Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

Harris, Audray K.; Borgnia, Mario J.; Shi, Dan; Bartesaghi, Alberto; He, Haifeng; Pejchal, Robert; Kang, Yun; Depetris, Rafael S.; Marozsan, Andre J.; Sanders, Rogier W.; Klasse, Per Johan; Milne, Jacqueline L.S.; Wilson, Ian A.; Olson, William C.; Moore, John P.; Subramaniam, Sriram

doi:10.1073/pnas.1101414108

Cited by 146 publications

(198 citation statements)

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“…Despite their definition as variable loops, the V2 and V3 regions of gp120 contain highly conserved domains (33). Because several lines of evidence suggest that V3 establishes direct interaction with V2 (12)(13)(14)(15)(16)(17)(18)(19)(20)(21), and the conserved base of V3 is the binding site for the N-terminal region of the CCR5 coreceptor (34), we looked for potential structural homology between V2 and CCR5. We recognized that the conserved central region of V2 contains two tyrosine residues (Tyr173 and Tyr177) with spacing identical to that of two tyrosines (Tyr10 and Tyr14) present in the N-terminal region of CCR5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Functional and antigenic interactions between V2 and V3 have long been recognized (21)(22)(23)(24)(25)(26)(27), and spatial proximity between these two loops has been documented by both cryo-EM studies (12)(13)(14)(15)(16)(17) and the recently solved crystal structure of a stabilized soluble SOSIP trimer at 4.7-Å resolution (18). Despite the lack of sulfated tyrosines (presumably because the soluble trimer was produced in HEK293 cells), the latter structure is consistent with our model because it shows the 173-177 segment of V2 directly juxtaposed to the CCR5-binding region at the base of V3, with the most conserved of the two sulfotyrosines, Tyr177, establishing van der Waals contacts with Ile420, Leu154, and Leu175, as well as an H-bond with Asn302, all residues that are proximal to the predicted binding pocket for the homologous sulfotyrosine (Tys14) of CCR5 (34).…”

Section: Discussionmentioning

confidence: 99%

“…Cryo-EM studies have provided evidence that in the prefusion conformation V2 and V3 are spatially contiguous and account for most of the density at the apex of the trimeric envelope spike (12)(13)(14)(15)(16)(17). Although various fragments of V2 and V3 were crystallized separately using antibody-complexed synthetic peptides (28,29), scaffolded chimeric constructs of the first and second variable loops (V1V2) (30,31), or a V3-containing gp120 core monomer (8), the only study in which the two loops were visualized simultaneously is the recent report of the BG505 SOSIP.664 trimer crystal structure (18).…”

mentioning

confidence: 99%

“…As a consequence, most of the available high-definition structures of gp120 have been obtained with deglycosylated, variable looptruncated core monomers in complex with stabilizing ligands such as soluble CD4 (sCD4) (6)(7)(8)(9)(10). Important information regarding the overall conformation and ligand interactions of the trimeric spike at intermediate resolution has emerged from the use of increasingly refined cryo-electron microscopy (cryo-EM) technologies (11)(12)(13)(14)(15)(16)(17). Moreover, the crystal structure of a stabilized, soluble, cleaved gp140 trimer (BG505 SOSIP.664) at 4.7-Å resolution was reported recently (18).…”

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confidence: 99%

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Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Cimbro¹,

Gallant²,

Dolan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4 + T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.T he development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic (1). However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate into the host chromosomes, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope in the native spikes that protrude from the virion surface (2). These spikes are composed of homotrimers of heterodimers of the envelope glycoprotein subunits gp120 and gp41 maintained in an energetically unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes dramatic conformational changes that lead to a low-energy state, creating permissive conditions for activation of the gp41 fusogenic mechanism (3). In the prefusion conformation, gp120 effectively conceals its highly conserved receptor-and coreceptor-binding sites from antibody recognition, imposing a high-entropy penalty for interaction with CD4 or antibodies to the coreceptor-binding site such as 17b; in contrast, in the open, l...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Cimbro¹,

Gallant²,

Dolan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…With the increasing understanding of the architecture of the viral spike (21,76,77), the possibility to generate stable soluble trimers that closely resemble the native spike (78,79), and the means to generate structurally arrested peptide mimetics of gp120 microdomains (31), a number of tools have become available which have promise to tailor future DARPin selections to specific domains of interest. As discussed above, based on our current data, this holds particular promise to improve envelope-specific DARPin identification and to harness the distinctive binding properties of DARPins for HIV inhibitor development.…”

Section: Discussionmentioning

confidence: 99%

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Mann

Friedrich

Krarup

et al. 2013

J Virol

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Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

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Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding

et al. 2016

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The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies. KeywordsPeptides; Membrane Proteins; Nanostructures; Antibodies; HIV The membrane proximal external region (MPER) is a portion of the envelope glycoprotein gp41 on the surface of HIV virions. The MPER is critical to membrane fusion and infection, and is one of the most highly conserved regions of HIV [2] . A number of broadly neutralizing antibodies including 4E10, Z13e1, 2F5, and 10E8 have been identified that target overlapping regions of the MPER, and a broad analysis of patient sera suggest that MPER-directed antibodies are more common than previously thought [3] . Neutralizing antibodies identified from HIV+ individuals have been shown to provide protection against Correspondence to: Philip E. Dawson. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript intravenous and mucosal challenge in passive transfer animal studies, suggesting that a neutralizing antibody response could protect against HIV [4] . Despite the apparent linear nature of described MPER epitopes, the design of effective MPER immunogens has had limited success [5] . Thus the MPER is ripe for a reverse vaccinology approach to elucidate the interactions between known broadly neutralizing antibodies and their epitopes in order to direct the iterative design of immunogens.The structure of the MPER is largely unknown in the context of Env trimers, despite highresolution structural information regarding Env trimers both on the virion surface and in soluble forms. Cryo-EM data of virion surfaces show low density and low signal to noise at the MPER, suggesting less protein mass or greater disorder in this region [6] . Recent cryo-EM [1] and x-ray crystallography structures [7] of stabilized soluble gp41-gp120 trimers required removal of the MPER to obtain stable constructs. The conformation of the MPER is still an open question of great interest and import to antigen design and general understanding of HIV.Recognizing the importance of the membrane in understanding the MPER, the MPER has been presented in association ...

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Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures

Cited by 146 publications

References 30 publications

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding

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