Human immunodeficiency virus type 1 envelope glycoproteins gp120 and gp160 induce interleukin-6 production in CD4+ T-cell clones.

Polyclonal B-cell activation is a characteristic feature of AIDS and of the AIDS-related complex. Since the immunoregulatory cytokine interleukin-6 (IL-6) plays a major role in inducing B-cell differentiation, we examined the effects of native human immunodeficiency virus type 1 envelope glycoproteins gp120 and gp160 on IL-6 induction. In this study, we have demonstrated that both gp120 and gp160 have the ability to induce IL-6 mRNA and biologically active IL-6 protein secretion in peripheral blood mononuclear cells in vitro. The envelope protein preparations had no detectable endotoxin as tested by the Limulus amebocyte lysate assay, and hence we can rule out the effect of contaminating endotoxin, which is a potent inducer of IL-6 in monocyte/macrophage cell cultures. In addition, we have shown that the envelope glycoproteins act directly on CD4(+)-cloned T cells to induce IL-6 production in the absence of monocytes. These findings indicate that monocytes and T cells both contribute to the secretion of IL-6, which plays an important role in the pathogenesis of B-cell activation in human immunodeficiency virus infection.     

Effects of changes in gp120-CD4 binding affinity on human immunodeficiency virus type 1 envelope glycoprotein function and soluble CD4 sensitivity.

Mutant gp120 glycoproteins exhibiting a range of affinities for CD4 were tested for ability to form syncytia and to complement an env-defective provirus for replication. Surprisingly, gp120 mutants that efficiently induced syncytia and/or complemented virus replication were identified that exhibited marked (up to 50-fold) reductions in CD4-binding ability. Temperature-dependent changes in gp120, which result in a seven- to ninefold increase in affinity for CD4, were shown not to be necessary for subsequent membrane fusion or virus entry events. Mutant glycoproteins demonstrating even relatively small decreases in CD4-binding ability exhibited reduced sensitivity to soluble CD4. The considerable range of CD4-binding affinities tolerated by replication-competent HIV-1 variants has important implications for antiviral strategies directed at the gp120-CD4 interaction.     

Human immunodeficiency virus type 1 envelope glycoprotein molecules containing membrane fusion-impairing mutations in the V3 region efficiently undergo soluble CD4-stimulated gp120 release.

The ability of soluble forms of CD4 to induce gp120 release from the human immunodeficiency virus type 1 envelope glycoprotein complex may reflect molecular events associated with membrane fusion. The third hypervariable (V3) region of gp120 appears to play a role in fusion independent of CD4 binding. We demonstrate herein that envelope glycoprotein molecules rendered fusion defective by mutations in conserved residues within the V3 region nevertheless undergo efficient soluble CD4-induced gp120 release.     

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

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

Differential regulation of cellular tropism and sensitivity to soluble CD4 neutralization by the envelope gp120 of human immunodeficiency virus type 1.

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.     

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4⁺ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4⁺ T cells.     

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

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

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Interaction with CD4 and antibodies to CD4-induced epitopes of the envelope gp120 from a microglial cell-adapted human immunodeficiency virus type 1 isolate

We previously showed that the envelope glycoprotein from an in vitro microglia-adapted human immunodeficiency virus type 1 isolate (HIV-1(Bori-15)) is able to use lower levels of CD4 for infection and demonstrates greater exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody than the envelope of its parental, peripheral isolate (HIV-1(Bori)). We investigated whether these phenotypic changes were related to a different interaction of their soluble monomeric gp120 proteins with CD4 or 17b. Equilibrium binding analyses showed no difference between Bori and Bori-15 gp120s. However, kinetic analysis of surface plasmon resonance-based, real-time binding experiments showed that while both proteins have similar association rates, Bori-15 gp120 has a statistically significant, 3-fold-lower dissociation rate from immobilized CD4 than Bori and a statistically significant, 14-fold-lower dissociation rate from 17b than Bori in the absence of soluble CD4. In addition, using the sensitivity to inhibition by anti-CD4 antibodies as a surrogate for CD4:trimeric envelope interaction, we found that Bori-15 envelope-pseudotyped viruses were significantly less sensitive than Bori pseudotypes, with four- to sixfold-higher 50% inhibitory concentration values for the three anti-CD4 antibodies tested. These differences, though small, suggest that adaptation to microglia correlates with the generation of a gp120 that forms a more stable interaction with CD4. Nonetheless, the observation of limited binding changes leaves open the possibility that HIV-1 adaptation to microglia and HIV-associated dementia may be related not only to diminished CD4 dependence but also to changes in other molecular factors involved in the infection process.     

Increase in soluble CD4 binding to and CD4-induced dissociation of gp120 from virions correlates with infectivity of human immunodeficiency virus type 1.

Mutations in the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41, previously shown to confer an enhanced replicative capacity and broadened host range to the ELI1 strain of HIV-1, were analyzed for their biochemical effects on envelope structure and function. The tendency of purified virions to release their extracellular gp120 component, either spontaneously or after interacting with soluble CD4 (CD4-induced shedding) was assessed. A single amino acid substitution in part of the CD4 binding site of gp120 (Gly-427 to Arg) enhanced both spontaneous and CD4-induced shedding of gp120 from virions, while a single change in the fusogenic region of gp41 (Met-7 to Val) affected only CD4-induced shedding. Although each codon change alone conferred increased growth ability, virus with both mutations exhibited the most rapid replication kinetics. In addition, when both of these mutations were present, virions had the highest tendency to shed gp120, both spontaneously and after exposure to soluble CD4. Analysis of CD4 binding to virion-associated gp120 showed that the changes in both gp120 and gp41 contributed to increased binding. These results demonstrated that the increased replicative capacity of the ELI variants in human CD4+ cell lines was associated with altered physical and functional properties of the virion envelope glycoproteins.     

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

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

CD4 binding site antibodies inhibit human immunodeficiency virus gp120 envelope glycoprotein interaction with CCR5

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.     

Kinetics of soluble CD4 binding to cells expressing human immunodeficiency virus type 1 envelope glycoprotein.

The high-affinity interaction between the envelope glycoprotein (gp120-gp41) of the human immunodeficiency virus type 1 and its receptor, CD4, is important for viral entry into cells and therapeutical approaches based on the soluble form of CD4 (sCD4). Using flow cytometry, we studied the kinetics of binding of sCD4 to gp120-gp41 expressed on the cell surface. sCD4 binding was dependent on sCD4 concentration and temperature and exhibited bimolecular reaction kinetics. Binding was very slow at low sCD4 concentrations (below 0.2 micrograms/ml) and low temperatures (below 13 degrees C) but increased sharply with increasing temperature. The rate constant for association at 37 degrees C (1.5 x 10(5) M-1 s-1) was 14-fold higher than at 4 degrees C, but the affinity of sCD4 to membrane-bound gp120-gp41 was not significantly affected. The activation energy at higher temperatures (28 to 37 degrees C) was less than at lower temperatures (4 to 13 degrees C). After long periods of incubation, we observed a decrease of surface-bound sCD4 and gp120, even at low temperatures, which was attributed to sCD4-induced shedding of gp120. The rate of gp120 shedding was much lower than the rate of sCD4 binding and was dependent on sCD4 concentration and temperature. The finding that sCD4 binding is slow, especially at low sCD4 concentrations, can be of critical importance for efficient blocking of viral infection by sCD4 and should be considered when designing new protocols in the therapy of AIDS patients.     

Complex determinants in human immunodeficiency virus type 1 envelope gp120 mediate CXCR4-dependent infection of macrophages

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.     

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.     

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

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

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

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

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120

Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.     

Recombinant CD4-selected human immunodeficiency virus type 1 variants with reduced gp120 affinity for CD4 and increased cell fusion capacity.

Variants of molecularly cloned human immunodeficiency virus type 1 (HIV-1) were analyzed following selection for the ability to replicate after exposure to soluble, recombinant CD4 protein (rCD4). Two variants, 4/1 and 16/2, show 8-fold and 16-fold reduced sensitivity to rCD4 neutralization yet remain as sensitive as the parental wild-type (wt) virus to neutralization by rCD4-immunoglobulin G (IgG) chimeric molecules and to inhibition of cellular infection by anti-CD4 antibody. The 4/1 variant is more cytopathic, with faster cell fusion and replication kinetics than the wt virus. The gp120s derived from the 4/1 and 16/2 variants have 3-fold and 30-fold reduced binding affinities to rCD4, respectively. The 4/1 variant exhibits diminished shedding of virion gp120 induced by rCD4. The binding of and neutralization by V3 loop antibodies and other anti-gp120 antibodies is reduced for 4/1 but not for 16/2. Sequence analysis revealed a codon change at amino acid residue 435 in the C4 region of the gp120 of 16/2. This accounts for its rCD4 insensitivity, since the insertion of this mutation in the wt gp120 yields the same phenotype. The 4/1 variant has a codon change in the V3 region of gp120 (amino acid 311), which accounts for its reduced sensitivity to some neutralizing antibodies but not to rCD4. The ready selection of rCD4-resistant variants has obvious relevance for rCD4-based therapeutic stratagems.