Stable,uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure

The HIV-1 envelope spike [trimeric (gp160)₃, cleaved to (gp120/gp41)₃] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41)₃ complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160)₃, have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties “dangling” from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.The HIV-1 envelope glycoprotein mediates initial steps of virus infection by engaging cellular receptors and facilitating fusion of viral and target-cell membranes (1). Biosynthesis of the virus-encoded envelope glycoprotein yields a precursor, gp160, which following trimerization undergoes cleavage by a furin-like protease into two noncovalently associated fragments: the receptor-binding fragment, gp120, and the fusion fragment, gp41 (1). Three copies each of gp120 and gp41 form the mature envelope spikes (gp120/gp41)₃, the major viral surface antigen. Binding, through a site on gp120, to the host primary receptor, CD4, and then, through a second site, to a coreceptor (e.g., CCR5 or CXCR4) triggers large conformational changes that include reduced interaction between gp120 and gp41 (probably leading to dissociation of the former) and a cascade of ensuing gp41 refolding events (2, 3). Within the precursor gp160, gp41, with its C-terminal transmembrane (TM) segment anchored in the viral membrane, folds into a prefusion conformation. Cleavage of gp160 makes this prefusion conformation metastable with respect to a rearranged, postfusion conformation. Thus, the loss of constraint on gp41 that accompanies coreceptor binding to gp120 triggers a transition in gp41 to an extended, membrane-bridging conformation (sometimes called a “prehairpin” conformation) (4) with a hydrophobic “fusion peptide” at its N terminus inserted into the target-cell membrane and the TM segment in the viral membrane. This relatively long-lived, transient conformation is the target of fusion inhibitors, such as enfuvirtide (5), and of several broadly neutralizing antibodies (bnAbs) (6–8). Folding back of each chain into an α-helical hairpin creates a stable, six-helix bundle—the “postfusion conformation”—placing the fusion peptide and TM segment at the same end of the molecule. This irreversible refolding of gp41 brings the two membranes together, leading to bilayer fusion and viral entry. Thus, during the fusion process, there are at least three distinct conformational states of the envelope protein: the prefusion conformation of (gp120/gp41)₃, the extended intermediate of gp41, and the postfusion conformation of gp41 (with release of free gp120). Moreover, conformational changes of the prefusion form occur upon CD4 and perhaps also coreceptor binding.The envelope glycoprotein is also the primary target of humoral responses in HIV-1–infected individuals. Studies of human monoclonal antibodies (mAbs) have identified a subset members that neutralize a wide range of HIV isolates (see Table S1 for a partial catalog and original references) (9, 10). These bnAbs are of particular interest, because they may guide a search for immunogens to elicit them in vaccinees. Epitopes on gp120 recognized by human bnAbs include the CD4-binding site, a trimer-specific epitope in the relatively invariant parts of the V2 and V3 loops, and a site near the base of the V3 loop involving an N-linked glycan at position 332. A glycan-dependent epitope spans both gp120 and gp41. The membrane-proximal external region (MPER) of gp41 binds a set of bnAbs that were among the earliest broad neutralizers discovered. The domain-swapped, dimeric antibody, 2G12, recognizes only glycans at defined positions, and its reactivity therefore depends on specific glycosylation patterns but not on many other aspects of the gp120 amino acid sequence.Interesting groups of nonneutralizing antibodies, or with a very narrow range of isolates neutralized, include those that bind the so-called CD4-induced (CD4i) epitope, which overlaps the coreceptor site, on the bridging sheet of gp120, when the epitope becomes exposed by the conformational changes that accompany CD4 binding (Table S1). Nonneutralizing antibodies that interact with gp41 fall into two “clusters”: those in cluster I, which recognize the “immunodominant” C-C loop of gp41, and those in cluster II, which bind strongly with a segment just preceding the MPER in the gp41 polypeptide chain, but only when gp41 is in the postfusion conformation. Most of the antibodies listed in Table S1 recognize conformation-dependent epitopes and thus are excellent molecular probes for defining the conformational state of the envelope trimer.A form of gp140, stabilized by a disulfide crosslink between gp120 and gp41 (perhaps related to the disulfide between surface and TM subunits in many oncoretroviruses) was introduced over a decade ago (11) and subsequently modified by introducing an Ile-to-Pro mutation in gp41, to retard formation of the six-helix bundle (12). The product, known as SOSIP (SOS to designate the double cysteine mutations and IP to denote the isoleucine to proline change), is possible only with certain isolates, and the most widely studied has been BG505 SOSIP.664 (13–16). This modified gp140 trimer, which can be cleaved with furin without compromising stability, has greatly facilitated structural analysis, probably by eliminating large-scale conformational fluctuations (13, 14). In this variant, the MPER has been deleted, and the furin site has been replaced with a string of six arginines. Its structure, determined by both electron cryomicroscopy and X-ray crystallography, shows that (as expected from other fusion proteins) the conformation of gp41 in the prefusion state is distinct from the postfusion six-helix bundle (3, 17). The SOSIP.664 structure, which is an extremely important contribution to our understanding of envelope trimer molecular architecture, has some puzzling features. Docking CD4 onto the model suggests that it can bind all three gp120 sites with no clashes. This observation is at odds with a large body of evidence, including a recent biophysical study of the same trimer, showing that CD4 binding induces a substantial structural rearrangement (18–22). Moreover, uncleaved BG505 gp140 without the SOSIP modifications is unstable and heterogeneous. Can one really conclude, as suggested (14), that all HIV-1 uncleaved gp140 trimers are misfolded products resembling three gp120 moieties flexibly linked to a trimer gp41 in the postfusion, six-helix bundle conformation? The answer to this question is of considerable consequence, as it relates directly to design and production of candidate HIV-1 vaccine immunogens.In the work reported here, we have inserted a flexible, 20-residue linker between gp120 and gp41 in the context of a previously characterized, stable gp140. The linker releases the tight covalent constraint between gp120 and gp41, and it should therefore mimic to some extent the furin cleavage (which is effectively a linker of infinite length). If an uncleaved gp140 trimer truly resembles “three balls on a string,” as asserted (14), addition of a linker that can extend as much as 70 Å should exaggerate this property, causing the trimer with the inserted linker to have antigenic properties resembling three monomeric gp120s and a very large hydrodynamic radius. We find, to the contrary, that gp140 with the flexible linker, 20-residue insert (gp140–FL20) has antigenic properties essentially identical to those of the unmodified trimer and very different from those of free gp120 or of a construct with three gp120s held together by a heterologous trimerization tag. Moreover, the linker barely affects the hydrodynamic radius of the trimer, which is even slightly smaller than that of SOSIP. These results show that the stable, uncleaved gp140 trimers we have characterized previously are compact and native-like, and they support our suggestion that they are promising, envelope-based immunogens for clinical testing in vaccine development.