No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4

CD4, a cell-surface glycoprotein expressed on a subset of T-cells and macrophages, serves as the receptor for the human immunodeficiency virus (HIV) (reviewed in ref. 1), binding to the HIV envelope glycoprotein, gp120 with high affinity. Attempts to block infection in vivo by raising antibodies against gp120 have failed, probably because these antibodies have insufficient neutralizing activity. In addition, because of the extensive polymorphism of gp120 in different isolates of HIV, antibodies raised against one HIV isolate are only weakly effective against others. Because interaction with CD4 is essential for infectivity by all isolates of HIV, an agent that could mimic CD4 in its ability to bind to gp120, such as a peptide or monoclonal antibody, might block infection by a wide spectrum of isolates. To aid the identification of such a ligand we have defined regions of CD4 that are required for binding to gp120. Although human CD4 is similar to mouse CD4 in amino-acid sequence (55% identity, ref. 6) and structure, we have found that the murine protein fails to bind detectably to gp120 and have exploited this finding to study binding of gp120 to mouse-human chimaeric CD4 molecules. These studies show that amino-acid residues within the amino-terminal immunoglobulin-like domain of human CD4 are involved in binding to gp120 as well as to many anti-CD4 monoclonal antibodies.     

Structure of an unliganded simian immunodeficiency virus gp120 core

Envelope glycoproteins of human and simian immunodeficiency virus (HIV and SIV) undergo a series of conformational changes when they interact with receptor (CD4) and co-receptor on the surface of a potential host cell, leading ultimately to fusion of viral and cellular membranes. Structures of fragments of gp120 and gp41 from the envelope protein are known, in conformations corresponding to their post-attachment and postfusion states, respectively. We report the crystal structure, at 4 A resolution, of a fully glycosylated SIV gp120 core, in a conformation representing its prefusion state, before interaction with CD4. Parts of the protein have a markedly different organization than they do in the CD4-bound state. Comparison of the unliganded and CD4-bound structures leads to a model for events that accompany receptor engagement of an envelope glycoprotein trimer. The two conformations of gp120 also present distinct antigenic surfaces. We identify the binding site for a compound that inhibits viral entry.     

A soluble CD4 protein selectively inhibits HIV replication and syncytium formation

The CD4 (T4) molecule is expressed on a subset of T lymphocytes involved in class II MHC recognition, and is probably the physiological receptor for one or more monomorphic regions of class II MHC (refs 1-3). CD4 also functions as a receptor for the human immunodeficiency virus (HIV) exterior envelope glycoprotein (gp120) (refs 4-9), being essential for virus entry into the host cell and for membrane fusion, which contributes to cell-to-cell transmission of the virus and to its cytopathic effects. We have used a baculovirus expression system to generate mg quantities of a hydrophilic extracellular segment of CD4. Concentrations of soluble CD4 in the nanomolar range, like certain anti-CD4 monoclonal antibodies, inhibit syncytium formation and HIV infection by binding gp120-expressing cells. Perhaps more importantly, class II specific T-cell interactions are uninhibited by soluble CD4 protein, whereas they are virtually abrogated by equivalent amounts of anti-T4 antibody. This may reflect substantial differences in CD4 affinity for gp120 and class II MHC.     

Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4

Binding of the human immunodeficiency virus (HIV) to infectable host cells, such as B and T lymphocytes, monocytes and colorectal cells, is mediated by a high-affinity interaction between the gp120 component of the viral envelope glycoprotein and the CD4 receptor. Upon binding, it is thought that the second component of the envelope, gp41, mediates fusion between the viral envelope and host cell membranes. However, the early steps of HIV infection have not yet been thoroughly elucidated. Viral entry was first reported to be mediated by pH-dependent receptor-mediated endocytosis; subsequent studies have shown entry to be pH-independent. Although direct fusion of virus to plasma membranes of infected cells has been observed by electron microscopy, it is still formally possible that the infectious path of the virus involves receptor-mediated endocytosis. To gain a better understanding of receptor function in viral entry, we have analysed the ability of several altered or truncated forms of CD4 to serve as effective viral receptors. Our results indicate that domains beyond the HIV-binding region of CD4 are not required for viral infection. Some of the altered forms of CD4 that serve as effective HIV receptors are severely impaired in their ability to be endocytosed. These experiments therefore support the notion that viral fusion to the plasma membrane is sufficient for infection.     

Molecular architecture of native HIV-1 gp120 trimers

The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.     

Single amino-acid changes in HIV envelope affect viral tropism and receptor binding

Infection by the human immunodeficiency virus (HIV) is initiated by the binding of its extracellular envelope glycoprotein, gp120, to the CD4 antigen on target cells. To map the residues of the HIV-1 glycoprotein that are critical for binding and to analyse the effects of binding on viral infectivity, we created 15 mutations in a region of gp120 that is important for binding to CD4 (refs 4,5). We find that substitution of a single amino acid (tryptophan at position 432) can abrogate CD4 binding and that virus carrying this mutation is non-infectious. By contrast, other amino-acid changes in the same region do not affect CD4 binding but restrict viral tropism: virions containing isoleucine substitutions at position 425 lose their ability to infect a monocyte cell line (U937 cells) but can still infect T-lymphocyte cell lines (CEM, SUP-T1) and activated human peripheral blood lymphocytes. These results indicate that cellular tropism of HIV can be influenced by a single amino-acid change in gp120.     

Prevention of HIV-1 glycoprotein transport by soluble CD4 retained in the endoplasmic reticulum

The envelope glycoprotein (gp120/41) of the human immunodeficiency virus (HIV-1) attaches the virus to the cellular CD4 receptor and mediates virus entry into the cytoplasm. In addition to being required for formation of infectious HIV, expression of gp120/41 at the plasma membrane causes the cytopathic fusion of cells carrying the CD4 antigen. The expression of gp120/41 is therefore an ideal target for therapeutic strategies designed to combat AIDS. Here we show that expression of a soluble CD4 molecule, mutated to contain a specific retention signal for the endoplasmic reticulum, blocks secretion of gp120 and surface expression of gp120/41, but does not interfere with transport of wild-type CD4. By blocking transport of the HIV glycoprotein, this retained CD4 molecule prevents the fusion of CD4 cells that is normally caused by the HIV glycoprotein. Expression of the retained CD4 molecule in human T cells might therefore be useful in the intracellular immunization procedure suggested by Baltimore.     

HIV requires multiple gp120 molecules for CD4-mediated infection

Binding of glycoprotein gp120 to the T cell-surface receptor CD4 is a crucial step in CD4-dependent infection of a target cell by the human immunodeficiency virus (HIV). Blocking some or all gp120 molecules on the viral surface should therefore inhibit infection. Consequently, competitive receptor inhibitors, such as soluble synthetic CD4 (sCD4), synthetic CD4 peptides and immunoglobulins, have been investigated in vitro and in vivo, but little is known about the molecular mechanisms of these inhibitors. We have now quantitatively examined blocking by soluble CD4 in the hope of gaining insight into the complex process of viral binding, adsorption and penetration. At low sCD4 concentrations, the inhibition in three HIV strains is proportional to the binding of gp120. The biological association constant (gp120-sCD4 Kassoc) for HIV-2NIHZ is (8.5 +/- 0.5) x 10(7) M-1, whereas Kassoc for HIV-1HXB3 (1.4 +/- 0.2) and HIV-1MN (1.7 +/- 0.1) x 10(9) M-1 are 15-20-fold larger. For all three viral strains, the biological Kassoc from infectivity assays is comparable to the chemical Kassoc. The inhibitory action of sCD4 at high concentrations, however, is not fully explained by simple proportionality with the binding to gp120. Positive synergy in blocking of infection occurs after about half the viral gp120s molecules are occupied, and is identical for all three viral strains, despite the large differences in Kassoc. Our method of measuring the viral-cell receptor Kassoc directly from infectivity assays is applicable to immunoglobulins, to other viruses and to assays using primary or transformed cell lines.     

Identification of human CD4 residues affecting class II MHC versus HIV-1 gp120 binding

Interactions of CD4 with the class II major histocompatibility complex (MHC) are crucial during thymic ontogeny and subsequently for helper and cytotoxic functions of CD4+CD8- T lymphocytes. CD4 is the receptor for the T-lymphotropic human immunodeficiency virus and binds its envelope glycoprotein, gp120. The residues involved in gp120 binding have been localized to a region within the immunoglobulin-like domain I of CD4, which corresponds to CDR2 of an immunoglobulin variable region, but the CD4 residues important in MHC class II interaction have not been characterized. Here, using a cell-binding assay dependent specifically on the CD4-MHC class II association, we analyse the effects of mutations in CD4 on class II versus gp120 binding. Mutations in CDR2 that destroy gp120 binding affect CD4-MHC class II binding similarly. In addition, binding of soluble gp120 to CD4-transfected cells abrogates their ability to interact with class II-bearing B lymphocytes. In contrast, other mutations within domains I or II that have no effect on gp120 binding eliminate or substantially decrease class II interaction. Thus, the CD4 binding site for class II MHC is more complex than the gp120 binding site, possibly reflecting a broader area of contact with the former ligand and a requirement for appropriate juxtaposition of the two N-terminal domains. The ability of gp120 to inhibit the binding of class II MHC to CD4 could be important in disrupting normal T-cell physiology, acting both to inhibit immune responses and to prevent differentiation of CD4+CD8+ thymocytes into CD4+CD8- T lymphocytes.     

HIV infection is blocked in vitro by recombinant soluble CD4

The T-cell surface glycoprotein, CD4 (T4), acts as the cellular receptor for human immunodeficiency virus, type 1 (HIV-1), the first member of the family of viruses that cause acquired immunodeficiency syndrome. HIV recognition of CD4 is probably mediated through the virus envelope glycoprotein (gp120) as shown by co-immunoprecipitation of CD4 and gp120 (ref.5) and by experiments using recombinant gp120 as a binding probe. Here we demonstrate that recombinant soluble CD4(rsT4) purified from the conditioned medium of a stably transfected Chinese hamster ovary cell line is a potent inhibitor of both virus replication and virus-induced cell fusion (syncytium formation). These results suggest that rsT4 is sufficient to bind HIV, and that it represents a potential anti-viral therapy for HIV infection.     

Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide

The clinical manifestations of AIDS (acquired immune deficiency syndrome) often include neuropsychiatric and neurological deficits, including early memory loss and progressive dementia. HIV (human immunodeficiency virus), the aetiological agent of AIDS, is probably carried by infected macrophages in the central nervous system. The virus enters cells by binding its envelope glycoprotein gp120 to the CD4 antigen present on brain and immune cells. From the data reported in this paper, we now suggest that the neuronal deficits associated with HIV may not be entirely a result of infectivity, but that gp120 shed from HIV could directly produce the neuropathology as a result of its interference with endogenous neurotrophic substances. It is known that an analogue of a sequence contained in vasoactive intestinal peptide (VIP) occurs in all known sequenced gp120 isolates and that VIP is important for neuronal survival in cell culture. Here we show that purified gp120 from two diverse HIV isolates and a recombinant gp120 from a third isolate were all potent in specifically producing significant neuronal cell death in dissociated hippocampal cultures derived from fetal mice, and that this could be reduced by monoclonal antibodies against the murine CD4 antigen and completely antagonized by VIP.     

Antibody and HIV-1 gp120 recognition of CD4 undermines the concept of mimicry between antibodies and receptors.

It has been proposed that antibodies can mimic the binding of a receptor to its ligand and that anti-idiotype antibodies raised against such antibodies can be used to identify the receptor. A large number of antibodies have been raised against CD4, the receptor on T cells for the envelope glycoprotein gp120 of the human immunodeficiency virus, and the site at which gp120 binds to CD4 has been delineated. It has therefore become possible to contrast the fine specificities of a natural ligand (gp120) and antibodies that interact with the receptor at the same site. Here we report that out of a panel of 225 anti-CD4 antibodies, only one showed fine binding specificity that was broadly like that of gp120, but the evidence was against this being an exact mimic. Thus the data indicate that the production of antibody mimics will occur very rarely or not at all and that the anti-idiotype approach is unlikely to be useful. This contention is supported by a review of the results of attempts to use this approach. Taking strict criteria for success, there is no example for which the anti-idiotype approach has led to the discovery of a previously undescribed receptor or other protein of interest.     

Interference with HIV-induced syncytium formation and viral infectivity by inhibitors of trimming glucosidase

Human immunodeficiency virus (HIV), the causative agent of AIDS, infects human lymphocytes and monocytes. An interaction between the viral envelope gp 120 and CD4 protein is required to initiate an infectious cycle. HIV infection in vitro induces syncytium formation by cell-to-cell fusion; this aspect of viral cytopathogenicity is even more dependent on gp120-CD4 interactions. That gp120 is extremely heavily glycosylated (31-36 N-linked glycans per molecule), suggests involvement of N-linked glycans in the gp120-CD4 interaction. We therefore investigated the effects of castanospermine, 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), three trimming glycosidase inhibitors which perturb N-linked glycan structure, on induction of the formation of syncytium between HIV-infected and CD4-expressing cells. The glucosidase inhibitors castanospermine and dNM, but not the mannosidase inhibitor dMM, inhibited syncytium formation and interfered with infectivity. The potential of glucosidase inhibitors as anti-HIV therapeutic agents deserves further investigation, especially because dNM and related compounds show little toxicity in vitro and in vivo.     

Structural definition of a conserved neutralization epitope on HIV-1 gp120

The remarkable diversity, glycosylation and conformational flexibility of the human immunodeficiency virus type 1 (HIV-1) envelope (Env), including substantial rearrangement of the gp120 glycoprotein upon binding the CD4 receptor, allow it to evade antibody-mediated neutralization. Despite this complexity, the HIV-1 Env must retain conserved determinants that mediate CD4 binding. To evaluate how these determinants might provide opportunities for antibody recognition, we created variants of gp120 stabilized in the CD4-bound state, assessed binding of CD4 and of receptor-binding-site antibodies, and determined the structure at 2.3 A resolution of the broadly neutralizing antibody b12 in complex with gp120. b12 binds to a conformationally invariant surface that overlaps a distinct subset of the CD4-binding site. This surface is involved in the metastable attachment of CD4, before the gp120 rearrangement required for stable engagement. A site of vulnerability, related to a functional requirement for efficient association with CD4, can therefore be targeted by antibody to neutralize HIV-1.     

Soluble CD4 molecules neutralize human immunodeficiency virus type 1

Human immunodeficiency virus (HIV) infection can bring about total collapse of the immune system by infecting helper T lymphocytes which express CD4, the molecule which mediates interaction between the cell surface and viral envelope glycoprotein gp120 (refs 3-10). HIV apparently escapes the effects of neutralizing antibodies in vivo by generating new variants which must still interact with CD4 to maintain a cycle of infection. One route to block HIV infection, therefore, could use solubilized CD4 protein to inhibit attachment of the virus to its target cell. We have used recombinant DNA techniques to generate soluble forms of CD4, and show here that these are potent inhibitors of HIV infection in vitro.     

Substitution of murine for human CD4 residues identifies amino acids critical for HIV-gp120 binding

Human CD4 is the receptor for the gp120 envelope glycoprotein of human immunodeficiency virus and is essential for virus entry into the host cell. Sequence analysis of CD4 has suggested an evolutionary origin from a structure with four immunoglobulin-related domains. Only the two NH2-terminal domains are required to mediate gp120 binding. The extracellular segment of murine CD4 has an overall 50% identity with its human counterpart at the amino-acid level, but fails to bind gp120. To define those residues of human CD4 critical for gp120 binding, we have taken advantage of this species difference and substituted all non-conserved murine for human CD4 residues between amino-acid positions 27-167. We used oligonucleotide-directed mutagenesis to create each of 16 individual mutant human CD4 molecules containing from 1-4 amino-acid substitutions. Introduction of as few as three amino acids into corresponding positions of human CD4 abrogates gp120 binding. Furthermore, these critical residues are located in domain I with a contribution from domain II. Modelling studies using the three-dimensional coordinates of the V kappa Bence-Jones REI homodimer localize the site in domain I to the C" beta strand within CDR2 but projecting away from the homologues of principle antigen-binding regions CDR 1 and 3.     

Prevention of HIV-1 IIIB infection in chimpanzees by CD4 immunoadhesin

The first step in infection by the human immunodeficiency virus (HIV) is the specific binding of gp120, the envelope glycoprotein of HIV, to its cellular receptor, CD4. To inhibit this interaction, soluble CD4 analogues that compete for gp120 binding and block HIV infection in vitro have been developed. To determine whether these analogues can protect an uninfected individual from challenge with HIV, we used the chimpanzee model system of cell-free HIV infection. Chimpanzees are readily infected with the IIIB strain of HIV-1, becoming viraemic within about 4-6 weeks of challenge, although they do not develop the profound CD4+ T-cell depletion and immunodeficiency characteristic of HIV infection in humans. CD4 immunoadhesin (CD4-IgG), a chimaeric molecule consisting of the N-terminal two immunoglobulin-like regions of CD4 joined to the Fc region of human IgG1, was selected as the CD4 analogue for testing because it has a longer half-life than CD4, contributed by the IgG Fc portion of the molecule. In humans, this difference results in a 25-fold increased concentration of CD4-IgG in the blood compared with recombinant CD4. Here we report that pretreatment with CD4-IgG can prevent the infection of chimpanzees with HIV-1. The need for a preventative agent is particularly acute in perinatal HIV transmission. As recombinant CD4-IgG, like the parent IgG molecule, efficiently crosses the primate placenta, it may be possible to set up an immune state in a fetus before HIV transfer occurs, thus preventing infection.     

Molecular motions and conformational transition between different conformational states of HIV-1 gp 120 envelope glycoprotein

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations,the details of molecular motions involved in conformational transition that are crucial to intervention remain elusive. We presented comprehensive comparative analyses of the dynamics behaviors of the gp120 in its CD4-complexed,CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops by means of CONCOORD computer simulation to generate ensembles of feasible protein structures that were sub-sequently analysed by essential dynamics analyses to identify preferred concerted motions. The re-vealed collective fluctuations are dominated by complex modes of combinational motions of the rota-tion/twisting,flexing/closure,and shortness/elongation between or within the inner,outer,and bridg-ing-sheet domains,and these modes are related to the CD4 association and HIV neutralization avoid-ance. Further essential subspace overlap analyses were performed to quantitatively distinguish the preference for conformational transitions between the three states,revealing that the unliganded gp120 has a greater potential to translate its conformation into the conformational state adopted by the CD4-complexed gp120 than by the CD4-free gp120,whereas the CD4-free gp120 has a greater potential to translate its conformation into the unliganded state than the CD4-complexed gp120 does. These dynamics data of gp120 in its different conformations are helpful in understanding the relationship between the molecular motion/conformational transition and the function of gp120,and in gp120-structure-based subunit vaccine design.     

Biological properties of a CD4 immunoadhesin

Molecular fusions of CD4, the receptor for human immunodeficiency virus (HIV), with immunoglobulin (termed CD4 immunoadhesins) possess both the gp120-binding and HIV-blocking properties of recombinant soluble CD4, and certain properties of IgG, notably long plasma half-life and Fc receptor binding. Here we show that a CD4 immunoadhesin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) towards HIV-infected cells, although, unlike natural anti-gp120 antibodies, it does not allow ADCC towards uninfected CD4-expressing cells that have bound soluble gp120 to the CD4 on their surface. In addition, CD4 immunoadhesin, like natural IgG molecules, is efficiently transferred across the placenta of a primate. These observations have implications for the therapeutic application of CD4 immunoadhesins, particularly in the area of perinatal transmission of HIV infection.