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1
McKenna, Philip M., Roger J. Pomerantz, Bernhard Dietzschold, James P. McGettigan, and Matthias J. Schnell. "Covalently Linked Human Immunodeficiency Virus Type 1 gp120/gp41 Is Stably Anchored in Rhabdovirus Particles and Exposes Critical Neutralizing Epitopes." Journal of Virology 77, no. 23 (December 1, 2003): 12782–94. http://dx.doi.org/10.1128/jvi.77.23.12782-12794.2003.
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ABSTRACT Rabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Because the gp120 and gp41 portions are covalently attached in the gp140 SOS molecule, the protein is maintained on the surface of the RV virion throughout purification. Surface immunostaining and fluorescence-activated cell sorting analysis with anti-envelope antibodies show that the gp140 SOS protein is stably expressed on the surface of infected cells and maintains CD4 binding capabilities. Furthermore, Western blot and immunoprecipitation experiments with infected-cell lysates and purified virions show that a panel of neutralizing anti-envelope antibodies efficiently recognize the gp140 SOS protein. The antigenic properties of this recombinant RV particle containing covalently attached Env, as well as the ability to present Env in a membrane-bound form, suggest that this approach could be a useful component of a HIV-1 vaccine strategy.
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Earl, Patricia L., Wataru Sugiura, David C. Montefiori, Christopher C. Broder, Susan A. Lee, Carl Wild, Jeffrey Lifson, and Bernard Moss. "Immunogenicity and Protective Efficacy of Oligomeric Human Immunodeficiency Virus Type 1 gp140." Journal of Virology 75, no. 2 (January 15, 2001): 645–53. http://dx.doi.org/10.1128/jvi.75.2.645-653.2001.
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ABSTRACT The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015–3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.
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Sanders, Rogier W., Mika Vesanen, Norbert Schuelke, Aditi Master, Linnea Schiffner, Roopa Kalyanaraman, Maciej Paluch, et al. "Stabilization of the Soluble, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1." Journal of Virology 76, no. 17 (September 1, 2002): 8875–89. http://dx.doi.org/10.1128/jvi.76.17.8875-8889.2002.
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ABSTRACT The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer (N. Schülke, M. S. Vesanen, R. W. Sanders, P. Zhu, D. J. Anselma, A. R. Villa, P. W. H. I. Parren, J. M. Binley, K. H. Roux, P. J. Maddon, J. P. Moore, and W. C. Olson, J. Virol. 76:7760-7776, 2002). Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.
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Cai, Yongfei, Selen Karaca-Griffin, Jia Chen, Sai Tian, Nicholas Fredette, Christine E. Linton, Sophia Rits-Volloch, et al. "Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike." Proceedings of the National Academy of Sciences 114, no. 17 (April 10, 2017): 4477–82. http://dx.doi.org/10.1073/pnas.1700634114.
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The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160)3, retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.
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Ringe, Rajesh P., Anila Yasmeen, Gabriel Ozorowski, Eden P. Go, Laura K. Pritchard, Miklos Guttman, Thomas A. Ketas, et al. "Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers." Journal of Virology 89, no. 23 (August 26, 2015): 12189–210. http://dx.doi.org/10.1128/jvi.01768-15.
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ABSTRACTWe have investigated factors that influence the production of native-like soluble, recombinant trimers based on theenvgenes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on theenvgenes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the sameenvgenes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTOwas also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components.IMPORTANCESoluble, recombinant multimeric proteins based on the HIV-1envgene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8envgenes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.
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Doranz, Benjamin J., Sarah S. W. Baik, and Robert W. Doms. "Use of a gp120 Binding Assay To Dissect the Requirements and Kinetics of Human Immunodeficiency Virus Fusion Events." Journal of Virology 73, no. 12 (December 1, 1999): 10346–58. http://dx.doi.org/10.1128/jvi.73.12.10346-10358.1999.
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ABSTRACT Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated theKd of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.
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Rao, Mangala, Kristina Peachman, Ousman Jobe, Alice Kuaban, Erik Billings, Guofen Guao, Lindsay Wieczorek, et al. "Induction of linear and conformational V2-specific antibodies using HIV-1 gp145 clade B envelope protein as the immunogen (P4505)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 179.15. http://dx.doi.org/10.4049/jimmunol.190.supp.179.15.
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Abstract TThe potential importance of binding antibodies (Abs) in preventing HIV-1 acquisition was recently demonstrated by the case control analysis of the RV144 phase III HIV-1 vaccine trial, which showed that binding Abs to a gp70 scaffolded V1V2 loop of gp120 correlated inversely with infection. We examined if immunization with a HIV-1 env trimer in the absence of any priming vector could induce functional V2-specific Abs. The clade B HIV-1 gp145 env protein consisting of gp120, gp41 ectodomain, and MPER, trimerized with foldon was expressed in 293F cells and purified. The protein adjuvanted with liposomes containing lipid A was used to immunize New Zealand white rabbits. Strong Ab binding titers (50,000-400,000) were induced to env epitopes including V2 and V3 peptides (linear) and MuLV gp70-scaffolded V1V2 protein (conformational) as measured by ELISA and Biacore. The sera neutralized infectious molecular clones and pseudoviruses in PBMC, monocyte-derived macrophage, and TZM-bl assays. Purified IgG neutralized primary US-1 in a dose-dependent manner in the macrophage assay. Depletion of linear V2 or V3 epitope-specific Abs retained the conformation-specific gp70 V1V2 binding Abs and did not completely abolish neutralization. Further depletion of gp70 V1V2-specific Abs significantly reduced neutralization. These data indicate that gp145 trimers induce linear as well as conformational V2-specific antibodies and might be a good candidate for designing a preventative HIV-1 vaccine.
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Kalia, Vandana, Surojit Sarkar, Phalguni Gupta, and Ronald C. Montelaro. "Antibody Neutralization Escape Mediated by Point Mutations in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41." Journal of Virology 79, no. 4 (February 15, 2005): 2097–107. http://dx.doi.org/10.1128/jvi.79.4.2097-2107.2005.
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ABSTRACT The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.
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Kwong, Peter. "HIV-1 Env structure and vaccine design." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C117. http://dx.doi.org/10.1107/s2053273314098829.
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Roughly one third of the HIV-1 genome is devoted to the HIV-1 envelope (Env) glycoprotein spike, which comprises three gp120 and three gp41 subunits. Structural characterization of the HIV-1 Env by electron microscopy, NMR, and X-ray crystallography reveals considerable conformational alterations, not only between trimeric ground state, CD4 receptor-bound conformation, and postfusion conformation of the spike, but also between monomeric and trimeric configurations of the subunits as well as between free- and antibody–bound states. One important structure, however, that of the prefusion HIV-1 spike, has resisted atomic level determination. This structure has been on the 10 list of most wanted structure for more than 20 years, because it is the target of the majority of broad HIV-1-neutralizing antibodies – and therefore of importance to vaccine design. In late 2013, the structure of a prefusion HIV-1 spike, based on a BG505 SOSIP.R6.664 construct, was reported by both X-ray crystallography (4.7 Å) and electron microscopy (5.8 Å). While these structures described the trimeric configuration of most of the HIV-1 gp120 subunit, the description of the gp41 subunit was limited to two helical regions comprising only about half the gp41 ectodomain, and the sequence register for the alpha helices was not reported. Recently, we were able to obtain x-ray diffraction data to 3.5 Å resolution on a prefusion crystal structure of the entire HIV-1 spike. The structure utilizes the same BG505 SOSIP.R6.664 construct as previously published, but crystallized in space group P6(3) with the antigen-binding fragments (Fab) of two antibodies, PGT122 and 35O22. The new structure provides atomic-level details for the complete prefusion structure of gp120 and the majority of the trimeric ectodomain of gp41 (up to residue 664). Also visualized are details of the gp120-gp41 interface and of antibodies such as 35O22. In addition to the complete HIV-1 Env ectodomain structure, implications for HIV-1 vaccine design will be described.
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Steckbeck, Jonathan D., Anne-Sophie Kuhlmann, and Ronald C. Montelaro. "C-terminal tail of human immunodeficiency virus gp41: functionally rich and structurally enigmatic." Journal of General Virology 94, no. 1 (January 1, 2013): 1–19. http://dx.doi.org/10.1099/vir.0.046508-0.
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The human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) pandemic is amongst the most important current worldwide public health threats. While much research has been focused on AIDS vaccines that target the surface viral envelope (Env) protein, including gp120 and the gp41 ectodomain, the C-terminal tail (CTT) of gp41 has received relatively little attention. Despite early studies highlighting the immunogenicity of a particular CTT sequence, the CTT has been classically portrayed as a type I membrane protein limited to functioning in Env trafficking and virion incorporation. Recent studies demonstrate, however, that the Env CTT has other important functions. The CTT has been shown to additionally modulate Env ectodomain structure on the cell and virion surface, affect Env reactivity and viral sensitivity to conformation-dependent neutralizing antibodies, and alter cell–cell and virus–cell fusogenicity of Env. This review provides an overview of the Env structure and function with a particular emphasis on the CTT and recent studies that highlight its functionally rich nature.
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McInerney, Tracey L., Walid El Ahmar, Bruce E. Kemp, and Pantelis Poumbourios. "Mutation-Directed Chemical Cross-Linking of Human Immunodeficiency Virus Type 1 gp41 Oligomers." Journal of Virology 72, no. 2 (February 1, 1998): 1523–33. http://dx.doi.org/10.1128/jvi.72.2.1523-1533.1998.
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ABSTRACT The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS3 generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic α-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal α-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal α-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.
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Edwards, Terri G., Stéphanie Wyss, Jacqueline D. Reeves, Susan Zolla-Pazner, James A. Hoxie, Robert W. Doms, and Frédéric Baribaud. "Truncation of the Cytoplasmic Domain Induces Exposure of Conserved Regions in the Ectodomain of Human Immunodeficiency Virus Type 1 Envelope Protein." Journal of Virology 76, no. 6 (March 15, 2002): 2683–91. http://dx.doi.org/10.1128/jvi.76.6.2683-2691.2002.
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ABSTRACT We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.
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Labrosse, Béatrice, Carole Treboute, and Marc Alizon. "Sensitivity to a Nonpeptidic Compound (RPR103611) Blocking Human Immunodeficiency Virus Type 1 Env-Mediated Fusion Depends on Sequence and Accessibility of the gp41 Loop Region." Journal of Virology 74, no. 5 (March 1, 2000): 2142–50. http://dx.doi.org/10.1128/jvi.74.5.2142-2150.2000.
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ABSTRACT The triterpene RPR103611 is an efficient inhibitor of membrane fusion mediated by the envelope proteins (Env, gp120-gp41) of CXCR4-dependent (X4) human immunodeficiency virus type 1 (HIV-1) strains, such as HIV-1LAI (LAI). Other X4 strains, such as HIV-1NDK (NDK), and CCR5-dependent (R5) HIV-1 strains, such as HIV-1ADA (ADA), were totally resistant to RPR103611. Analysis of chimeric LAI-NDK Env proteins identified a fragment of the NDK gp41 ectodomain determining drug resistance. A single difference at position 91, leucine in LAI and histidine in NDK, apparently accounted for their sensitivity or resistance to RPR103611. We had previously identified a mutation of isoleucine 84 to serine in a drug escape LAI variant. Both I84 and L91 are located in the “loop region” of gp41 separating the proximal and distal helix domains. Nonpolar residues in this region therefore appear to be important for the antiviral activity of RPR103611 and are possibly part of its target. However, another mechanism had to be envisaged to explain the drug resistance of ADA, since its gp41 loop region was almost identical to that of LAI. Fusion mediated by chimeric Env consisting of LAI gp120 and ADA gp41, or the reciprocal construct, was fully blocked by RPR103611. The gp120-gp41 complex of R5 strains is stable, relative to that of X4 strains, and this stability could play a role in their drug resistance. Indeed, when the postbinding steps of ADA infection were performed under mildly acidic conditions (pH 6.5 or 6.0), a treatment expected to favor dissociation of gp120, we achieved almost complete neutralization by RPR103611. The drug resistance of NDK was partially overcome by preincubating virus with soluble CD4, a gp120 ligand inducing conformational changes in the Env complex. The antiviral efficacy of RPR103611 therefore depends on the sequence of the gp41 loop and the stability of the gp120-gp41 complex, which could limit the accessibility of this target.
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McKenna, Philip M., Pyone Pyone Aye, Bernhard Dietzschold, David C. Montefiori, Louis N. Martin, Preston A. Marx, Roger J. Pomerantz, Andrew Lackner, and Matthias J. Schnell. "Immunogenicity Study of Glycoprotein-Deficient Rabies Virus Expressing Simian/Human Immunodeficiency Virus SHIV89.6P Envelope in a Rhesus Macaque." Journal of Virology 78, no. 24 (December 15, 2004): 13455–59. http://dx.doi.org/10.1128/jvi.78.24.13455-13459.2004.
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ABSTRACT Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (ΔG) expressing a simian/human immunodeficiency virus SHIV89.6P Env ectodomain and transmembrane domain fused to the RV G CD (ΔG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with ΔG-89.6P-RVG developed SHIV89.6P virus-neutralizing antibodies and SHIV89.6P-specific cellular immune responses after challenge with SHIV89.6P. There was no evidence of CD4+ T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.
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Bär, Séverine, and Marc Alizon. "Role of the Ectodomain of the gp41 Transmembrane Envelope Protein of Human Immunodeficiency Virus Type 1 in Late Steps of the Membrane Fusion Process." Journal of Virology 78, no. 2 (January 15, 2004): 811–20. http://dx.doi.org/10.1128/jvi.78.2.811-820.2004.
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ABSTRACT The membrane fusion process mediated by the gp41 transmembrane envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) was addressed by a flow cytometry assay detecting exchanges of fluorescent membrane probes (DiI and DiO) between cells expressing the HIV-1 envelope proteins (Env) and target cells. Double-fluorescent cells were detected when target cells expressed the type of chemokine receptor, CXCR4 or CCR5, matching the type of gp120 surface envelope protein, X4 or R5, respectively. Background levels of double-fluorescent cells were observed when the gp120-receptor interaction was blocked by AMD3100, a CXCR4 antagonist. The L568A mutation in the N-terminal heptad repeat (HR1) of gp41 resulted in parallel inhibition of the formation of syncytia and double-fluorescent cells, indicating that gp41 had a direct role in the exchange of fluorescent probes. In contrast, three mutations in the loop region of the gp41 ectodomain, located on either side of the Cys-(X)5-Cys motif (W596 M and W610A) or at the distal end of HR1 (D589L), had limited or no apparent effect on membrane lipid mixing between Env+ and target cells, while they blocked formation of syncytia and markedly reduced the exchanges of cytoplasmic fluorescent probes. The loop region could therefore have a direct or indirect role in events occurring after the merging of membranes, such as the formation or dilation of fusion pores. Two types of inhibitors of HIV-1 entry, the gp41-derived peptide T20 and the betulinic acid derivative RPR103611, had limited effects on membrane exchanges at concentrations blocking or markedly reducing syncytium formation. This finding confirmed that T20 can inhibit the late steps of membrane fusion (post-lipid mixing) and brought forth an indirect argument for the role of the gp41 loop region in these steps, as mutations conferring resistance to RPR103611V were mapped in this region (I595S or L602H).
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BAHBOUHI, Bouchaib, Nabil Georges SEIDAH, and Elmostafa BAHRAOUI. "Replication of HIV-1 viruses in the presence of the Portland α1-antitrypsin variant (α1-PDX) inhibitor." Biochemical Journal 360, no. 1 (November 8, 2001): 127–34. http://dx.doi.org/10.1042/bj3600127.
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The Portland α1-antitrypsin variant (α1-PDX) inhibits gp160 cleavage into gp120 and gp41 by different prohormone convertases (PCs) including furin, PC5 and PC7. Jurkat cells stably transfected with this inhibitor (J-PDX cells) and, as controls, Jurkat cells transfected with the empty vector (J-pcDNA3) were tested for their susceptibility to HIV-1 infection. We found that HIV-1 replication was significantly impaired in J-PDX cells. However, the analysis of the infectivity of HIV-1 viruses produced in J-PDX cells on different days during the infection indicated that they recovered infectivity starting from 13 days post-infection. The sequencing of viruses collected earlier and later from J-PDX cells revealed no mutations in envelope-glycoprotein precursor (Env) maturation sites or in the N-terminal sequence of gp41 fusion peptide, which plays a key role in membrane fusion. Although conserved mutations were detected at the C-terminus of the gp41 fusion peptide and ectodomain, the replication of mutant HIV-1 viruses produced on day 20 in J-PDX cells was inhibited at a similar level to wild-type viruses after a second passage in J-PDX cells. We then investigated the expression of the α1-PDX protein, and found that HIV-1 replication activated its proteolysis since the 54kDa cleaved form became predominant later on in the infection. In contrast, the expression of PC7, a protein that is transported through the secretory pathway, was unaltered in HIV-1 infected cells. We conclude that recovered HIV-1 infectivity in J-PDX cells was due to a loss of α1-PDX activity via its extensive processing by intracellular proteases that cleave it through the substrate pathway.
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Finnegan, Catherine M., Werner Berg, George K. Lewis, and Anthony L. DeVico. "Antigenic Properties of the Human Immunodeficiency Virus Transmembrane Glycoprotein during Cell-Cell Fusion." Journal of Virology 76, no. 23 (December 1, 2002): 12123–34. http://dx.doi.org/10.1128/jvi.76.23.12123-12134.2002.
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ABSTRACT Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIVHXB2 envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19°C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.
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York, Joanne, Kathryn E. Follis, Meg Trahey, Phillipe N. Nyambi, Susan Zolla-Pazner, and Jack H. Nunberg. "Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1." Journal of Virology 75, no. 6 (March 15, 2001): 2741–52. http://dx.doi.org/10.1128/jvi.75.6.2741-2752.2001.
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ABSTRACT The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly cloned env genes. By using complementary techniques of flow cytometry and virion binding assays, we show that monoclonal antibodies targeting the V3 loop, CD4-binding site, CD4-induced determinant of gp120, or the ectodomain of gp41 bind equally well to PI and TCLA Env complexes, despite large differences in neutralization outcome. The data suggest that the differential neutralization sensitivity of PI and TCLA viruses may derive not from differences in the initial antibody binding event but rather from differences in the subsequent functioning of the PI and TCLA Envs during virus entry. An understanding of these as yet undefined differences may enhance our ability to generate broadly neutralizing HIV vaccine immunogens.
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Labrosse, Béatrice, Laurence Morand-Joubert, Armelle Goubard, Séverine Rochas, Jean-Louis Labernardière, Jerôme Pacanowski, Jean-Luc Meynard, Allan J. Hance, François Clavel, and Fabrizio Mammano. "Role of the Envelope Genetic Context in the Development of Enfuvirtide Resistance in Human Immunodeficiency Virus Type 1-Infected Patients." Journal of Virology 80, no. 17 (September 1, 2006): 8807–19. http://dx.doi.org/10.1128/jvi.02706-05.
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ABSTRACT Acquired human immunodeficiency virus type 1(HIV-1) resistance to the fusion inhibitor enfuvirtide (ENF) is primarily associated with mutations within the highly conserved first heptad repeat (HR1) region of gp41. Viral env sequences, however, are remarkably variable, and the envelope genetic background could have an important impact on optimal expression of HR1 mutations. We have examined the genetic evolution of env sequences, ENF susceptibility, and Env replicative capacity in patients failing ENF treatment. Sequential plasma-derived virus populations, obtained from six patients initiating ENF treatment as part of a salvage therapy, were studied using a recombinant phenotypic assay evaluating the entire gp120 and the gp41 ectodomains. Regardless of major differences in the baseline ENF susceptibilities, viral populations with similar phenotypic ENF resistance (50% inhibitory concentration, >3,000 ng/ml) were selected under treatment in four of six patients. As expected, in all patients ENF-resistant viruses harbored one or more HR1 mutations (positions 36, 38, and 43). Interestingly, in five patients the emergence of resistance mutations was not associated with reduced Env replicative capacity. Phylogenetic analysis of env sequences in sequential samples from two patients showed that the HR1 mutations had emerged in the context of env quasi-species that were different from those prevalent at baseline. Thus, the envelope genetic context appears to play a critical role in the selection of HR1 mutations and the expression of ENF resistance, thereby conditioning the evolution of HIV-1 under fusion inhibitor selective pressure.
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Foley, Heather D., Miguel Otero, Jan M. Orenstein, Roger J. Pomerantz, and Matthias J. Schnell. "Rhabdovirus-Based Vectors with Human Immunodeficiency Virus Type 1 (HIV-1) Envelopes Display HIV-1-Like Tropism and Target Human Dendritic Cells." Journal of Virology 76, no. 1 (January 1, 2002): 19–31. http://dx.doi.org/10.1128/jvi.76.1.19-31.2002.
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ABSTRACT We describe replication-competent, vaccine strain-based rabies viruses (RVs) that lack their own single glycoprotein and express, instead, a chimeric RV-human immunodeficiency virus type 1 (HIV-1) envelope protein composed of the ectodomain and transmembrane domains of HIV-1 gp160 and the cytoplasmic domain of RV G. The envelope proteins from both X4 (NL4-3)- and R5X4 (89.6)-tropic HIV-1 strains were utilized. These recombinant viruses very closely mimicked an HIV-1- like tropism, as indicated by blocking experiments. Infection was inhibited by SDF-1 on cells expressing CD4 and CXCR4 for both viruses, whereas RANTES abolished infection of cells expressing CCR5 in addition to CD4 in studies of the RV expressing HIV-189.6 Env. In addition, preincubation with soluble CD4 or monoclonal antibodies directed against HIV-1 gp160 blocked the infectivity of both G-deficient viruses but did not affect the G-containing RVs. Our results also indicated that the G-deficient viruses expressing HIV-1 envelope protein, in contrast to wild-type RV but similar to HIV-1, enter cells by a pH-independent pathway. As observed for HIV-1, the surrogate viruses were able to target human peripheral blood mononuclear cells, macrophages, and immature and mature human dendritic cells (DC). Moreover, G-containing RV-based vectors also infected mature human DC, indicating that infection of these cells is also supported by RV G. The ability of RV-based vectors to infect professional antigen-presenting cells efficiently further emphasizes the potential use of recombinant RVs as vaccines.
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Desmezieres, Emmanuel, Nidhi Gupta, Russell Vassell, Yong He, Keith Peden, Lev Sirota, Zhongning Yang, Paul Wingfield, and Carol D. Weiss. "Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120." Journal of Virology 79, no. 8 (April 15, 2005): 4774–81. http://dx.doi.org/10.1128/jvi.79.8.4774-4781.2005.
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ABSTRACT Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.
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Johnson, Welkin E., Hannah Sanford, Linda Schwall, Dennis R. Burton, Paul W. H. I. Parren, James E. Robinson, and Ronald C. Desrosiers. "Assorted Mutations in the Envelope Gene of Simian Immunodeficiency Virus Lead to Loss of Neutralization Resistance against Antibodies Representing a Broad Spectrum of Specificities." Journal of Virology 77, no. 18 (September 15, 2003): 9993–10003. http://dx.doi.org/10.1128/jvi.77.18.9993-10003.2003.
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ABSTRACT Simian immunodeficiency virus (SIV) of macaques isolate SIVmac239 is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, a property that it shares with most primary isolates of human immunodeficiency virus type 1 (HIV-1). This resistance is important for the ability of the virus to persist at high levels in vivo. To explore the physical features of the viral envelope complex that contribute to the neutralization-resistant phenotype, we examined a panel of SIVmac239 derivatives for sensitivity to neutralization by a large collection of monoclonal antibodies (MAbs). These MAbs recognize both linear and conformational epitopes throughout the viral envelope proteins. The variant viruses included three derivatives of SIVmac239 with substitutions in specific N-linked glycosylation sites of gp120 and a fourth variant that lacked the100 amino acids that encompass the V1 and V2 loops. Also included in this study was SIVmac316, a variant of SIVmac239 with distributed mutations in env that confer significantly increased replicative capacity in tissue macrophages. These viruses were chosen to represent a broad range of neutralization sensitivities based on susceptibility to pooled, SIV-positive plasma. All three of these very different kinds of mutations (amino acid substitutions, elimination of N-glycan attachment sites, and a 100-amino-acid deletion spanning variable loops V1 and V2) dramatically increased sensitivity to neutralization by MAbs from multiple competition groups. Thus, the mutations did not simply expose localized epitopes but rather conferred global increases in neutralization sensitivity. The removal of specific N-glycan attachment sites from V1 and V2 led to increased sensitivity to neutralization by antibodies recognizing epitopes from both within and outside of the V1-V2 sequence. Surprisingly, while most of the mutations that gave rise to increased sensitivity were located in the N-terminal half of gp120 (surface subunit [SU]), the greatest increases in sensitivity were to MAbs recognizing the C-terminal half of gp120 or the ectodomain of gp41 (transmembrane subunit [TM]). This reagent set and information should now be useful for defining the physical, structural, thermodynamic, and kinetic factors that influence relative sensitivity to antibody-mediated neutralization.
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Chen, Steve S. L., Sheau-Fen Lee, and Chin-Tien Wang. "Cellular Membrane-Binding Ability of the C-Terminal Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope Transmembrane Protein gp41." Journal of Virology 75, no. 20 (October 15, 2001): 9925–38. http://dx.doi.org/10.1128/jvi.75.20.9925-9938.2001.
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ABSTRACT The amphipathic α-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli β-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the β-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated β-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.
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Weissenhorn, W., S. A. Wharton, L. J. Calder, P. L. Earl, B. Moss, E. Aliprandis, J. J. Skehel, and D. C. Wiley. "The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide." EMBO Journal 15, no. 7 (April 1996): 1507–14. http://dx.doi.org/10.1002/j.1460-2075.1996.tb00494.x.
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Liu, Yuhang, Junhua Pan, Yongfei Cai, Nikolaus Grigorieff, Stephen C. Harrison, and Bing Chen. "Conformational States of a Soluble, Uncleaved HIV-1 Envelope Trimer." Journal of Virology 91, no. 10 (March 1, 2017). http://dx.doi.org/10.1128/jvi.00175-17.
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ABSTRACT The HIV-1 envelope spike [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers—the uncleaved ectodomains of (gp160)3—from a few selected viral isolates adopt a compact conformation with many antigenic properties of native Env spikes. One is currently being evaluated in a clinical trial. We report here low-resolution (20 Å) electron cryomicroscopy (cryoEM) structures of this gp140 trimer, which adopts two principal conformations, one closed and the other slightly open. The former is indistinguishable at this resolution from those adopted by a stabilized, cleaved trimer (SOSIP) or by a membrane-bound Env trimer with a truncated cytoplasmic tail (EnvΔCT). The latter conformation is closer to a partially open Env trimer than to the fully open conformation induced by CD4. These results show that a stable, uncleaved HIV-1 gp140 trimer has a compact structure close to that of native Env. IMPORTANCE Development of any HIV vaccine with a protein component (for either priming or boosting) requires production of a recombinant form to mimic the trimeric, functional HIV-1 envelope spike in quantities suitable for clinical studies. Our understanding of the envelope structure has depended in part on a cleaved, soluble trimer, known as SOSIP.664, stabilized by several modifications, including an engineered disulfide. This construct, which is difficult to produce in large quantities, has yet to induce better antibody responses than those to other envelope-based immunogens, even in animal models. The uncleaved ectodomain of the envelope protein, called gp140, has also been made as a soluble form to mimic the native Env present on the virion surface. Most HIV-1 gp140 preparations are not stable, however, and have an inhomogeneous conformation. The results presented here show that gp140 preparations from suitable isolates can adopt a compact, native-like structure, supporting its use as a vaccine candidate.
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Sullivan, Jonathan T., Chidananda Sulli, Alberto Nilo, Anila Yasmeen, Gabriel Ozorowski, Rogier W. Sanders, Andrew B. Ward, P. J. Klasse, John P. Moore, and Benjamin J. Doranz. "High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers." Journal of Virology 91, no. 22 (September 6, 2017). http://dx.doi.org/10.1128/jvi.00862-17.
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ABSTRACT Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies. IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.
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Das, Raksha, Rohini Datta, and Raghavan Varadarajan. "Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis." Journal of Virology 94, no. 21 (August 19, 2020). http://dx.doi.org/10.1128/jvi.01426-20.
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ABSTRACT HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens. IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.
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Pacheco, Beatriz, Nirmin Alsahafi, Olfa Debbeche, Jérémie Prévost, Shilei Ding, Jean-Philippe Chapleau, Alon Herschhorn, et al. "Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors." Journal of Virology 91, no. 5 (December 21, 2016). http://dx.doi.org/10.1128/jvi.02219-16.
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ABSTRACT Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations. IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.
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Ringe, Rajesh P., Philippe Colin, Jonathan L. Torres, Anila Yasmeen, Wen-Hsin Lee, Albert Cupo, Andrew B. Ward, P. J. Klasse, and John P. Moore. "SOS and IP Modifications Predominantly Affect the Yield but Not Other Properties of SOSIP.664 HIV-1 Env Glycoprotein Trimers." Journal of Virology 94, no. 1 (October 16, 2019). http://dx.doi.org/10.1128/jvi.01521-19.
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ABSTRACT Soluble recombinant native-like (NL) envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design, designated BG505 SOSIP.664, incorporates an intersubunit disulfide bond (SOS) to covalently link the gp120 and gp41 ectodomain (gp41ECTO) subunits and a point substitution, I559P (IP), to further stabilize the gp41ECTO components. Without the SOS and IP changes, proteolytically cleaved trimers tend to disintegrate into their constituent gp120 and gp41ECTO subunits. We show, however, that NL trimers lacking the SOS and/or IP change can be affinity purified in amounts sufficient for analyses of their antigenicity and thermal stability. In general, these trimer variants have properties highly comparable to those of the fully stabilized SOSIP.664 version. We conclude that the major effect of the SOS and IP changes is to substantially increase trimer stability during and after the expression process, thereby allowing useful amounts to be produced. However, once the trimers have been purified, the SOS and IP changes have only subtle impacts on thermostability and the antigenicity of bNAb and other epitopes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. One vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. A commonly used design is designated SOSIP.664, a term reflecting the sequence changes that are used to stabilize the trimers and allow their production in practically useful amounts. Here, we show that these stabilizing changes act to increase trimer yield during the biosynthesis process within the producer cell but have little impact on the properties of purified trimers.