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 Å 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.The human immunodeficiency virus type 1 (HIV-1) crossed from chimpanzees to humans early in the twentieth century and has since infected ~1% of the world's adult population 1,2 . ThisCorrespondence and requests for materials should be addressed to P.D.K. (pdkwong@nih.gov). Author Contributions T.Z. and P.D.K. carried out structure-based stabilization, SPR analyses and structural determinations; L.X. and G.J.N. constructed gp120 substitutions and developed and implemented a high-throughput gp120-production system suitable for crystallization; B.D. and R.W. carried out ITC characterizations; A.J.H., M.B.Z. and D.R.B. provided b12, b3, b6, b11 and b13, and mutant b12 binding; D.V.R. and J.A. provided D1D2-Igαtp and associated SPR analyses; S.-H.X., X.Y. and J.S. provided OD1 and preliminary design and antigenic analyses; and M.-Y.Z. and D.S.D. provided m6, m14 and m18. All authors contributed to the manuscript preparation.Author Information Coordinates and structure factors have been deposited in the Protein Data Bank and may be obtained from the authors (accession codes 2nxy-2ny6 for the nine variant gp120 molecules with CD4 and 17b; accession code 2ny7 for the b12-gp120 complex). Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. spread and the absence of an effective vaccine are to a large degree a consequence of the ability of HIV-1 to evade antibody-mediated neutralization 3-5 . On HIV-1, the only viral target available for neutralizing antibodies is the envelope spike, which is composed of three copies of the gp120 exterior envelope glycoprotein and three gp41 transmembrane glyco-protein molecules 6,7 . Genetic, immunological and structural studies of the HIV-1 envelope glycoproteins have revealed extraordinary diversity, manifest in a variety of immunodominant loops, as well as multiple overlapping mechanisms of humoral evasion, including se...

The CCR5 co-receptor binds to the HIV-1 gp120 envelope glycoprotein and facilitates HIV-1 entry into cells. Its N terminus is tyrosine-sulfated, as are many antibodies that react with the co-receptor binding site on gp120. We applied nuclear magnetic resonance and crystallographic techniques to analyze the structure of the CCR5 N terminus and that of the tyrosine-sulfated antibody 412d in complex with gp120 and CD4. The conformations of tyrosine-sulfated regions of CCR5 (alpha-helix) and 412d (extended loop) are surprisingly different. Nonetheless, a critical sulfotyrosine on CCR5 and on 412d induces similar structural rearrangements in gp120. These results now provide a framework for understanding HIV-1 interactions with the CCR5 N terminus during viral entry and define a conserved site on gp120, whose recognition of sulfotyrosine engenders posttranslational mimicry by the immune system.

SUMMARY The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (“Layers 1 and 2”) in the gp120 inner domain. Both layers apparently contribute to the non-covalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, Layer 1-Layer 2 interactions strengthen gp120-CD4 binding by reducing the off-rate. Layer 1-Layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.Over 35 million people are currently infected with human immunodeficiency virus type 1 (HIV-1), the major cause of AIDS (4, 24). The development of a preventive vaccine, which optimally should elicit both virus-neutralizing antibodies and cellular immune responses, is of high priority and urgency (25,32).Neutralizing antibodies must bind the HIV-1 envelope glycoproteins, which mediate the entry of the virus into the target cell (97). The trimeric envelope glycoprotein complex is anchored in the host cell or viral membrane by the gp41 transmembrane glycoprotein, which is noncovalently attached to the gp120 exterior envelope glycoprotein. Most of the surfaceexposed elements of the trimeric envelope glycoprotein complex are contained on the gp120 exterior envelope glycoprotein (52). Comparison of the gp120 glycoproteins from different HIV-1 strains reveals regions of conservation interrupted by long regions of variability (V1 to V5) (46, 80). Intramolecular disulfide bonds in the gp120 glycoprotein result in the incorporation of the first four variable regions (V1 to V4) into large, surface-exposed loops (43, 52). The conserved gp120 regions fold into a core, which contains many of the gp120 elements important for receptor binding (6,65,98).CD4 and the chemokine receptors CCR5 and CXCR4 serve as HIV-1 receptors (14, 17-19, 22, 31). The binding of the HIV-1 gp120 glycoprotein to CD4 contributes to the attachme...

Summary Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells, and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogues and gp120 mutants suggest that: 1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4-bound conformation; 2) the NBD-556 phenyl ring projects into the Phe 43 cavity; 3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and 4) increased affinity of NBD-556 analogues for gp120 improves antiviral potency during infection of CD4-expressing cells.

The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S؉S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S؉S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a "spring-loaded" conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.H uman immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus that causes AIDS (1, 2). To enter host cells, HIV-1 uses a metastable, trimeric envelope glycoprotein (Env) spike to engage cellular receptors and to fuse the viral and target cell membranes (3-5). During synthesis and folding in the endoplasmic reticulum of the virus-producing cell, the Env precursor [glycoprotein (gp)160] is heavily modified by N-linked glycosylation and assembles into trimers (6). In most HIV-1 strains, more than 27 potential N-linked glycosylation sites are present in each gp160 protomer. After further glycan processing in the Golgi apparatus, the gp160 Env trimer precursors are proteolytically cleaved and transported to the cell surface for incorporation into virions (7). Each protomer composing the trimeric Env spike thus consists of a gp120 exterior subunit and a gp41 transmembrane subunit. The sequential binding of gp120 to the target cell receptors, CD4 and chemokine receptor (either CCR5 or CXCR4), allows the metastable Env complex to transit into fusion-active conformations (3,(8)(9)(10)(11). These conformational transitions expose the hydrophobic gp41 N-terminal fusion peptide, promoting its insertion into the target cell membrane, and further permit the formation of a highly stable gp41 six-helix bundle that mediates viral-cell membrane fusion (12)(13)(14). The Env spike is the only virus-specific component potentially accessible to neutralizing antibodies and thus has evolved a protective "glycan shield" and a high degree of interstrain variability.High-resolution structures are available for monomeric HIV-1 gp120 core fragments in the CD4-boun...

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5␣. We modeled the structure of the B-box 2 domain of TRIM5␣ based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5␣ hu or rhesus monkey TRIM5␣ rh . However, alteration of arginine 119 of TRIM5␣ hu or the corresponding arginine 121 of TRIM5␣ rh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5␣ proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5␣ and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.