Interaction of CD4 with HLA class II antigens and HIV gp120

We have developed a cellular adhesion assay in which B lymphocytes expressing HLA class II antigens form rosettes with COS cells expressing high levels of cell surface CD4 upon transient transfection with a CDM8-CD4 plasmid construct. The assay is specific, quantitative, and overcomes the difficulties encountered with a previously described system using an SV40 viral vector. Rosette formation was inhibited by a series of CD4- and HLA-DR-specific antibodies, as well as by human immunodeficiency virus (HIV) gp 120, and a synthetic peptide derived from part of its binding site for CD4 (amino acid residues 414-434), but not by a variety of other effectors, including several soluble CD4 derivatives. The comparison of this pattern of inhibition with those observed in other systems further emphasizes the great similarity, but incomplete identity, in the CD4 binding sites for HLA class II antigens and HIV gp120, and supports a model in which CD4 is considered as an allosteric servomodulator of T-cell adhesion and function which probably is induced to interact with HLA class II antigens when associated with the Tcr/CD3 complex.     

The Protein Tyrosine Kinase p56lck Is Required for Triggering NF-κB Activation upon Interaction of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 with Cell Surface CD4

We have previously shown that NF-κB nuclear translocation can be observed upon human immunodeficiency virus type 1 (HIV-1) binding to cells expressing the wild-type CD4 molecule, but not in cells expressing a truncated form of CD4 that lacks the cytoplasmic domain (M. Benkirane, K.-T. Jeang, and C. Devaux, EMBO J. 13:5559–5569, 1994). This result indicated that the signaling cascade which controls HIV-1-induced NF-κB activation requires the integrity of the CD4 cytoplasmic tail and suggested the involvement of a second protein that binds to this portion of the molecule. Here we investigate the putative role of p56^lck as a possible cellular intermediate in this signal transduction pathway. Using human cervical carcinoma HeLa cells stably expressing CD4, p56^lck, or both molecules, we provide direct evidence that expression of CD4 and p56^lck is required for HIV-1-induced NF-κB translocation. Moreover, the fact that HIV-1 stimulation did not induce nuclear translocation of NF-κB in cells expressing a mutant form of CD4 at position 420 (C420A) and the wild-type p56^lck indicates the requirement for a functional CD4-p56^lck complex.     

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.     

Use of Human CD4 Transgenic Mice for Studying Immunogenicity of HIV-1 Envelope Protein gp120

HIV-1 envelope protein, gp120, is a major immunogenic protein of the AIDS virus. A specific feature of this protein is its interaction with the receptor protein, human CD4, an important component of the immune system. This interaction might affect the immunogenic properties of the gp120 and modulate the immune response towards HIV. To test this hypothesis we used human CD4-transgenic mice for immunization with gp120. The dynamics of the immune response towards gp120, CD4 and other proteins was followed. The results show that the primary immune response to gp120 (two weeks) developed somewhat faster in CD4-transgenic mice versus non-transgenic mice. Both animals, however, ultimately mounted the same level of response over time. The primary immune response to gp120 when complexed with soluble CD4 before the immunization, developed similarly in both groups. The secondary immune response was earlier and markedly stronger in non-transgenic mice compared with the transgenic mice where a less efficient memory response to gp120 was observed. The ability of gp120 to directly interact with CD4+ helper lymphocytes appears to affect the humoral response towards this antigen. Moreover, these effects illustrate how viral modulation of these cells may in turn lead to potentially different states of immunological equilibrium.     

Structure of an HIV-2 gp120 in Complex with CD4

HIV-2 is a nonpandemic form of the virus causing AIDS, and the majority of HIV-2-infected patients exhibit long-term nonprogression. The HIV-1 and HIV-2 envelope glycoproteins, the sole targets of neutralizing antibodies, share 30 to 40% identity. As a first step in understanding the reduced pathogenicity of HIV-2, we solved a 3.0-Å structure of an HIV-2 gp120 bound to the host receptor CD4, which reveals structural similarity to HIV-1 gp120 despite divergence in amino acid sequence.     

Disulfide Reduction in CD4 Domain 1 or 2 Is Essential for Interaction with HIV Glycoprotein 120 (gp120), which Impairs Thioredoxin-driven CD4 Dimerization

Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.     

Human Immunodeficiency Virus Type 1 Attachment to HeLa CD4 Cells Is CD4 Independent and gp120 Dependent and Requires Cell Surface Heparans

The binding of human immunodeficiency virus type 1 (HIV-1) (Hx10) virions to two different cell lines was analyzed by using a novel assay based on the detection, by anti-HLA-DR-specific antibodies, of HLA-DR⁺ virus binding to HLA-DR⁻ cells. Virion attachment to the CD4⁺-T-cell line A3.01 was highly CD4 dependent in that it was potently inhibited by CD4 monoclonal antibodies (MAbs), and little virus binding to the CD4⁻ sister A2.01 line was observed. By contrast, virion binding to HeLa cells expressing moderate or high levels of CD4 was equivalent to, or lower than, binding to wild-type CD4⁻ HeLa cells. Moreover, several CD4 MAbs did not reduce, but enhanced, HIV-1 attachment to HeLa-CD4 cells. CD4 was required for infection of HeLa cells, however, demonstrating a postattachment role for this receptor. MAbs specific for the V2 and V3 loops and the CD4i epitope of gp120 strongly inhibited virion binding to HeLa-CD4 cells, whereas MAbs specific for the CD4bs and the 2G12 epitopes enhanced attachment. Despite this, all gp120- and gp41-specific MAbs tested neutralized infectivity on HeLa-CD4 cells. HIV-1 attachment to HeLa cells was only partially inhibited by MAbs specific for adhesion molecules present on the virus or target cells but was completely blocked by polyanions such as heparin, dextran sulfate, and pentosan sulfate. Treatment of HeLa-CD4 cells with heparinases completely eliminated HIV attachment and infection, strongly implicating cell surface heparans in the attachment process. CD4 dependence for HIV-1 attachment to target cells is thus highly cell line specific and may be replaced by other ligand-receptor interactions.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation

Membrane nanotubes are thin membranous projections that physically connect two cells. While nanotubes have been studied in human natural killer (NK) cells and are implicated in aiding NK cell cytotoxic function, requirements for their formation to susceptible target cells remain incompletely understood. Here we demonstrate that the CD2-CD58/48 receptor-ligand interaction promotes and is required for nanotube formation in human NK cells. In the CD2⁻ NK cell line YTS, a stable CD2 expression variant enabled effective nanotube formation, and was associated with better cytotoxic function. Importantly, only interactions between an NK cell and a susceptible target cell were associated with multiple nanotubes and the number of nanotubes was inversely correlated with their length. Quantitative live cell fluorescence microscopy of CD2 nanotubes revealed time-dependent enrichment and localization of CD2 to the nanotube tip, and blocking CD2 receptor-ligand interactions prevented nanotube formation. Increased nanotube formation was not simply a feature of receptor-ligand pairing, as a KIR-MHC interaction in the same cell line system failed to promote nanotube formation. Additionally, blocking LFA-1-ICAM and 2B4-CD48 receptor-ligand interactions failed to inhibit nanotube formation. Thus only specific receptor-ligand pairs promote nanotubes. CD2 also promoted nanotube formation in ex vivo NK cells suggesting that CD2 plays a crucial role in the generation of nanotubes between an NK cell and its target.     

Endothelial CD47 Promotes Vascular Endothelial-Cadherin Tyrosine Phosphorylation and Participates in T Cell Recruitment at Sites of Inflammation In Vivo

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein γ (SIRPγ) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47(-/-) mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α-induced inflammation. Reconstitution of CD47(-/-) mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47(-/-) endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(αi)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(αi), had no inhibitory effect, whereas G(αi) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(αi). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.     

Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.     

Spatial Structure Model of the CD4 Receptor-Binding Site of the HIV Envelope Protein gp120

Abstract

In this study we have undertaken attempt to predict 3D structure of the CD4 receptor-binding site of the HIV envelope protein gp120. The structure of this site has been constructed by the analysis of low-energy conformers of peptide T, an HIV reproduction inhibitor with amino acid sequence corresponding to the fragment Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr of protein gp120, ensuring the interaction of virus with T4 lymphocytes. To do this, the following researches have been carried out: i) the spatial structure models of peptide T and similar fragment 4–11 of an analogues of vasoactive intestinal peptide have been modeled by the restrained molecular mechanics method developed earlier, ii) conformational parameters of these models have been compared to geometrical characteristics of homologous segments of unrelated proteins with known spatial structures. The following major conclusions have been made based on the comparative analysis: i) the conformation of C-terminal fragment Thr-Thr-Asn-Tyr-Thr of peptide T, responsible for the biological activity of the molecule, does not undergo the essential distortions while embedding into the peptide chains of unrelated proteins; ii) this conformation, that is realized in isolated molecule and includes two consecutive reverse turns of the polypeptide chain, adequately describes the main conformational features of an appropriate site of the HIV protein gp120; iii) the fragment Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr of protein gp120 accepts one of six spatial forms which are characteristic for peptide T.     

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.     

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.     

Lineage-Specific Differences between Human and Simian Immunodeficiency Virus Regulation of gp120 Trimer Association and CD4 Binding

Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain β-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding.     

Epithelial Uptake and Transport of Cell-Free Human Immunodeficiency Virus Type 1 and gp120-Coated Microparticles

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4⁺ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and α-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.     

Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region.

The envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) binds the cellular protein CD4 with high affinity. By deletion we show that 62 N- and 20 C-terminal residues along with the V1, V2 and V3 variable regions of gp 120 are unnecessary for CD4 binding. A 287 residue variant (ENV59), missing those 197 amino acids, binds to CD4 with high affinity. A polyclonal antibody failed to efficiently precipitate ENV59 which is consistent with the loss of immunodominant antigenic structures in the regions deleted. This suggests that ENV59 may have potential as an immunogen, able to elicit antibodies against more conserved regions of gp120. Additionally, complementing co-expressed gp120 fragments as well as a circularly permuted molecule bind CD4, and suggest either that the molecular termini are adjacent in the folded structure, or that an N-terminal region folds into the structure unconstrained by its method of attachment to the rest of the molecule.