted chimeric strains of human immunodeficiency virus type 1 differ in their susceptibilities to neutralization by soluble CD4 at different temperatures.

Molecular clones of three macrophage-tropic and three T-cell line-adapted strains of human immunodeficiency virus type 1 (HIV-1) were used to explore the mechanism of HIV-1 resistance to neutralization by soluble CD4 (sCD4). The three macrophage-tropic viruses, each possessing the V3 and flanking regions of JR-FL, were all resistant to sCD4 neutralization under the standard conditions of a short preincubation of the virus and sCD4 at 37 degrees C prior to inoculation of peripheral blood mononuclear cells. In contrast, the three T-cell line-adapted viruses, NL4-3 and two chimeras possessing the V3 and flanking regions of NL4-3 in the envelope background of JR-FL, were all sCD4 sensitive under these conditions. Sensitivity to sCD4 neutralization at 37 degrees C corresponded with rapid, sCD4-induced gp120 shedding from the viruses. However, when the incubation temperature of the sCD4 and virus was reduced to 4 degrees C, the three macrophage-tropic viruses shed gp120 and became more sensitive to sCD4 neutralization. In contrast, the rates of sCD4-induced gp120 shedding and virus neutralization were reduced for the three T-cell line-adapted viruses at 4 degrees C. Thus, HIV resistance to sCD4 is a conditional phenomenon; macrophage-tropic and T-cell line-adapted strains can be distinguished by the temperature dependencies of their neutralization by sCD4. The average density of gp120 molecules on the macrophage-tropic viruses exceeded by about fourfold that on the T-cell line-adapted viruses, suggesting that HIV growth in T-cell lines may select for a destabilized envelope glycoprotein complex. Further studies of early events in HIV-1 infection should focus on primary virus strains.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.     

Mapping genetic determinants for human immunodeficiency virus type 1 resistance to soluble CD4.

Neutralization of human immunodeficiency virus type 1 (HIV-1) infection with soluble CD4 (sCD4) can be achieved over a broad range of concentrations for different virus strains. Laboratory virus strains passaged in transformed T-cell lines are typically sensitive to sCD4 neutralization, whereas primary virus isolates require over 100-fold-higher sCD4 concentrations. Using recombinant viruses generated from a laboratory strain, HIV-1NL4-3, and a primary macrophagetropic strain, HIV-1JR-FL, we mapped a region of gp120 important for determining sensitivity to sCD4 neutralization. This same region has previously been defined as important for macrophage and transformed T-cell line tropism and includes the V3 neutralization domain but does not include regions of gp120 that have been shown to be most important for CD4 binding.     

Chemically induced infection of CD4-negative HeLa cells with HIV-1

Infection with human immunodeficiency virus type-1 (HIV-1) requires the presence of a CD4 molecule and chemokine receptors such as CXCR4 or CCR5 on the surface of target cells. However, it is still not clear how the virus enters the cells. Although CD4 was initially identified as the primary receptor for HIV-1, the expression of CD4 or one of the chemokine receptors alone is not sufficient to render susceptibility to infection with the virus. To ascertain whether or not adsorption of the virus needs charge-to-charge interaction between viral envelope and host cell membrane protein(s) and if binding alone promotes penetration of the virus into the cells, we have developed a chemically induced infection system targeting a CD4-negative and CXCR4-positive HeLa cell clone (N7 HeLa) which is usually not susceptible to infection with the LAI strain of HIV-1. Use of a poly-L-lysine (PLL)-coated culture plate to enhance the attachment of the virus to the cells made N7 HeLa cells infectable with HIV-1 at very low efficiency. PLL alone cannot fully substitute for the function of the CD4 molecule. However, trypsin-treated viruses, which have largely lost infectivity to CD4-positive MT-4 cells that are highly susceptible to HIV-1 infection, enhanced infectivity against N7 HeLa cells when the PLL-coated plate was used. These results provide evidence that infection with HIV-1 requires both high binding affinity between viruses and cells, and then needs a modification of the viral envelope such as cleavage of gp120/160 to enhance the infection, probably resulting in exposure of the hydrophobic fusion domain of gp41. HIV-1 infection of N7 HeLa cells was also enhanced by treatment with low pH, 12-O-tetradecanoylphorbol-13-acetate (TPA) and some factor(s) from the MT-4 cell culture supernatant. Not only tight viral adsorption with cleavage of the viral envelope but also some activated status of the cells may be required for sufficient HIV-1 infection in this artificial condition.     

Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants

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

Subunit stoichiometry of human immunodeficiency virus type 1 envelope glycoprotein trimers during virus entry into host cells

The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope glycoproteins, we found that two wild-type subunits within an envelope glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes.