Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.     

Direct measurement of soluble CD4 binding to human immunodeficiency virus type 1 virions: gp120 dissociation and its implications for virus-cell binding and fusion reactions and their neutralization by soluble CD4.

We have analyzed the binding of soluble CD4 (sCD4) to human immunodeficiency virus type 1 (HIV-1) virions (isolates IIIB and RF) at 4 and 37 degrees C by using a combination of gel exclusion chromatography and enzyme-linked immunosorbent assay detection systems. The sCD4 binding curve at 37 degrees C indicates that the affinity of the interaction of sCD4 with gp120 on the virion surface is indistinguishable from the affinity of sCD4 for the equivalent concentration of soluble gp120. At 4 degrees C, however, the affinity of sCD4 for virion-bound gp120 but not for soluble gp120 is reduced by about 20-fold. Binding of sCD4 (greater than 0.2 microgram/ml) to virions at 37 degrees C but not 4 degrees C induces the rapid dissociation of a major proportion of gp120 from gp41 on the virion surface. This dissociation requires occupancy by sCD4 of multiple (probably two) binding sites on a gp120-gp41 oligomer. At 37 degrees C there are two components to the neutralizing action of sCD4 on HIV-1; reversible, competitive inhibition at low sCD4 concentrations (less than 0.2 microgram/ml) and essentially irreversible inhibition due to gp120 loss at higher sCD4 concentrations. At 4 degrees C, sCD4 neutralizes HIV infectivity by competitive inhibition alone. These findings may have implications for the HIV-CD4+ cell binding and fusion reactions and the mechanism by which sCD4 blocks infectivity.     

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

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

Peptide mimic of the HIV envelope gp120-gp41 interface

The human immunodeficiency virus envelope glycoprotein (Env) is composed of surface (gp120) and transmembrane (gp41) subunits, which are noncovalently associated on the viral surface. Human immunodeficiency virus Env mediates viral entry after undergoing a complex series of conformational changes induced by interaction with cellular CD4 and a chemokine coreceptor. These changes propagate from gp120 to gp41 via the gp120-gp41 interface, ultimately exposing gp41 and allowing it to form the trimer-of-hairpins structure that provides the driving force for membrane fusion. Key unresolved questions about the gp120-gp41 interface include the specific regions of gp41 and gp120 involved, the mechanism by which receptor and coreceptor-binding-induced conformational changes in gp120 are communicated to gp41, how trimer-of-hairpins formation is prevented in the prefusogenic gp120-gp41 complex, and, ultimately, the structure of the prefusion gp120-gp41 complex. Here, we develop a biochemical model system that mimics a key portion of the gp120-gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and C-peptide region binds primarily to the gp120 C5 region and that this interaction is incompatible with trimer-of-hairpins formation. Based on these data, we propose that in prefusogenic Env, gp120 sequesters the gp41 C-peptide region away from the N-trimer region, preventing trimer-of-hairpins formation until coreceptor binding disrupts this interface. This model system is a valuable tool for studying the gp120-gp41 complex, conformational changes induced by CD4 and coreceptor binding, and the mechanism of membrane fusion.     

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.     

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4⁺ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.     

Two mechanisms of soluble CD4 (sCD4)-mediated inhibition of human immunodeficiency virus type 1 (HIV-1) infectivity and their relation to primary HIV-1 isolates with reduced sensitivity to sCD4.

Two assays for measuring inhibition of human immunodeficiency virus type 1 (HIV-1) infection by soluble CD4 (sCD4) are described. Experiments in which sCD4, HIV-1, and cell concentrations and sequence of combination, noninfectious/infectious particle ratio, and temperature were varied produced results that support the conclusion that sCD4 inhibits HIV-1 infection by two mechanisms: reversible blockage of receptor binding and irreversible inactivation of infectivity. Fresh isolates obtained from HIV-1-infected persons were tested in both assays and found to be more resistant to both mechanisms of sCD4-mediated inhibition than multiply passaged laboratory strains. Binding studies revealed similar affinities for sCD4 in detergent lysates of sensitive and resistant strains at both 4 and 37 degrees C. The avidity of intact virions for sCD4 was lower at 4 than at 37 degrees C, and in the presence of excess sCD4, less sCD4 was bound at 4 than at 37 degrees C. The avidity differences were similar for fresh isolates and laboratory strains. However, fresh isolates were more resistant to sCD4-induced shedding of envelope glycoprotein gp120 from intact virions than was the laboratory strain. Relative resistance to sCD4 by certain isolates does not represent a lower intrinsic affinity of their envelope for sCD4 or a lower capacity for sCD4 binding. Rather, an event that occurs after binding may account for the differences. This postbinding event or feature may be determined by regions of the envelope outside the CD4 binding site.     

Equal levels of gp120 retention and neutralization resistance of phenotypically distinct primary human immunodeficiency virus type 1 variants upon soluble CD4 treatment.

Human immunodeficiency virus type 1 (HIV-1) variants passaged in T-cell lines, often called laboratory isolates, are potently neutralized by soluble CD4 (sCD4), whereas primary HIV-1 variants are highly resistant to sCD4 neutralization. Previously, it was demonstrated that the domain from V1 to V3 of the HIV-1 gp120 molecule contains one of the major determinants of sCD4 neutralization sensitivity, and the same region has also been implicated as influencing syncytium-inducing (SI) capacity and T-cell-line tropism. To determine possible differences in sCD4 neutralization sensitivity between phenotypically distinct primary HIV-1 variants, a panel of non-syncytium-inducing (NSI) and SI HIV-1 variants was studied. Primary NSI and SI HIV-1 variants appeared to be equally resistant to sCD4 neutralization. Consistent with this observation, sCD4 did not induce gp120 shedding from either primary NSI or SI HIV-1 variants at 37 degrees C. Thus, it is not the potential of certain primary HIV-1 variants to infect T-cell lines but rather their adaptation to T-cell lines that is reflected in specific properties of the viral envelope which influence sCD4 neutralization sensitivity.     

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.     

Mode of action for linear peptide inhibitors of HIV-1 gp120 interactions

The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.     

Functional evolution of the HIV-1 envelope glycoprotein 120 association site of glycoprotein 41

Protein-protein interaction surfaces can exhibit structural plasticity, a mechanism whereby an interface adapts to mutations as binding partners coevolve. The HIV-1 envelope glycoprotein gp120-gp41 complex, which is responsible for receptor attachment and membrane fusion, represents an extreme example of a coevolving complex as up to 35% amino acid sequence divergence has been observed in these proteins among HIV-1 isolates. In this study, the function of conserved gp120 contact residues, Leu593, Trp596, Gly597, Lys601, and Trp610 within the disulfide-bonded region of gp41, was examined in envelope glycoproteins derived from diverse HIV-1 isolates. We found that the gp120-gp41 association function of the disulfide-bonded region is conserved. However, the contribution of individual residues to gp41 folding and/or stability, gp120-gp41 association, membrane fusion function, and viral entry varied from isolate to isolate. In gp120-gp41 derived from the dual-tropic isolate, HIV-189.6, the importance of Trp596 for fusion function was dependent on the chemokine receptor utilized as a fusion cofactor. Thus, the engagement of alternative chemokine receptors may evoke distinct fusion-activation signals involving the site of gp120-gp41 association. An examination of chimeric glycoproteins revealed that the isolate-specific functional contributions of particular gp120-contact residues are influenced by the sequence of gp120 hypervariable regions 1, 2, and 3. These data indicate that the gp120-gp41 association site is structurally and functionally adaptable, perhaps to maintain a functional glycoprotein complex in a setting of host selective pressures driving the rapid coevolution of gp120 and gp41.     

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.     

Functional links between the fusion peptide-proximal polar segment and membrane-proximal region of human immunodeficiency virus gp41 in distinct phases of membrane fusion

The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp(666), Trp(672), Phe(673), and Ile(675) in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by approximately 10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu(537) with Trp(666), Trp(672), Phe(673), or Ile(675) abolished entry with 50-80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an approximately 80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.     

Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1_QH1549.13 blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.     

Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01

The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike.     

Infectious properties of human immunodeficiency virus type 1 mutants with distinct affinities for the CD4 receptor.

Recent evidence suggests that primary patient isolates of T-cell-tropic human immunodeficiency virus type 1 (HIV-1 ) have lower affinities for CD4 than their laboratory-adapted derivatives, that this may partly result from tighter gp120-gp41 bonds that constrain the CD4 binding sites of the primary viruses, and that selection for increased CD4 affinity may be the principal factor in laboratory adaptation of HIV-1 (S. L. Kozak, E. J. Platt, N. Madani, F. E. Ferro, Jr., K. Peden, and D. Kabat, J. Virol. 71:873-882, 1997). These conclusions were based on studies with a panel of HeLa-CD4 cell clones that differ in CD4 levels over a broad range, with laboratory-adapted viruses infecting all clones with equal efficiencies and primary T-cell-tropic viruses infecting the clones in proportion to cellular CD4 levels. Additionally, all of the primary and laboratory-adapted T-cell-tropic viruses efficiently used CXCR-4 (fusin) as a coreceptor. To test these conclusions by an independent approach, we studied mutations in the laboratory-adapted virus LAV/IIIB that alter the CD)4 binding region of gp120 and specifically reduce CD4 affinities of free gp 120 by 85 to 98% (U. Olshevsky et al., J. Virol. 64:5701-5707, 1990). These mutations reduced virus titers to widely varying extents that ranged from severalfold to several orders of magnitude and converted infectivities on the HeLa-CD4 panel from CD4 independency to a high degree of CD4 dependency that resembled the behavior of primary patient viruses. The relative infectivities of the mutants correlated closely with their sensitivities to inactivation by soluble CD4 but did not correlate with the relative CD4 affinities of their free gp120s. Most of the mutations did not substantially alter envelope glycoprotein synthesis, processing, expression on cell surfaces, incorporation into virions, or rates of gp120 shedding from virions. However, one mutation (D457R) caused a decrease in gp160 processing by approximately 80%. The fact that several mutations increased rates of spontaneous viral inactivation (especially D368P) suggests that HIV-1 life spans may be determined by structural stabilities of viral envelope glycoproteins. All of the wild-type and mutant viruses were only slowly and inefficiently adsorbed onto cultured CD4-positive cells at 37 degrees C, and the gradual declines in viral titers in the media were caused almost exclusively by spontaneous inactivation rather than by adsorption. The extreme inefficiency with which infectious HIV-1 is able to infect cultured susceptible CD4-positive cells in standard assay conditions casts doubt on previous inferences that the vast majority of retrovirions produced in cultures are noninfectious. Apparent infectivity of T-cell-tropic HIV-1 in culture is limited by productive associations with CD4 and is influenced in an interdependent manner by CD4 affinities of viral gp120-gp41 complexes and quantities of cell surface CD4.     

The disulfide loop of gp41 is critical to the furin recognition site of HIV gp160

The importance of the HIV gp41 conserved disulfide loop to envelope function has been examined by mutational and functional analyses. Based on a luciferase-reporter entry assay, mutants gp41-CC/AA (C598A/C604A) and gp41-Delta (deletion of residues 596-606) result in a nonfunctional envelope protein. Western blot analysis shows both mutants to be properly expressed but not processed to form gp120 and gp41, which explains their nonfunctionality. The presence of mutant gp160 on the cell surface, as well as their ability to bind to sCD4, suggests that the mutations have disrupted processing at the furin recognition site encoded within the gp120 conserved domain 5, without resulting in an overall misfolding of the protein. With respect to the furin recognition site, the mutations are sequentially distant, which implies that the gp41 disulfide loop is interacting with gp120 C5 in gp160. In addition, we have modeled the gp120-gp41 interaction in unprocessed precursor gp160 using structural data available for gp120 and gp41 domains in isolation, supplemented by mutagenesis data. We suggest that the mutations have altered the interaction between gp120 C5 and the gp41 disulfide loop, resulting in decreased accessibility of the furin recognition site and implying that the interaction between the gp120 C5 and gp41 loop is a conformational requirement for gp160 processing. The sensitivity of this interaction could be exploited in future antivirals designed to disrupt HIV pathogenesis by disrupting gp160 processing.     

Phorbol ester-induced down modulation of tailless CD4 receptors requires prior binding of gp120 and suggests a role for accessory molecules.

The entry of human immunodeficiency virus type 1 into cells proceeds via a fusion mechanism that is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. Species- and tissue-specific restrictions to viral entry suggested the participation of additional membrane components in the postbinding fusion events. In a previous study (H. Golding, J. Manischewitz, L. Vujcic, R. Blumenthal, and D. Dimitrov, J. Virol. 68:1962-1968, 1994), it was found that phorbol myristate acetate (PMA) inhibits human immunodeficiency virus type 1 envelope-mediated cell fusion by inducing down modulation of an accessory component(s) in the CD4-expressing cells. The fusion inhibition was seen in a variety of cells, including T-cell transfectants expressing engineered CD4 receptors (CD4.401 and CD4.CD8) which are not susceptible to down modulation by PMA treatment. In the current study, it was found that preincubation of A2.01.CD4.401 cells with soluble monomeric gp120 for 1 h at 37 degrees C primed them for PMA-induced down modulation (up to 70%) of the tailless CD4 receptors. The gp120-priming effect was temperature dependent, and the down modulation may have occurred via clathrin-coated pits. Importantly, nonhuman cell lines expressing tailless CD4 molecules did not down modulate their CD4 receptors under the same conditions. The gp120-dependent PMA-induced down modulation of tailless CD4 receptors could be efficiently blocked by the human monoclonal antibodies 48D and 17B, which bind with increased avidity to gp120 that was previously bound to CD4 (M. Thali, J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski, J. Virol. 67:3978-3988, 1993). These findings suggest that gp120 binding to cellular CD4 receptors induces conformational changes leading to association of the gp120-CD4 complexes with accessory transmembrane molecules that are susceptible to PMA-induced down modulation and can target the virions to clathrin-coated pits.     

Mutation-Directed Chemical Cross-Linking of Human Immunodeficiency Virus Type 1 gp41 Oligomers

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS³) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS³ generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic α-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal α-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal α-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.     

Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³⁶ in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³⁹INN sequence, which ablates the overlapping Asn¹⁴¹-Asn¹⁴²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu⁵⁹³, Trp⁵⁹⁶ and Lys⁶⁰¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.