The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.The human immunodeficiency viruses HIV-1 and HIV-2 and the related simian immunodeficiency viruses (SIV) cause the destruction of CD4 + lymphocytes in their respective hosts, resulting in the development of acquired immunodeficiency syndromeCorrespondence and requests for materials should be addressed to W. A.H. (wayne@convex.hhmi.columbia.edu).Coordinates have been deposited in the Brookhaven Protein Data Bank (accession code 1gc1) and maybe obtained from the authors. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (AIDS) 1,2 . The entry of HIV into host cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric spikes displayed on the surface of the virion. These envelope complexes are anchored in the viral membrane by the gp41 transmembrane envelope glycoprotein. The surface of the spike is composed primarily of the exterior envelope glycoprotein, gp120, associated by non-covalent interactions with each subunit of the trimeric gp41 glycoprotein complex 3,4 . Comparison of the gp120 sequences of different primate immunodeficiency viruses identified five variable regions (V1-V5) (ref. 5 ). The first four variable regions form surface-exposed loops that contain disulphide bonds at their bases 6 . The conserved gp120 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. Both conserved and variable gp120 regions are extensively glycosylated 6 . The variability and glycosylation of the gp120 surface probably modulate the immunogenicity and antigenicity of the gp120 glycoprotein, which is the main target for neutralizing antibodies elicited during natural infection 7 .Entry of primate immunodeficiency viruses into the host cell involves the binding of the gp120 envelope glycoprotein to the CD4 glycoprotein, which serves as the primary receptor. The gp120 glycoprotein binds to the most amino-terminal of the four immunoglobulin-like domains of CD4. S...

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.

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HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical proteinprotein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system. E ntry of enveloped viruses into cells requires transformation of the protective envelope into a fusion-competent state. In the case of the human immunodeficiency virus (HIV-1), infection is initiated by the selective interaction between the viral exterior envelope glycoprotein, gp120, and receptors on the target cell, CD4, and obligatory chemokine receptors (CCR5 or CXCR4). Accumulating biochemical and structural evidence indicates that, in addition to contributing to viral attachment, CD4 triggers conformational alterations in the HIV envelope that promote recognition of the chemokine receptors and ultimately lead to membrane fusion (1, 2). Evidence of CD4-induced conformational changes includes enhanced protease sensitivity in gp120 variable loops (3) and release of gp120 from virus and virus-infected cells (4, 5), as well as exposure or formation of the chemokine receptor site (1, 2) and of the epitopes for neutralizing antibodies that can block chemokine receptor binding (1, 2, 6, 7). By their nature, these biochemical studies primarily probe the peripheral regions of gp120 and indicate that CD4-induced movement of variable loops occurs.Recently, a structure was solved for a truncated core gp120 protein in a ternary complex with soluble CD4 and a Fab fragment of the neutralizing antibody 17b (8). In this complex, gp120 is organized into an inner and outer domain connected by a bridging sheet. Several elements of this structure appear to depend on association with CD4 for their stabilization. In particular, the extended conformation of the bridging sheet, which constitutes key elements of the chemokine receptorbinding site, is stabilized by direct contacts with CD4. In contrast, the structure of CD4 (D1D2) (9, 10) is virtually unchanged in the presence of gp120.Although the crystallographic data provi...

CD4 glycoprotein on the surface of T cells helps in the immune response and is the receptor for HIV infection. The structure of a soluble fragment of CD4 determined at 2.3 Å resolution reveals that the molecule has two intimately associated immunoglobulin-like domains. Residues implicated in HIV recognition by analysis of mutants and antibody binding are salient features in domain D1. Domain D2 is distinguished by a variation on the β-strand topologies of antibody domains and by an intra-sheet disulphide bridge.CD4, a cell-surface glycoprotein found primarily on T lymphocytes, is required to shape the T-cell repertoire during thymic development and to permit appropriate activation of mature T cells 1 . T cells that recognize antigens associated with class II major histocompatibility complex (MHC) molecules, mainly T helper cells, express CD4. Evidence is accumulating that CD4 and the T-cell receptor coordinately engage class II molecules on antigenpresenting cells to mediate an efficient cellular immune response, and that engaged CD4 may transmit a signal to an associated cytoplasmic tyrosine kinase, p56 lck .CD4 belongs to the immunoglobulin superfamily of molecules which generally serve in recognition processes 2,3 . The sequence of CD4 4,5 indicates that it consists of a large (~370 residues) extracellular segment composed of four tandem immunoglobulin-like domains, a single transmembrane span, and a short (38 residues) C-terminal cytoplasmic tail. The first domain (D1) shares several features with immunoglobulin variable domains, but the sequence similarities between immunoglobulins and the other extracellular domains (D2, D3 and D4) are more remote.In humans, CD4 can be subverted from its normal immuno-supportive role to become the receptor for infection by the human immunodeficiency virus (HIV) 1,6,7 . Recombinant soluble CD4 proteins bind to the HIV envelope glycoprotein gp120, and can thus inhibit viral infection and virus-mediated cell fusion in vitro (refs 8, 9 and references therein). (refs 21-23 and unpublished results), the main flexibility seems to be at the D2 to D3 junction. We have now crystallized a truncated derivative of CD4 that diffracts well, and here we report its atomic structure. This recombinant fragment 8 as secreted from Chinese hamster ovary (CHO) cells consists of residues 1-183 of human CD4 plus two missense residues, Asp-Thr; and it is unglycosylated. This molecule, which we refer to as D1D2, is as active as sCD4 in binding to gp120 (dissociation constant K d ≃ 3 nM) and retains all antibody epitopes mapped to these domains of CD4 (ref. 8 and unpublished results). Others have crystallized similar fragments from the N-terminal half of sCD4 24,25 and the structure of one is reported in the accompanying paper 25 . HHS Public AccessHere we describe the D1D2 structure in comparison with that of immunoglobulin domains, provide a geometrical definition for HIV recognition sites, and discuss implications of the structure for normal CD4 function and evolution of the immunoglobu...