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

Abstract

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...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

“…For example, surface plasmon resonance or immunoprecipitation assays using conformation specific monoclonal antibodies (mAbs), rely either on monomeric soluble or solubilized Env molecules which are known to be immunogenetically different from their trimeric forms 20,21 . Recent studies also suggest that cleavage affects Env conformations resulting in the exposure of epitopes mainly recognized by nonneutralizing antibodies 14,22,23 .…”

Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env

“…Designing soluble gp140s that can act as structural and antigenic mimics of the closed state of mature prefusion Env, however, has proven difficult. The current best soluble gp140 molecule, named BG505.SOSIP, is a derivative of the clade A HIV-1 strain BG505, with a number of stabilizing mutations that allow for proper structural and antigenic mimicry of the closed state of mature prefusion Env (35,36,44,45). Specifically, BG505.SOSIP is truncated at residue 664 in gp41 and includes the trimer-stabilizing gp120-gp41 disulfide bridge between residues 501 and 605 (termed SOS) and an Ile-to-Pro mutation at gp41 residue 559 (termed IP), as well as a Thr-to-Asn mutation at residue 332 to introduce a glycosylation site within the epitope for broadly neutralizing antibody PGT122 and a modification of the native 508 REKR 511 furin cleavage site to six Arg residues for improved cleavage (36).…”

“…Specifically, BG505.SOSIP is truncated at residue 664 in gp41 and includes the trimer-stabilizing gp120-gp41 disulfide bridge between residues 501 and 605 (termed SOS) and an Ile-to-Pro mutation at gp41 residue 559 (termed IP), as well as a Thr-to-Asn mutation at residue 332 to introduce a glycosylation site within the epitope for broadly neutralizing antibody PGT122 and a modification of the native 508 REKR 511 furin cleavage site to six Arg residues for improved cleavage (36). When the cleavage site is mutated to the inactive 508 SEKS 511 without the incorporation of the SOSIP mutations, the resulting construct is a poor mimic of the closed state of mature prefusion Env (35,45). While the addition of the SOSIP mutations partially restores the antigenic and structural properties of the 508 SEKS 511 molecule, only the cleaved BG505.SOSIP appears to be fully able to mimic the closed state of mature prefusion Env, with the major differences observed in the proportions of aberrant structures for cleaved versus uncleaved BG505.SOSIP (35,45).…”

“…When the cleavage site is mutated to the inactive 508 SEKS 511 without the incorporation of the SOSIP mutations, the resulting construct is a poor mimic of the closed state of mature prefusion Env (35,45). While the addition of the SOSIP mutations partially restores the antigenic and structural properties of the 508 SEKS 511 molecule, only the cleaved BG505.SOSIP appears to be fully able to mimic the closed state of mature prefusion Env, with the major differences observed in the proportions of aberrant structures for cleaved versus uncleaved BG505.SOSIP (35,45).…”

Vaccine Nanoparticles for Protection Against HIV Infection

“…In particular, BG505.SOSIP [24][25][26][27][28] self-assembles to form a trimer ( Figure 1B), resembling the native Env trimer on HIV-1 virions. BG505.SOSIP trimer is recognized by a large panel of bNAbs, including PGV04, PGT145, PGT128, PGT151 and 2G12, but not by non-neutralizing antibodies against gp120 and gp41 epitopes on trimers, such as b6 and F240, respectively [24][25][26][27][28]. BG505.SOSIP trimer immunizations have been shown to generate HIV-1 neutralizing responses in preclinical models as discussed below.…”