The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is immunodominant and contains features essential for coreceptor binding. We determined the structure of V3 in the context of an HIV-1 gp120 core complexed to the CD4 receptor and to the X5 antibody at 3.5 angstrom resolution. Binding of gp120 to cell-surface CD4 would position V3 so that its coreceptor-binding tip protrudes 30 angstroms from the core toward the target cell membrane. The extended nature and antibody accessibility of V3 explain its immunodominance. Together, the results provide a structural rationale for the role of V3 in HIV entry and neutralization.

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug-and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibodybound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded β-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate-and structurally plastic-layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated β-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a β-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.HIV-1 viral spike | molecular motion | protein architecture | receptortriggered entry | type 1 fusion protein T he viral spike (gp120/gp41) of HIV type 1 (HIV-1) uses substantial conformational changes to facilitate viral entry (reviewed in ref. 1). Receptor binding by gp120 triggers a series of conformational changes in gp41, which in the unliganded envelope spike possesses a high potential energy that will ultimately be used to fuse the viral and target cell membranes. Binding of the initial receptor, CD4, induces changes in gp120 conformation that allow high-affinity interaction with the coreceptor, CCR5 or CXCR4, and the formation of a gp41 prehairpin intermediate. Subsequent engagement of coreceptor is thought to promote additional conformational changes in gp41 that create an energetically stable sixhelix bundle coincident with the fusion of viral and cell membranes.The gp120 and gp41 glycoproteins are not linked by disulfide bonds, and the noncovalent association of these spike subunits presents significant challenges. The gp120 glycoprotein must be flexible to allow conformational change, yet retain sufficient contact with gp41 to maintain the integrity of the unliganded trimer and, after CD4 binding, to...

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates.To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.Human immunodeficiency virus type 1 (HIV-1) entry into host cells is initiated by the binding of the gp120 subunit of the viral envelope glycoprotein (Env) complex to the host cell receptor (CD4) (8,20). This interaction induces conformational changes in gp120 resulting in the exposure of a conserved high-affinity binding site for the coreceptor (the chemokine receptors CCR5 or CXCR4) (46,47,54,56,59). A second obligatory binding step between the gp120-CD4 complex and the coreceptor is then thought to induce additional conformational changes that ultimately result in the fusion of viral and host cell membranes (9, 18).Neutralizing antibodies are believed to act, at least in part, by binding to the exposed Env surface and obstructing the initial interaction between a trimeric array of gp120 molecules on the virion surface and receptor molecules on the target cell (36,37,57). In response, HIV-1 has evolved a number of strategies to evade recognition by neutralizing antibodies, particularly those directed to the conserved CD4 and coreceptor binding sites of Env. The extent of protection of these sites from antibody recognition is limited by the necessity to preserve the accessibility for receptor interaction. In the case of the CD4bs this has led to the following structural features: (i) it is partially obscured from antibody recognition by the V1/V2 loop and associated carbohydrate structures; (ii) the flanking residues are variable and modified by glycosylation; (iii) it is recessed to an extent that limits direct access by an antibody variable region; (iv) clusters of residues within the CD4bs that do not directly interact with CD4 are subject to variation...