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1.
Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain. 总被引:2,自引:1,他引:1
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E Fenouillet I Jones B Powell D Schmitt M P Kieny J C Gluckman 《Journal of virology》1993,67(1):150-160
To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed. 相似文献
2.
Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex 总被引:3,自引:0,他引:3
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Fouts TR Tuskan R Godfrey K Reitz M Hone D Lewis GK DeVico AL 《Journal of virology》2000,74(24):11427-11436
The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication. 相似文献
3.
Conformational changes affecting the V3 and CD4-binding domains of human immunodeficiency virus type 1 gp120 associated with env processing and with binding of ligands to these sites. 总被引:8,自引:7,他引:1
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Two neutralizing human monoclonal antibodies (HuMAbs) directed against epitopes located near the tip of the V3 loop of human immunodeficiency virus type 1 env protein recognized solubilized gPr160, but not gp120, in radioimmunoprecipitation assays. Efficient immunoprecipitation of solubilized gp120 by these antibodies did occur in the presence of HuMAb 1125H, directed against a conformational epitope overlapping the CD4-binding site, or its F(ab')2 fragment. In contrast to the inability of the anti-V3 antibodies to immunoprecipitate solubilized gp120, these HuMAbs did bind to gp120 in intact virions; this level of binding increased severalfold in the presence of the F(ab')2 fragment of 1125H. These results demonstrate that neutralization epitopes in the V3 loop are sequestered in soluble gp120 but partly exposed in gPr160 and in virion-associated gp120 and that binding of antibodies to the discontinuous CD4-binding site leads to conformational changes that result in the exposure of V3 epitopes in soluble gp120 and their enhanced accessibility in gPr160 and in virion-associated gp120. Enhanced binding of suboptimal concentrations of 1125H to soluble gp120 was also induced by the presence of an anti-V3 HuMAb, indicating the occurrence of reciprocal allosteric interactions between the V3 loop and the CD4-binding site. It is likely that these effects contribute to the synergistic neutralization of human immunodeficiency virus type 1 previously reported for antibodies directed against these two regions. 相似文献
4.
Azimi I Matthias LJ Center RJ Wong JW Hogg PJ 《The Journal of biological chemistry》2010,285(51):40072-40080
A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4. 相似文献
5.
The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site. 总被引:8,自引:5,他引:3
We have compared the expression of full-length gp160 envelope protein from human immunodeficiency virus type 1 with that of a deletion mutant lacking the N-terminal 31 amino acids of the mature protein (gp160 delta 32). The gp160 and gp160 delta 32 proteins are processed to yield gp41 and gp120 or gp120 delta 32, respectively. In contrast to full-length gp120, gp120 delta 32 failed to associate with gp41 at the cell surface, despite conformational integrity as judged by soluble CD4 binding. Thus, the N-terminal 31 amino acids of gp120, which contain hyperconserved sequences, are likely involved in forming a contact site for gp41. 相似文献
6.
Véronique Robert Mariana Resnicoff Jean-Claude Chermann Christian Devaux 《Molecular and cellular biochemistry》1991,102(2):115-123
Summary Several hybridoma cell lines were raised against the highly cytopathic Zairian isolate of Human Immunodeficiency Virus (HIV), HIV1-NDK.The specificity of the secreted monoclonal antibodies (mAb) was demonstrated by immunoblotting, radioimmunoprecipitation and immunofluorescence. Two hybridoma cell lines secreted mAb reacting with independent epitopes of the NDK p17 capsid protein and its precursors. One, RL16.24.5, is specific for the NDK isolate whereas the other, RL16.45.1, along with anti-p25 RL16.30.1 mAb, bind all HIV1 isolates but not HIV2. Together with the previously described mAb RL4.72.1 those reagents define lentivirus subfamily (HIV1, HIV2, SIV) type/subtype (HIV1) and strain (HIVI-NDK) specific epitopes expressed on HIVl-NDK core proteins. The last mAb RL16.76.1 binds the env gene products gp160 and gp120. 相似文献
7.
Effects of anti-gp120 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1. 总被引:50,自引:33,他引:17
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Monoclonal antibodies (MAbs) to defined peptide epitopes on gp120 from human immunodeficiency virus type 1 were used to investigate the involvement of their epitopes in gp120 binding to the CD4 receptor. Recombinant vaccinia viruses were constructed that expressed either full-length gp120 (v-ED6), or a truncated gp120 lacking 44 amino acids at the carboxyl terminus (v-ED4). Binding of these glycoproteins to the CD4 receptor was detected directly with metabolically labeled gp120 or indirectly with the gp120 MAbs. Truncated gp120 from v-ED4 bound to CD4-positive cells less than 1/12 as well as gp120 from v-ED6, indicating that the C-terminal region of gp120, which is conserved in numerous isolates of human immunodeficiency virus type 1, is critical for CD4 binding. However, MAb 110-1, which recognizes a peptide contained in the region deleted from v-ED4 (amino acids 489 through 511), did not inhibit binding of gp120 to CD4. MAb 110-1 also reacted with gp120 bound to the CD4 receptor, indicating that the epitope for this antibody does not directly interact with CD4. A second MAb, 110-4, which recognizes a peptide epitope located between amino acids 303 and 323 and has potent viral neutralizing activity, also bound to gp120 on the CD4 receptor. Furthermore, pretreatment of gp120 with MAb 110-4 at concentrations approximately 1,000-fold higher than those required for complete virus neutralization inhibited subsequent CD4 binding by only about 65%. Taken together, these data suggest that neutralization mediated by antibody 110-4 does not result from binding of this MAb to the CD4-binding site of gp120. 相似文献
8.
Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane. 总被引:37,自引:32,他引:5
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The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. Using a transient-expression system, we investigated the interaction of the HIV-1 envelope glycoprotein with CD4 during their movement through the intracellular membrane traffic. In singly transfected cells, the envelope glyprotein gp160 was synthesized, glycosylated, and localized predominantly in the endoplasmic reticulum. Only a minor fraction of gp160 was proteolytically cleaved, producing gp120 and gp41, and gp120 was secreted into the medium. On the other hand, the CD4 molecule, when expressed alone, was properly glycosylated and transported efficiently to the cell surface. However, when gp160 and CD4 were coexpressed in the same cell, the cell surface delivery of CD4 was greatly reduced. In coexpressing cells, CD4 formed a specific intracellular complex with gp160 as both proteins could be immunoprecipitated by antibodies against either the gp160 or CD4 (OKT4) but not by OKT4A, a blocking antibody against CD4. The specific gp160-CD4 complex was localized predominantly in the endoplasmic reticulum, and the CD4 in the complex did not acquire endoglycosidase H resistance. The present studies demonstrated that a specific intracellular interaction between gp160 and CD4 was responsible for the cell surface down-regulation of CD4 in cells expressing both the envelope glycoprotein of HIV-1 and its receptor, CD4. 相似文献
9.
Lewis GK Fouts TR Ibrahim S Taylor BM Salkar R Guan Y Kamin-Lewis R Robinson JE Devico AL 《Journal of virology》2011,85(24):13097-13104
Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection. 相似文献
10.
Improved antigenicity of the HIV env protein by cleavage site removal 总被引:25,自引:0,他引:25
M P Kieny R Lathe Y Rivière K Dott D Schmitt M Girard L Montagnier J Lecocq 《Protein engineering》1988,2(3):219-225
The HIV env glycoprotein mediates virus infection and cell fusion through an interaction with the CD4 molecule present at the surface of T4+ lymphocytes. Although env presents a major antigenic target, vaccinia recombinants expressing env elicit low titres of anti-env antibody (Kieny et al., Bio/Technology, 4, 790-795, 1986). To delimit the functional domains of env and to improve the immunogenicity of the vaccinia recombinants we constructed variants expressing env proteins in which the site permitting cleavage of the gp160 precursor to yield gp120 and gp41 was removed, the gp120 and gp41 moieties separated or in which the signal sequence and hydrophobic domains were replaced by equivalents from rabies virus G. Analysis of variants revealed that the gp120 moiety is alone capable of interacting with CD4 and of provoking aggregation of T4+ lymphocytes, whereas cell-associated gp41 liberated by gp160 cleavage was essential for cell fusion. The identity of the signal and transmembrane zones however appeared unimportant. Although removal of the consensus sequence permitting cleavage of gp160 prevented syncytium formation but not aggregation of T4+ lymphocytes, significant cleavage continued to take place. Removal of a second potential cleavage site blocked gp160 cleavage. The live viruses were examined for immunogenicity: recombinant 1139 which lacks both putative cleavage sites was found to elicit a 10-fold higher antibody response in experimental animals than the parental recombinant. 相似文献
11.
Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein. 总被引:10,自引:9,他引:1
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T M Richardson Jr B L Stryjewski C C Broder J A Hoxie J R Mascola P L Earl R W Doms 《Journal of virology》1996,70(2):753-762
The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120. 相似文献
12.
P Botarelli B A Houlden N L Haigwood C Servis D Montagna S Abrignani 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(9):3128-3132
The HIV envelope protein gp120 is heavily glycosylated, having 55% of its molecular mass contributed by N-linked carbohydrates. We investigated the role of N-glycosylation in presentation of HIV-gp120 to T cells. T cell clones obtained from humans immunized with a recombinant nonglycosylated form of HIV-gp120 (env 2-3) were studied for their ability to recognize both env 2-3 and glycosylated gp120. We found that 20% of CD4+ T cell clones specific for env 2-3 fail to respond to glycosylated gp120 of the same HIV isolate. Using synthetic peptides, we mapped one of the epitopes recognized by such clones to the sequence 292-300 (NESVAINCT), which contains two asparagines that are glycosylated in the native gp120. These findings suggest that N-linked carbohydrates within an epitope can function as hindering structures that limit Ag recognition by T lymphocytes. 相似文献
13.
Saha P Bhattacharyya S Kesavardhana S Miranda ER Ali PS Sharma D Varadarajan R 《Biochemistry》2012,51(9):1836-1847
Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ~20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface. 相似文献
14.
Conserved and exposed epitopes on intact, native, primary human immunodeficiency virus type 1 virions of group M
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Nyambi PN Mbah HA Burda S Williams C Gorny MK Nádas A Zolla-Pazner S 《Journal of virology》2000,74(15):7096-7107
We have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates. 相似文献
15.
Antonio Facchiano Francesco Facchiano Jos van Renswoude 《Journal of molecular evolution》1993,36(5):448-457
Summary A local sequence similarity of HIV envelope proteins (gp120 and gp41) to immunoglobulins suggests that a mimicry phenomenon
may form the basis of the HIV-cell membrane interaction and of HIV-induced autoimmune reaction. We explored the hypothesis
of any deeper relationship between HIV env proteins and immunoglobulin family members. An overall DNA sequence similarity
between gp41 coding region of env gene and the HIV-receptor CD4 gene was observed and a 14-base-long oligonucleotide, almost
unique in the GenBank, was found in gp41 and CD4 genes. The alignment of env gene to CD4 gene and to 84 different sequences
showed a significantly higher homology score and a nonrandom similarity in the CD4-env alignment. A significant similarity
was also found between the env protein and the sequence encoded by an alternate reading frame of CD4 gene. Our observations
suggest that gp41 coding region might have a different origin than the gp120 coding region of the env gene, and that a divergent
evolution might link gp41 to CD4 or immunoglobulin family members. In this study the analysis of alternate-reading-frame products
is also proposed as a novel approach to investigate evolutionary links and structure-function relationships. 相似文献
16.
Clara Larcher Michael Bröker Hartwig P. Huemer Brigitte Sölder Thomas F. Schulz Johanna M. Hofbauer Helmut Wachter Manfred P. Dierich 《FEMS microbiology letters》1990,64(2):103-110
Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins. 相似文献
17.
Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160. 总被引:3,自引:7,他引:3
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P W Berman L Riddle G Nakamura O K Haffar W M Nunes P Skehel R Byrn J Groopman T Matthews T Gregory 《Journal of virology》1989,63(8):3489-3498
The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation. 相似文献
18.
A Amadori G De Silvestro R Zamarchi M L Veronese M R Mazza G Schiavo M Panozzo A De Rossi L Ometto J Mous 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(9):2709-2716
The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency. 相似文献
19.
Li H Chien PC Tuen M Visciano ML Cohen S Blais S Xu CF Zhang HT Hioe CE 《Journal of immunology (Baltimore, Md. : 1950)》2008,180(6):4011-4021
The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120. 相似文献
20.
W Chen Y Feng Y Wang Z Zhu DS Dimitrov 《Biochemical and biophysical research communications》2012,425(4):931-937
Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion. 相似文献