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.     

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.     

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.     

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.     

Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex

The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.     

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.     

Biochemical and genetic characterizations of a novel human immunodeficiency virus type 1 inhibitor that blocks gp120-CD4 interactions

BMS-378806 is a recently discovered small-molecule human immunodeficiency virus type 1 (HIV-1) attachment inhibitor with good antiviral activity and pharmacokinetic properties. Here, we demonstrate that the compound targets viral entry by inhibiting the binding of the HIV-1 envelope gp120 protein to cellular CD4 receptors via a specific and competitive mechanism. BMS-378806 binds directly to gp120 at a stoichiometry of approximately 1:1, with a binding affinity similar to that of soluble CD4. The potential BMS-378806 target site was localized to a specific region within the CD4 binding pocket of gp120 by using HIV-1 gp120 variants carrying either compound-selected resistant substitutions or gp120-CD4 contact site mutations. Mapping of resistance substitutions to the HIV-1 envelope, and the lack of compound activity against a CD4-independent viral infection confirm the gp120-CD4 interactions as the target in infected cells. BMS-378806 therefore serves as a prototype for this new class of antiretroviral agents and validates gp120 as a viable target for small-molecule inhibitors.     

An aptamer that neutralizes R5 strains of human immunodeficiency virus type 1 blocks gp120-CCR5 interaction

We recently described the isolation and structural characterization of 2'-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency.     

CD4-induced T-20 binding to human immunodeficiency virus type 1 gp120 blocks interaction with the CXCR4 coreceptor

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.     

Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gp120.

A previously reported amino acid substitution within the second conserved domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope results in the production of noninfectious particles. Molecular characterization of spontaneous revertant viruses, which arose during long-term cocultures of this env mutant, revealed that an amino acid change within another region of gp120 could functionally compensate for the mutation and restore infectivity. In the current study, we have introduced a conservative amino acid substitution at this second-site revertant codon and observed a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared which contained an amino acid change within a variable region of gp120 which restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the HIV-1 envelope, suggest that at least three discrete regions of gp120 may interact during the establishment of a productive viral infection. This critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to or concomitant with the fusion of viral and cellular membranes.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.     

CD4-independent use of Rhesus CCR5 by human immunodeficiency virus Type 2 implicates an electrostatic interaction between the CCR5 N terminus and the gp120 C4 domain

Envelope glycoproteins (Envs) of human immunodeficiency virus type 2 (HIV-2) are frequently able to use chemokine receptors, CXCR4 or CCR5, in the absence of CD4. However, while these Envs are commonly dual-tropic, no isolate has been described to date that is CD4 independent on both CXCR4 and CCR5. In this report we show that a variant of HIV-2/NIHz, termed HIV-2/vcp, previously shown to utilize CXCR4 without CD4, is also CD4 independent on rhesus (rh) CCR5, but requires CD4 to fuse with human (hu) CCR5. The critical determinant for this effect was an acidic amino acid at position 13 in the CCR5 N terminus, which is an asparagine in huCCR5 and an aspartic acid in rhCCR5. Transferring the huCCR5 N terminus with an N13D substitution to CCR2b or CXCR2 was sufficient to render these heterologous chemokine receptors permissive for CD4-independent fusion. Chimeric Envs between HIV-2/vcp and a CD4-dependent clone of HIV-2/NIHz as well as site-directed Env mutations implicated a positively charged amino acid (lysine or arginine) at position 427 in the C4 region of the HIV-2/vcp env gene product (VCP) gp120 as a key determinant for this phenotype. Because CD4-independent use of CCR5 mapped to a negatively charged amino acid in the CCR5 N terminus and a positively charged amino acid in the gp120 C4 domain, an electrostatic interaction between these residues or domains is likely. Although not required for CD4-dependent fusion, this interaction may serve to increase the binding affinity of Env and CCR5 and/or to facilitate subsequent conformational changes that are required for fusion. Because the structural requirements for chemokine receptor use by HIV are likely to be more stringent in the absence of CD4, CD4-independent viruses should be particularly useful in dissecting molecular events that are critical for viral entry.     

Dextran sulfate blocks antibody binding to the principal neutralizing domain of human immunodeficiency virus type 1 without interfering with gp120-CD4 interactions.

The mechanism of the antiviral activity of sulfated polysaccharides on human immunodeficiency virus type 1 (HIV-1) was investigated by determining the effect of dextran sulfate on the binding of CD4 and several anti-gp120 monoclonal antibodies to both recombinant and cell surface gp120. Dextran sulfate did not interfere with the binding of sCD4 to rgp120 on enzyme-linked immunosorbent assay (ELISA) plates or in solution and did not block sCD4 binding to HIV-1-infected cells expressing gp120 on the cell surface. Dextran sulfate had minimal effects on rgp120 binding to CD4+ cells at concentrations which effectively prevent HIV replication. In contrast, it potently inhibited the binding of both rgp120 and cell surface gp120 to several monoclonal antibodies directed against the principal neutralizing domain of gp120 (V3). In an ELISA format, dextran sulfate enhanced the binding of monoclonal antibodies against amino-terminal regions of gp120 and had no effect on antibodies directed to other regions of gp120, including the carboxy terminus. The inhibitory effects of polyanionic polysaccharides on viral binding, viral replication, and formation of syncytia therefore appear mediated by interactions with positively charged amino acids concentrated in the V3 region. This high local positive charge density, unique to the V3 loop, leads us to propose that this property is critical to the function of the V3 region in mediating envelope binding and subsequent fusion between viral and cell membranes. The specific interaction of dextran sulfate with this domain suggests that structurally related molecules on the cell surface, such as heparan sulfate, may be additional targets for HIV binding and infection.     

Antibody-mediated enhancement of human immunodeficiency virus type 1 infectivity is determined by the structure of gp120 and depends on modulation of the gp120-CCR5 interaction

In this study, we characterized the viral determinants of coreceptor usage in relation to susceptibility to antibody-mediated neutralization or enhancement of infectivity by using chimeras of three highly related human immunodeficiency virus type 1 (HIV-1) isolates of different phenotypes. We found that the V3 region was the main determinant of antibody-mediated enhancement and coreceptor specificity but that the overall structure of gp120 was also important for these properties. Constructs susceptible to antibody-mediated enhancement preferentially use CCR5 as a coreceptor, in contrast to constructs that were neutralized or not affected. Using monoclonal antibodies directed against CD4 or CCR5, we were able to show that antibody-mediated enhancement was CD4 dependent. Altogether, our results suggest that the modulation of the interaction of gp120 with CCR5 is the mechanism underlying antibody-mediated enhancement of HIV-1 infectivity.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

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

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

Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to CCR5

The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs' abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events.