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1.
Maraviroc (MVC) is a CCR5 antagonist that inhibits HIV-1 entry by binding to the coreceptor and inducing structural alterations in the extracellular loops. In this study, we isolated MVC-resistant variants from an HIV-1 primary isolate that arose after 21 weeks of tissue culture passage in the presence of inhibitor. gp120 sequences from passage control and MVC-resistant cultures were cloned into NL4-3 via yeast-based recombination followed by sequencing and drug susceptibility testing. Using 140 clones, three mutations were linked to MVC resistance, but none appeared in the V3 loop as was the case with previous HIV-1 strains resistant to CCR5 antagonists. Rather, resistance was dependent upon a single mutation in the C4 region of gp120. Chimeric clones bearing this N425K mutation replicated at high MVC concentrations and displayed significant shifts in 50% inhibitory concentrations (IC50s), characteristic of resistance to all other antiretroviral drugs but not typical of MVC resistance. Previous reports on MVC resistance describe an ability to use a drug-bound form of the receptor, leading to reduction in maximal drug inhibition. In contrast, our structural models on K425 gp120 suggest that this resistant mutation impacts CD4 interactions and highlights a novel pathway for MVC resistance. 相似文献
2.
Binding of full-length HIV-1 gp120 to CD4 induces structural reorientation around the gp120 core
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Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor, and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding. 相似文献
3.
Tumanova O. Yu. Kuvshinov V. N. Il'ichev A. A. Nekrasov B. G. Ivanisenko V. A. Kozlov A. P. Sandakhchiev L. S. 《Molecular Biology》2002,36(4):517-521
A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface. 相似文献
4.
Jean-Philippe Julien Devin Sok Reza Khayat Jeong Hyun Lee Katie J. Doores Laura M. Walker Alejandra Ramos Devan C. Diwanji Robert Pejchal Albert Cupo Umesh Katpally Rafael S. Depetris Robyn L. Stanfield Ryan McBride Andre J. Marozsan James C. Paulson Rogier W. Sanders John P. Moore Dennis R. Burton Pascal Poignard Andrew B. Ward Ian A. Wilson 《PLoS pathogens》2013,9(5)
5.
Johannes S. Gach Heribert Quendler Tommy Tong Kristin M. Narayan Sean X. Du Robert G. Whalen James M. Binley Donald N. Forthal Pascal Poignard Michael B. Zwick 《PloS one》2013,8(8)
Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs. 相似文献
6.
MY Zhang T Yuan J Li A Rosa Borges JD Watkins J Guenaga Z Yang Y Wang R Wilson Y Li VR Polonis SH Pincus RM Ruprecht DS Dimitrov 《PloS one》2012,7(9):e44241
Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics. 相似文献
7.
Sunita S. Balla-Jhagjhoorsingh Davide Corti Leo Heyndrickx Elisabeth Willems Katleen Vereecken David Davis Guido Vanham 《PloS one》2013,8(7)
Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb. 相似文献
8.
Young Do Kwon Judith M. LaLonde Yongping Yang Mark A. Elban Akihiro Sugawara Joel R. Courter David M. Jones Amos B. Smith III Asim K. Debnath Peter D. Kwong 《PloS one》2014,9(1)
Efforts to develop therapeutic agents that inhibit HIV-1 entry have led to the identification of several small molecule leads. One of the most promising is the NBD series, which binds within a conserved gp120 cavity and possesses para-halogen substituted aromatic rings, a central oxalamide linker, and a tetramethylpiperidine moiety. In this study, we characterized structurally the interactions of four NBD analogues containing meta-fluoro substitution on the aromatic ring and various heterocyclic ring replacements of the tetramethylpiperidine group. The addition of a meta-fluorine to the aromatic ring improved surface complementarity and did not alter the position of the analogue relative to gp120. By contrast, heterocyclic ring replacements of the tetramethylpiperidine moiety exhibited diverse positioning and interactions with the vestibule of the gp120 cavity. Overall, the biological profile of NBD-congeners was modulated by ligand interactions with the gp120-cavity vestibule. Herein, six co-crystal structures of NBD-analogues with gp120 provide a structural framework for continued small molecule-entry inhibitor optimization. 相似文献
9.
Ema T. Crooks Tommy Tong Bimal Chakrabarti Kristin Narayan Ivelin S. Georgiev Sergey Menis Xiaoxing Huang Daniel Kulp Keiko Osawa Janelle Muranaka Guillaume Stewart-Jones Joanne Destefano Sijy O’Dell Celia LaBranche James E. Robinson David C. Montefiori Krisha McKee Sean X. Du Nicole Doria-Rose Peter D. Kwong John R. Mascola Ping Zhu William R. Schief Richard T. Wyatt Robert G. Whalen James M. Binley 《PLoS pathogens》2015,11(5)
Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine. 相似文献
10.
Alexandre KB Gray ES Pantophlet R Moore PL McMahon JB Chakauya E O'Keefe BR Chikwamba R Morris L 《Journal of virology》2011,85(17):9039-9050
The glycans on HIV-1 gp120 play an important role in shielding neutralization-sensitive epitopes from antibody recognition. They also serve as targets for lectins that bind mannose-rich glycans. In this study, we investigated the interaction of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 to plates coated with anti-CD4bs antibodies b12 and b6 or the CD4 receptor mimetic CD4-IgG2. The average enhancement of b12 or b6 binding was higher for subtype B viruses than for subtype C, while for CD4-IgG2, it was similar for both subtypes, although lower than observed with antibodies. This GRFT-mediated enhancement of HIV-1 binding to b12 was reflected in synergistic neutralization for 2 of the 4 viruses tested. The glycan at position 386, which shields the CD4bs, was involved in both GRFT-mediated enhancement of binding and neutralization synergism between GRFT and b12. Although GRFT enhanced CD4bs exposure, it simultaneously inhibited ligand binding to the coreceptor binding site, suggesting that GRFT-dependent enhancement and neutralization utilize independent mechanisms. This study shows for the first time that GRFT interaction with gp120 exposes the CD4bs through binding the glycan at position 386, which may have implications for how to access this conserved site. 相似文献
11.
RAIMO FRANKE TATJANA HIRSCH JUTTA EICHLER 《Journal of receptor and signal transduction research》2013,33(5-6):453-460
Synthetic mimetics of the CD4-binding site of HIV-1 gp120 are promising candidates for HIV-1 entry inhibition, as well as immunogen candidates for the elicitation of virus-neutralizing antibodies. On the basis of the crystal structure of gp120 in complex with CD4, we have used a recently introduced strategy for the generation of structurally diverse scaffolds to design and synthesize a scaffolded peptide, in which three fragments, making up the sequentially discontinuous binding site of gp120 for CD4, are presented in a nonlinear and discontinuous fashion through a molecular scoffold, which restrains conformational flexibility. The affinities of this molecule to CD4, as well as to the broadly neutralizing antibody mAb b12, whose epitope overlaps the CD4-binding site of gp120, were determined in competitive binding assays. 相似文献
12.
Nanako Takeda-Hirokawa Lian-pin Neoh Hiroaki Akimoto Hiroshi Kaneko Takashi Hishikawa Iwao Sekigawa Hiroshi Hashimoto Shun-ichi Hirose Tsutomu Murakami Naoki Yamamoto Tohru Mimura Yutaro Kaneko 《Microbiology and immunology》1997,41(9):741-745
To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)-1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF-α by gp120-stimulated macrophages (which promotes HIV-1 replication). CRDS treatment of cells not only inhibited the binding of HIV-1 gp120 to CD4+ cells, but also inhibited TNF-α production induced by gp120. Inhibition of HIV-1 infection by CRDS may be related to these two actions. 相似文献
13.
Barna Dey Krisha Svehla Ling Xu Dianne Wycuff Tongqing Zhou Gerald Voss Adhuna Phogat Bimal K. Chakrabarti Yuxing Li George Shaw Peter D. Kwong Gary J. Nabel John R. Mascola Richard T. Wyatt 《PLoS pathogens》2009,5(5)
The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region. 相似文献
14.
Tumanova OIu Kuvshinov VN Il'ichev AA Nekrasov BG Ivanisenko VA Kozlov AP Sandakhchiev LS 《Molekuliarnaia biologiia》2002,36(4):657-663
A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Trh-297, Phe-383, Tyr-384, Arg-419, Ile-420, Thr-415, Leu-416, Pro-417, Lys-421, and Trp-112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface. 相似文献
15.
16.
Tahir Bashir Mandar Patgaonkar Selvaa Kumar C Achhelal Pasi Kudumula Venkata Rami Reddy 《PloS one》2015,10(4)
Human Immunodeficiency Virus (HIV-1) poses a serious threat to the developing world and sexual transmission continues to be the major source of new infections. Therefore, the development of molecules, which prevent new HIV-1 infections, is highly warranted. In the present study, a panel of human hemoglobin (Hb)-α subunit derived peptides and their analogues, with an ability to bind gp120, were designed in-silico and their anti-HIV-1 activity was evaluated. Of these peptides, HbAHP-25, an analogue of Hb-α derived peptide, demonstrated significant anti-HIV-1 activity. HbAHP-25 was found to be active against CCR5-tropic HIV-1 strains (ADA5 and BaL) and CXCR4-tropic HIV-1 strains (IIIB and NL4-3). Surface plasmon resonance (SPR) and ELISA revealed direct interaction between HbAHP-25 and HIV-1 envelope protein, gp120. The peptide prevented binding of CD4 to gp120 and blocked subsequent steps leading to entry and/or fusion or both. Anti-HIV activity of HbAHP-25 appeared to be specific as it failed to inhibit the entry of HIV-1 pseudotyped virus (HIV-1 VSV). Further, HbAHP-25 was found to be non-cytotoxic to TZM-bl cells, VK2/E6E7 cells, CEM-GFP cells and PBMCs, even at higher concentrations. Moreover, HbAHP-25 retained its anti-HIV activity in presence of seminal plasma and vaginal fluid. In brief, the study identified HbAHP-25, a novel anti-HIV peptide, which directly interacts with gp120 and thus has a potential to inhibit early stages of HIV-1 infection. 相似文献
17.
Robert J. Luallen Hu Fu Caroline Agrawal-Gamse Innocent Mboudjeka Wei Huang Fang-Hua Lee Lai-Xi Wang Robert W. Doms Yu Geng 《Journal of virology》2009,83(10):4861-4870
The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a Δpmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the Δmnn1 deletion to the Δpmr1 background, ensuring the exposure of terminal α1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man8GlcNAc2 structures in Δoch1 Δmnn1 Δmnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies.The human immunodeficiency virus (HIV) has evolved numerous means to evade the humoral immune response, including a two-receptor mechanism for entry that recesses and protects highly conserved binding sites in the gp120 subunit of the viral envelope (Env) protein, trimerization of Env to further protect neutralizing epitopes readily exposed on the monomer, and rapid and continual mutation in the face of immune selective pressure (8, 9). Another highly effective defense mechanism is found in the extensive array of oligosaccharides covering gp120, with approximately 25 N-linked glycosylation sites per gp120 monomer (26). These glycans facilitate HIV type 1 (HIV-1) escape from immune surveillance by presenting immunologically “self” molecules with highly variable glycoforms that mask polypeptide epitopes along the “silent face” of gp120 (46, 49). Additionally, high-mannose-type N-linked glycans on gp120 have been implicated in inducing immunosuppressive responses from dendritic cells (DCs) (40), and in helping viral dissemination by binding to DCs through C-type lectins, such as DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing nonintegrin) (18, 33, 34). The high affinity of DC-SIGN for mannose structures on gp120 (29, 41), and evidence that DC-SIGN+ mucosal cells assist trans infection of permissive T cells, imply a key role for DC-SIGN in early HIV infection after sexual transmission (19).The high-mannose-type glycans of gp120 also represent a vulnerability for HIV-1. Mannose-binding lectins, such as cyanovirin N (16), actinohivin (12), and human mannose-binding protein (17), can interact with gp120 and inhibit HIV-1 infection in vitro. More critically for vaccine studies, high-mannose glycans are also the target of 2G12, one of the few broadly neutralizing monoclonal antibodies (MAbs) isolated from HIV-1-infected patients (36, 37, 42). The potency of this MAb stems from its unique epitope on the exposed and relatively conserved “silent face” of gp120, comprised of a cluster of terminal Manα1,2-Man residues on the D1 and D3 arms of up to three high-mannose glycans (10, 11, 36, 37). 2G12 is thought to have a high affinity for these gp120 glycans due to a unique heavy chain variable (VH) domain-swapped configuration that forms a multivalent binding surface with a potential noncanonical binding site at the novel VH/VH interface in addition to the two conventional VH/light chain variable (VL) binding sites. This extended antigen binding surface is thought to allow 2G12 to interact with multiple clustered high-mannose glycans (11).Due to the broadly neutralizing activity of 2G12, the high-mannose glycans on gp120 have aroused interest in the design of glycoantigens that recapitulate the 2G12 epitope. Several such antigens have been created by using flexible linkers to cross-link natural or chemically synthesized high-mannose glycans to various molecular scaffolds, each showing that multivalency of high-mannose glycans is the key to higher 2G12 affinity (2, 25, 27, 43-45). An alternative approach is to express heterologous glycoproteins with natural high-mannose glycans able to support 2G12 binding (28, 38). The yeast Saccharomyces cerevisiae expresses many proteins with high densities of N-linked glycans, and the enzymes involved in its N-glycosylation pathway are easily manipulated to produce glycans with various high-mannose structures (3, 31). We previously showed that an engineered strain lacking the OCH1, MNN1, and MNN4 genes for carbohydrate-processing enzymes expressed at least four highly glycosylated proteins that supported 2G12 binding and that immunization of rabbits with whole yeast cells from this strain elicited antibodies that cross-reacted with the glycans of gp120 (28). Here, we describe a second approach to modify the glycosylation machinery of S. cerevisiae and the subsequent discovery of Pst1, a yeast glycoprotein able to bind MAb 2G12. We show that Pst1 displays increased 2G12 binding as the dominant glycans on the protein become more similar to the glycans on the 2G12 epitope of gp120. This form of Pst1, containing strictly Man8GlcNAc2 glycans, displayed high affinity for 2G12 and effectively blocked the interaction of gp120 with 2G12 and DC-SIGN. This identifies Pst1 as a candidate molecular scaffold for an effective presentation of the 2G12 epitope and as a potential immunogen to induce mannose-specific antibodies. 相似文献
18.
Sanchari Bhattacharyya Roshan Elizabeth Rajan Yalla Swarupa Ujjwal Rathore Anjali Verma Ranga Udaykumar Raghavan Varadarajan 《The Journal of biological chemistry》2010,285(35):27100-27110
The outer domain (OD) of the HIV-1 envelope glycoprotein gp120 is an important target for vaccine design as it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD-based immunogens in the past have met with little success. We report the design and characterization of an Escherichia coli-expressed OD-based immunogen (ODEC), based on the sequence of the HxBc2 strain. The ODEC-designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120. Biophysical studies showed that ODEC is folded and protease-resistant, whereas ODEC lacking the designed mutations is highly aggregation-prone. In contrast to previously characterized OD constructs, ODEC bound CD4 and the broadly neutralizing antibody b12 but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, ODEC was highly immunogenic, and the sera showed measurable neutralization for four subtype B and one subtype C virus including two b12-resistant viruses. In contrast, sera from rabbits immunized with gp120 did not neutralize any of the viruses. ODEC is the first example of a gp120 fragment-based immunogen that yields significant neutralizing antibodies. 相似文献
19.
Schön A Madani N Klein JC Hubicki A Ng D Yang X Smith AB Sodroski J Freire E 《Biochemistry》2006,45(36):10973-10980
NBD-556 and the chemically and structurally similar NBD-557 are two low-molecular weight compounds that reportedly block the interaction between the HIV-1 envelope glycoprotein gp120 and its receptor, CD4. NBD-556 binds to gp120 with a binding affinity of 2.7 x 10(5) M(-1) (K(d) = 3.7 muM) in a process characterized by a large favorable change in enthalpy partially compensated by a large unfavorable entropy change, a thermodynamic signature similar to that observed for binding of sCD4 to gp120. NBD-556 binding is associated with a large structuring of the gp120 molecule, as also demonstrated by CD spectroscopy. NBD-556, like CD4, activates the binding of gp120 to the HIV-1 coreceptor, CCR5, and to the 17b monoclonal antibody, which recognizes the coreceptor binding site of gp120. NBD-556 stimulates HIV-1 infection of CD4-negative, CCR5-expressing cells. The thermodynamic signature of the binding of NBD-556 to gp120 is very different from that of another viral entry inhibitor, BMS-378806. Whereas NBD-556 binds gp120 with a large favorable enthalpy and compensating unfavorable entropy changes, BMS-378806 does so with a small binding enthalpy change in a mostly entropy-driven process. NBD-556 is a competitive inhibitor of sCD4 and elicits a similar structuring of the coreceptor binding site, whereas BMS-378806 does not compete with sCD4 and does not induce coreceptor binding. These studies demonstrate that low-molecular-weight compounds can induce conformational changes in the HIV-1 gp120 glycoprotein similar to those observed upon CD4 binding, revealing distinct strategies for inhibiting the function of the HIV-1 gp120 envelope glycoprotein. Furthermore, competitive and noncompetitive compounds have characteristic thermodynamic signatures that can be used to guide the design of more potent and effective viral entry inhibitors. 相似文献
20.
The entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A "binding funnel" model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded "bridging sheet" structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies. 相似文献