Identification and characterization of an immunogenic hybrid epitope formed by both HIV gp120 and human CD4 proteins

Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection.     

A predicted three-dimensional structure for the carcinoembryonic antigen (CEA).

A three-dimensional model for the carcinoembryonic antigen (CEA) has been constructed by knowledge-based computer modelling. Each of the seven extracellular domains of CEA are expected to have immunoglobulin folds. The N-terminal domain of CEA was modelled using the first domain of the recently solved NMR structure of rat CD2, as well as the first domain of the X-ray crystal structure of human CD4 and an immunoglobulin variable domain REI as templates. The remaining domains were modelled from the first and second domains of CD4 and REI. Link conformations between the domains were taken from the elbow region of antibodies. A possible packing model between each of the seven domains is proposed. Each residue of the model is labelled as to its suitability for site-directed mutagenesis.     

Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.     

Structure of the Fab fragment of F105, a broadly reactive anti-human immunodeficiency virus (HIV) antibody that recognizes the CD4 binding site of HIV type 1 gp120

We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe(43) of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.     

Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.     

Protein minimization of the gp120 binding region of human CD4

CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.     

The HIV-1 envelope glycoprotein gp120 features four heparan sulfate binding domains, including the co-receptor binding site

It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg(419), Lys(421), and Lys(432), which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition.     

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.     

Role of CD4 epitopes outside the gp120-binding site during entry of human immunodeficiency virus type 1.

CD4 is the primary receptor for human immunodeficiency virus (HIV). The binding site for the surface glycoprotein of HIV type 1 (HIV-1), gp120, has been mapped to the C'-C" region of domain 1 of CD4. Previously, we have shown that a mutant of rat CD4, in which this region was exchanged for that of human CD4, is able to mediate infection of human cells by HIV-1, suggesting that essential interactions between HIV and CD4 are confined to this region. Our observations appeared to conflict with mutagenesis and antibody studies which implicate regions of CD4 outside the gp120-binding site in postbinding events during viral entry. In order to resolve this issue, we have utilized a panel of anti-rat CD4 monoclonal antibodies in conjunction with the rat-human chimeric CD4 to distinguish sequence-specific from steric effects. We find that several antibodies to rat CD4 inhibit HIV infection in cells expressing the chimeric CD4 and that this is probably due to steric hinderance. In addition, we demonstrate that replacement of the rat CDR3-like region with its human homolog does not increase the affinity of the rat-human chimeric CD4 for gp120 or affect the exposure of gp41 following binding to CD4, providing further evidence that this region does not play a crucial role during entry of virus.     

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.     

The V3 region of the envelope glycoprotein of human immunodeficiency virus type 1 binds sulfated polysaccharides and CD4-derived synthetic peptides.

gp120 is the envelope glycoprotein found on the surface of human immunodeficiency virus type 1 (HIV-1), and it binds to human cell surface CD4 receptors to initiate the HIV-1 infection process. It is now well-established that synthetic peptides from the V3 region on gp120 elicit antibodies that block HIV-1 infection and HIV-1-mediated cell fusion. Here we show that synthetic peptides derived from similar V3 regions of several isolates of HIV-1 bind [3H]heparin, and we also demonstrate that [3H]heparin binds to recombinant gp120 IIIB. The binding could be blocked by unlabeled heparin, dextran sulfate, and by a highly anionic benzylated synthetic peptide derived from human CD4 (amino acids 81-92). The nonbenzylated peptides from the same region were considerably less active. Unlabeled heparin, dextran sulfate, and the CD4-derived peptides were able to compete with the binding of soluble gp120 to immobilized antibodies against fragments of the V3 from isolate IIIB, but they had no effect on the binding of gp120 to anti-peptide antibodies targeted against another unrelated region of gp120. Biotin conjugated to the benzylated CD4-peptide bound to gp120 and was blocked from this binding by anti-V3 antibodies. These results indicate that the three materials that have been demonstrated by others to block HIV-1 infection in vitro, sulfated polysaccharides, certain CD4-derived synthetic peptides, and anti-V3 antibodies, may be acting through a common mechanism that includes binding to the V3 region of gp120 on HIV-1.     

Identification of the residues in human CD4 critical for the binding of HIV

The CD4 molecule is a T cell surface glycoprotein that interacts with high affinity with the envelope glycoprotein of the human immunodeficiency virus, HIV, thus serving as a cellular receptor for this virus. To define the sites on CD4 essential for binding to gp120, we produced several truncated, soluble derivatives of CD4 and a series of 26 substitution mutants. Quantitative binding analyses with the truncated proteins demonstrate that the determinants for high affinity binding lie solely with the first 106 amino acids of CD4 (the V1 domain), a region having significant sequence homology to immunoglobulin variable regions. Analysis of the substitution mutants further defines a discrete binding site within this domain that overlaps a region structurally homologous to the second complementarity-determining region of antibody variable domains. Finally, we demonstrate that the inhibition of virus infection and virus-mediated cell fusion by soluble CD4 proteins depends on their association with gp120 at this binding site.     

An efficient phage plaque screen for the random mutational analysis of the interaction of HIV-1 gp120 with human CD4.

A lambda phage expression methodology was adapted to dissect protein/ligand interactions efficiently through the creation and rapid screening of large numbers of mutants. Here we describe the method and its specific application to the interaction between the external envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, and the human cell surface protein CD4. Random substitutions were introduced throughout the gp120 binding region (amino acids 38-62) in the amino-terminal domain of CD4 by oligonucleotide mutagenesis. These mutations were expressed within phage plaques and directly screened for their effect on binding of gp120 using a modified phage plaque lift procedure. Plaques showing increased, decreased, and no effect on binding were identified and mutations were verified by sequence analysis. In this manner, 25 unique mutations were identified that altered CD4 binding to gp120. A new site was identified at which mutations reduced binding to gp120 and several novel amino acid substitutions were defined at sites previously implicated in binding. Of particular interest, this in vitro genetic approach identified a mutation which significantly increased binding to gp120. The phenotypes of several of these mutants were further characterized by quantitative measurement of their binding affinity. The results confirmed the accuracy of the phenotypic selection and demonstrated that the sensitivity of the system allowed detection of a 3-4-fold increase or decrease in affinity. In the context of the recently determined atomic structure of CD4, these results further implicate residues in the CDR2-like region and in an adjacent loop in recognition of gp120. This methodology should be generally applicable to other high affinity protein/ligand interactions that are compatible with expression in Escherichia coli.     

Is the V3 loop involved in HIV binding to CD4?

The entry of the human immunodeficiency virus into cells requires the interaction of the viral envelope glycoprotein gp120 with CD4 and a chemokine receptor. The gp120 binding site has been previously mapped to the Ig-CDR2-like region of CD4 first domain. A second area of this domain (Ig-CDR3-like region) is involved in gp120-CD4 interactions, but its gp120 counterpart remained so far unknown. Using a photoaffinity labeling experiment, we demonstrate that a peptide, mapping the (307-330)m region of HIV-MN-gp120 V3 loop, binds a sequence including a part of the Ig-CDR3-like region. These results may contribute to explain the complex mechanism of human immunodeficiency virus penetration, helping the development of new therapeutic agents.     

Synergistic inhibition of human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion and infection by an antibody to CD4 domain 2 in combination with anti-gp120 antibodies.

Antibodies to several epitopes of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41) can synergize in inhibiting HIV-1 infection. In the present study we tested the ability of a monoclonal antibody (MAb), 5A8, which interacts with CD4 domain 2, and other CD4-specific MAbs to synergize with antibodies against gp120. We have previously found that 5A8 inhibits HIV-1 entry without interfering with gp120 binding to CD4, presumably by affecting a postbinding membrane fusion event. Because antibodies to the gp120 V3 loop also affect post-CD4-gp120-binding events, 5A8 was first tested in combination with anti-V3 loop antibodies for possible synergy. The anti-V3 loop antibodies 0.5 beta, NEA-9205, and 110.5 acted synergistically with 5A8 in inhibiting syncytium formation between gp120-gp41- and CD4-expressing cells. A human MAb to an epitope of gp120 involved in CD4 binding, IAM 120-1B1, and another anti-CD4 binding site antibody, PC39.13, also exerted synergistic effects in combination with 5A8. Similarly, an antibody against the gp120 binding site on CD4, 6H10, acted synergistically with an anti-V3 loop antibody, NEA-9205. However, a control anti-CD4 antibody, OKT4, which does not significantly inhibit syncytium formation alone, produced only an additive effect when combined with NEA-9205. Serum from HIV-1-infected individuals, which presumably contains antibodies to the V3 loop and the CD4 binding site, exhibited a strong synergistic effect with 5A8 in inhibiting infection by a patient HIV-1 isolate (0104B) and in blocking syncytium formation. These results indicate that therapeutics based on antibodies affecting both non-gp120 binding and gp120 binding epitopes of the target receptor molecule, CD4, could be efficient in patients who already contain anti-gp120 antibodies and could also be used to enhance passive immunization against HIV-1 in combination with anti-gp120 antibodies.     

A proposed molecular model for the carboxy terminus of HIV-1 gp120 showing structural features consistent with the presence of a T-cell alloepitope.

A computer graphics molecular model of the C terminus of gp120 of HIV has been constructed using predicted secondary structure based on homologies with proteins for which X-ray crystallographic data have been published. The model shows sequences known to be important in CD4 binding in close proximity to regions with a high probability of forming alpha helical and beta strand motifs. The orientation adopted by these domains approximates to the known 3D structure of HLA-A2 alpha 2 chain without constraints based on HLA-A2 as a template being introduced. The model may therefore represent an energetically favourable conformation for a part of gp120 which mimics the binding domain for the T-cell receptor on MHC molecules. Recognition of gp120 as an alloepitope in high affinity association with CD4 would explain many of the sequelae of acquired immune deficiency on HIV infection.     

The Ixodes scapularis salivary protein, salp15, prevents the association of HIV-1 gp120 and CD4

Ixodes scapularis salivary protein, Salp15, inhibits CD4(+) T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interaction with the envelope glycoprotein. A phage display library screen provided the interacting residues in the C1 domain of gp120. These results provide a potential basis to define exposed gp120 epitopes for the generation of neutralizing vaccines.     

Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein

We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.