Structural Characterization of Cleaved, Soluble HIV-1 Envelope Glycoprotein Trimers

Khayat, Reza; Lee, Jeong Hyun; Julien, Jean-Philippe; Cupo, Albert; Klasse, Per Johan; Sanders, Rogier W.; Moore, John P.; Wilson, Ian A.; Ward, Andrew B.

doi:10.1128/jvi.01222-13

Cited by 74 publications

(86 citation statements)

References 50 publications

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“…S1). This density is far larger than can be accounted for by the additional 17 amino acids, but resembles a micelle that associated with similar KNH1144.681 trimers when a small amount of detergent was added to prevent aggregate formation (23,24). Although there was some aggregation of the MPER-containing BG505 trimers, the extent was much less than with the KNH1144 versions.…”

Section: Resultsmentioning

confidence: 80%

“…MPER Deletion Does Not Explain the Rarity of Native-Like gp140 UNC Trimers. The above experiments used gp140s from which the MPER was deleted to increase trimer homogeneity (23,24). To assess whether the absence of the MPER influenced trimer configuration, we made SOSIP.R6-MPER, SOSIP.SEKS-MPER, and WT.SEKS-MPER constructs that are identical to the corresponding variants described above except for truncation at residue 681, not residue 664.…”

Section: Resultsmentioning

confidence: 99%

“…Variants bearing a D7324 epitope tag at the gp41 ECTO C terminus were purified in the same way, and designated by appending "-D7324" to the gp140 descriptor (e.g., SOSIP.R6-D7324, WT.SEKS-D7324). MPER-containing SOSIP.R6-MPER, WT.SEKS-MPER, and SOSIP.SEKS-MPER constructs were based on the SOSIP.681 gene (23,24). See also SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

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Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

Ringe

Sanders

Yasmeen

et al. 2013

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

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We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120-gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120-gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41 ECTO ) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41 ECTO . Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structuredependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41 ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated.T rimeric envelope glycoprotein (Env gp) spikes on the HIV type 1 (HIV-1) surface mediate entry of the viral genome into the target cell (1, 2). When spikes interact with their cellsurface receptors, a series of conformational changes within the Env culminates in virus-cell membrane fusion. Neutralizing antibodies (NAbs) against various Env epitopes antagonize these events (2, 3). Hence, Env glycoproteins are a focus of vaccine design programs intended to induce NAbs and thereby prevent HIV-1 transmission (3, 4). Env trimers are composed of three gp120 surface glycoprotein subunits and three gp41 transmembrane glycoproteins, the six subunits all associated via noncovalent interactions (5, 6). A critical event in trimer assembly is proteolytic cleavage of the gp160 precursor into its gp120 and gp41 components, a process essential for HIV-1 entry not least because it liberates the fusion peptide (FP) at the gp41 N terminus (5, 6).Trimer-based vaccine strategies involve expressing soluble, recombinant versions of the virion-associated (i.e., native) spikes. To facilitate production and purification, the membrane-spanning and cytoplasmic domains that anchor spikes to the virion, but that are not NAb targets, are eliminated (7-12). However, the resulting proteins, known as gp140s, are highly unstable and disintegrate into their gp120 and gp41-ectodomain (gp41 ECTO ) components, making them useless as immunogens. Two fundamentally different protein-engineering strategies have been used to create gp140s t...

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

Ringe

Sanders

Yasmeen

et al. 2013

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

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170

303

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“…3A). The deletion of the membrane-proximal external region (MPER) and the occlusion of other hydrophobic areas of gp41 ECTO by the overlying gp120 subunits accounts for why SOSIP.664 proteins aggregate only minimally (1,8,45,56). In contrast, the SEC profiles of the Ni-NTA-purified gp140 UNC -Fd-His proteins revealed a quantitatively dominant single peak that contained high-molecularweight (MW) protein aggregates, followed by trimers, with a small amount (1 to 3% of the total) of later-eluting gp140 monomers also present (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

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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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“…An alternative approach to soluble trimer design involves actively promoting the natural gp120-gp41 ECTO cleavage event while also introducing specific stabilizing mutations, i.e., an engineered disulfide bond that covalently links the two subunits and an Ile-to-Pro change at residue 559 that helps maintain the gp41 ECTO moieties in the prefusion form (4,6). In addition, the truncation of gp41 ECTO at residue 664 eliminates a hydrophobic region that tends to cause trimer aggregation (34,35). The resulting trimers are designated SOSIP.664 gp140s.…”

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confidence: 99%

A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

Pugach

Ozorowski

Cupo

et al. 2015

J Virol

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238

408

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Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP. IMPORTANCEThe cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine. Immunogens capable of inducing protective titers of broadly neutralizing antibodies (bNAbs) are being widely sought for use in vaccine design strategies for human immunodeficiency virus type 1 (HIV-1) (1). The basis of this approach is that bNAbs can prevent globally diverse HIV-1 strains from infecting target cells. They do so via binding to the envelope glycoprotein (Env) complex on the virion surface, an event that is both necessary and sufficient to neutralize HIV-1 infectivity (2, 3). One of the more common strategies to induce bNAbs involves the design of soluble, recombinant protein mimics of the native Env complex, a meta-stable structure comprising three gp120 and three gp41 subunits. The production of soluble Env trimers involves introducing a stop codon to truncate the gp41 ectodomain (gp41 ECTO ) subunit prior to the transmembrane region to yield soluble gp140 proteins (4-6). The fragility of the Env complex is, however, a substantial problem from a protein engineering perspective, as the natural noncovalent interactions between the six subunits are not robust enough to allow so...

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Structural Characterization of Cleaved, Soluble HIV-1 Envelope Glycoprotein Trimers

Cited by 74 publications

References 50 publications

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

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

A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

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