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

Abstract

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.

Keywords: HIV-1; envelope; gp140; immunogen.

Fig. 1.

Design of envelope expression constructs. (A) Schematic representations of HIV-1 envelope glycoproteins: gp160, the full-length precursor. Various segments include gp120, gp41 ectodomain (gp41ecto), TM anchor, and cytoplasmic tail (CT). Expression constructs are gp140, the uncleaved ectodomain of gp160 with a trimerization foldon tag and a His-tag at its C terminus; gp140–FL20, the uncleaved ectodomain of gp160 with a 20-residue linker (SGGGG)₄ inserted between gp120 and gp41, as well as a foldon tag and a His-tag at its C terminus; and gp120-fd, gp120 fused to a foldon tag and a His-tag through a 20-residue linker (SGGGG)₄. Tree-like symbols represent glycans. (B) Diagrams represent alternative 3D organizations of each protein species. Gp120 and gp41 are shown in green and blue, respectively. Foldon is in gray and the flexible linker in yellow. The conformation in the top row for each construct is supported by the data presented in this work.

Fig. 2.

Production of gp140–FL20 and gp120-fd proteins in 293T cells. (A) His-tagged gp140 and gp140–FL20 proteins derived from the HIV-1 92UG037.8 were purified by Ni-NTA from supernatants of 293T cells transiently transfected with the expression constructs of these proteins. The purified gp140 (in black) and gp140–FL20 (in red) were resolved by gel filtration chromatography on a Superose 6 column. The molecular weight standards include thyoglobulin (670 kDa), ferritin (440 kDa), γ-globulin (158 kDa), and ovalbumin (44 kDa). Peak fractions were pooled and analyzed by Coomassie-stained SDS/PAGE (Insets). Purified gp140 and gp140–FL20 were also compared in a furin cleavage assay, and digested proteins were analyzed by SDS/PAGE as shown. (B) Purification of his-tagged gp120-fd. (C) Dynamic light scattering analysis of monomeric gp120, gp120-fd trimer, gp140 trimer, and gp140–FL20 trimer. ^aThe hydrodynamic radius for the BG505 SOSIP gp140 trimer was reported in ref. .

Fig. 3.

Interactions of gp140, gp140–FL20, and gp120-fd with CD4 binding site and CD4i antibodies. Envelope proteins or their purified complexes with two-domain CD4 were captured to the surface of a sensor chip coated with an anti-HIS antibody to avoid potential artifacts introduced by protein immobilization. CD4 binding site antibodies include bnAbs VRC01, 12A12, PGV04, and a nonneutralizing antibody b6. CD4i antibodies are 17b and 412d. Fab fragment of each antibody at various concentrations were passed over the envelope trimer surface individually without regeneration for single-cycle kinetic analysis. The recorded sensorgram for gp140 is in cyan, gp140–CD4 complex in blue, gp140–FL20 in magenta, gp140–FL20–CD4 complex in red, gp120-fd in black, and gp120-fd–CD4 complex in gray; the fitted curves are in green. Sensorgrams were fit using a 1:1 binding model; and binding constructs are summarized in Table S2.

Fig. 4.

Summary of the key differences between the gp140 and gp120 constructs. Molecular mass of the monomeric 92UG037.8 gp120 was reported previously in ref. . As a comparison, the hydrodynamic radius and molecular mass for the BG505 SOSIP gp140 trimer are 8.1 nm and 365 kDa, respectively (24, 44).