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

Finzi, Andrés; Pacheco, Beatriz; Zeng, Xin; Kwon, Young Do; Kwong, Peter D.; Sodroski, Joseph

doi:10.1016/j.jviromet.2010.05.008

Cited by 34 publications

(52 citation statements)

References 45 publications

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“…The data for the BG505 SOSIP.664 and 92UG037.8 gp120 proteins were derived from prior reports (41,48). units within a gp140 protein or cause these proteins to oligomerize and aggregate (48,71,(75)(76)(77); E. P. Go, A. Cupo, R. P. Ringe, P. Pugach, J. P. Moore, and H. Desaire, unpublished results). As an example of the latter problem, we show that the higher-MW gel bands that are visible in preparations of purified 92UG037.8 and CZA97.012 gp140 UNC -Fd-His proteins are DTT sensitive and, hence, contain disulfide-linked components (Fig.…”

Section: Resultsmentioning

confidence: 99%

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

166

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We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes 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 the env genes 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 (gp140 UNC -Fd-His), based on the same env genes, 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 (gp41 ECTO ) 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 gp41 ECTO was 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 ϳ20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140 UNC -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-1 env gene 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 92UG037.8 env genes 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. R ecombinant, soluble envelope glycoprotein (Env) gp140 trimers from...

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Section: Resultsmentioning

confidence: 99%

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

166

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“…They show only a slight increase in protection within the N-terminal extension and V2 relative to gp120 monomer. Both V1/V2 and the N-terminal extension regions have been shown to be involved in aberrant gp120 dimerization (35,56,57). A small degree of increased protection was also seen in layer 2 of the inner domain (HEDIISL), which is also likely to be involved in the oligomeric interface since mutations within this region have been reported to hinder dimerization (57).…”

Section: Figmentioning

confidence: 99%

“…Both V1/V2 and the N-terminal extension regions have been shown to be involved in aberrant gp120 dimerization (35,56,57). A small degree of increased protection was also seen in layer 2 of the inner domain (HEDIISL), which is also likely to be involved in the oligomeric interface since mutations within this region have been reported to hinder dimerization (57). Even the trimerization motif-stabilized, uncleaved clade C (92UG37) gp140, which indeed bears the molecular mass of a trimer, showed the same pattern in its HDX-MS profile (Fig.…”

Section: Figmentioning

confidence: 99%

A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric, Uncleaved HIV-1 Env gp140

Guttman

Lee

2013

J Virol

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The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.T he envelope glycoprotein (Env) is the sole target of neutralizing antibodies against HIV. Env is expressed as a single polypeptide (gp160), which oligomerizes into a trimer, is extensively glycosylated, and is proteolytically cleaved to produce the gp120 surface subunit containing the receptor binding sites and the membrane-anchored gp41 subunit. Engagement of the primary receptor, CD4, leads to conformational changes that expose elements necessary for binding the coreceptor. These receptor interactions trigger a series of conformational changes in Env that catalyze host-viral membrane fusion, with gp41 ultimately forming a stable six helix bundle in the postfusion state (1-3). Crystal structures have been determined for gp120 core in complex with stabilizing ligands such as CD4 or antibody Fabs (4-8); however, these generally have variable loops truncated and are heavily deglycosylated. The core of gp120 consists of an outer domain that contains the majority of glycosylation sites, an inner domain composed of three structural layers plus a ␤-sandwich motif, and a bridging sheet formed by strands from the inner and outer domains. Cryo-electron microscopy (cryo-EM) structures of both viral surface Env, detergent-solubilized trimer, and stabilized, soluble trimeric constructs have suggested an intimate association between gp120 and gp41 (9-12), and numerous biochemical studies have implicated residues involved in this interaction (13-20), ...

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“…2A). A faint band, at about 250 kDa, can also be seen and probably corresponds to a dimer of gp120, which is classically obtained by recombinant expression (39,40). Covalent complexes formed by gp120 and M64U1(Biot)-SH remained detectable in 8 M urea (Fig.…”

Section: Resultsmentioning

confidence: 86%

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

Martin¹,

Burke

Thaï³

et al. 2011

Journal of Biological Chemistry

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CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

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

Cited by 34 publications

References 45 publications

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric, Uncleaved HIV-1 Env gp140

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

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