Article

Conformational characterization of aberrant disulfide-linked HIV-1 gp120 dimers secreted from overexpressing cells

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Journal of virological methods (Impact Factor: 1.78). 09/2010; 168(1-2):155-61. DOI: 10.1016/j.jviromet.2010.05.008
Source: PubMed

ABSTRACT

The envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) mediate viral entry and are also the primary target of neutralizing antibodies. The gp160 envelope glycoprotein precursor undergoes proteolytic cleavage in the Golgi complex to produce the gp120 exterior glycoprotein and the gp41 transmembrane glycoprotein, which remain associated non-covalently in the trimeric Env complex. Monomeric soluble gp120 has been used extensively to investigate conformational states, structure, antigenicity and immunogenicity of the HIV-1 Env glycoproteins. Expression of gp120 alone (without gp41) leads to the accumulation not only of monomeric gp120 but also an aberrant dimeric form. The gp120 dimers were sensitive to reducing agents. The formation of gp120 dimers was disrupted by a single amino acid change in the inner domain, and was reduced by removal of the V1/V2 variable loops or the N and C termini. Epitopes on the gp120 inner domain and the chemokine receptor-binding surface were altered or occluded by gp120 dimerization. Awareness of the existence and properties of gp120 dimers should assist interpretation of studies of this key viral protein.

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Available from: Andres Finzi, May 12, 2015
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    • "Accordingly, in absence of ␤-mercaptoethanol, in addition to the expected band of monomeric gp120, a slow migrating band corresponding to disulfide-linked gp120 dimers can be detected for wt, N386A N397A N406A and V5 mutants but not for V1V2V3 and V1V2V3V5 (Fig. 1A). This is in line with previously reported observations that V1V2 and, to a lesser extent, V3 regions are involved in gp120 dimer formation (Center et al., 2000; Finzi et al., 2010a). However, upon addition of ␤-mercaptoethanol, which reduces disulfide links, all variants migrate as a single band (Fig. 1B). "
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    ABSTRACT: For several years, tools to study the conformational changes of HIV-1 envelope glycoproteins have been developed in order to comprehend those changes and their role in the fusion process and immunogenicity of HIV-1. To facilitate these studies, expression of the HIV-1 gp120 envelope glycoprotein has been done in several over-expression settings. However, over-expression of HIV-1 gp120 in mammalian cells leads to the formation of aberrant disulfide-linked dimers that can bias the results of experiments aimed at measuring gp120 affinity with different ligands. The presence of these gp120 dimers, generated in a widely-used gp120 expression system, affects the affinity of gp120 for CD4-induced ligands, as evaluated by surface plasmon resonance. Upon monomeric gp120 purification, neither the removal of potential glycosylation sites on V4 nor the removal of the V5 variable region affect the overall affinity of gp120 for 17b and A32 CD4-induced ligands. Removal of these aberrant disulfide-linked gp120 dimers by standard size exclusion chromatography is sufficient to restore the overall affinity of gp120 preparations for these ligands. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Feb 2015 · Journal of Virological Methods
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    • "The monomeric BG505 gp120 construct was created by introducing a stop codon into the SOSIP.664 gp140 gene at residue 512; the cleavage site was reverted to wild-type (REKR); C501 was reverted to A501; and the L111A substitution was introduced to prevent gp120 dimerization [45,87]. Furthermore, to allow capture by antibody D7324, substitutions R500K and G507Q were introduced into the C5 region. "
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    ABSTRACT: Background The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. Conclusions The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs.
    Full-text · Article · May 2014 · Retrovirology
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    • "The monomeric BG505 gp120 construct was created by introducing a stop codon into the SOSIP.664 gp140 gene at residue 512; the cleavage site was reverted to wild-type (REKR); C501 was reverted to A501; and the L111A substitution was introduced to prevent gp120 dimerization [45,87]. Furthermore, to allow capture by antibody D7324, substitutions R500K and G507Q were introduced into the C5 region. "

    Full-text · Conference Paper · Nov 2013
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