Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan

Abstract

The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines.

Importance: The HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.

FIG 1

Purification of WT and N197Q SOSIP trimers. (A) Size exclusion profile of purified fully glycosylated WT and N197Q BG505 SOSIP expressed in HEK293F cells. Abs, absorbance. (B) SDS-PAGE analysis of purified BG505 (left) and PVO.4 (right) WT and N197Q mutant trimers under reducing and nonreducing conditions. DTT, dithiothreitol. (C) BN-PAGE analysis of WT and N197Q mutant trimers. (D) Sequence alignment of BG505 and PVO.4 at the V1/V2 region of Env. Protein alignment was performed by using Vector NTI AlignX, and the color scheme represents amino acid identity (yellow) and blocks of similarity (green) (88).

FIG 2

Biochemical and biophysical comparison of the purified BG505 SOSIP.664 WT and the BG505 SOSIP.664 N197Q mutant. (A) Thermal stability of the BG505 SOSIP.664 WT and the BG505 SOSIP.664 N197Q mutant analyzed by DLS. The BG505 SOSIP.664 N197Q mutant presents slightly lower onset and midpoint temperatures of melting, indicating slightly less stability than the wild-type trimer. (B) SAXS parameters for the BG505 SOSIP.664 WT and the BG505 SOSIP.664 N197Q mutant. Radii of gyration (Rg) from Guinier approximation and from the P(r) plot calculated by the program GNOM were in good agreement. (C) 2D class averages of EM reconstruction of the BG505 SOSIP.664 WT and the N197Q mutant. The two display 3-fold symmetry and are consistent with trimeric, native-like morphology.

FIG 3

Local dynamic changes detected by HDX in the presence and absence of the N197 glycan. (A) Differences in deuterium exchange are plotted on the trimer crystal structure (PDB accession number 4TVP) (61). V1/V2 base and β20 bridging sheets are slightly less protected (red) in the BG505 SOSIP.664 N197Q trimer. The N197 glycan base is shown in yellow. (B) Individual deuterium uptake plots of selected peptides with error bars from replicate experiments. All uptake plots are shown in Fig. S1 in the supplemental material.

FIG 4

Biolayer interferometry to assess antibody binding kinetics. Sensograms show the BG505 SOSIP.664 WT or the BG505 SOSIP.664 N197Q mutant and the WT BG505 gp120 monomer or the N197Q mutant BG505 gp120 monomer for binding to and unbinding from immobilized CD4 and antibodies targeting different epitopes on Env. The full concentration series of the sensograms are shown in Fig. S2 in the supplemental material. All analyses were performed at least in duplicate.

FIG 5

Position of the N197 glycan in the Env trimer. (A and B) The spatial occupancy from the ensemble of glycosylated Env models shows the positions of the N197, N276, N301, N363, N386, and N463 glycans, which were found to limit access to the CD4bs (yellow surface) on trimeric Env (A) and monomeric gp120 (B). (C) Position of the N197 glycan relevant to various epitopes on the trimer. The binding surface is indicated in dashed lines for CD4, b12, and VRC01. The positions of other antibodies relevant to this study are shown, including PG9 (blue), PGT122 (which binds a similar epitope as PGT121) (orange), and 2G12 (yellow).

FIG 6

Effects of N197 glycan removal in the Env trimer or gp120 monomer. In the trimer (top), the N197 glycan partially occludes the CD4 binding site. Removal of the N197 glycan increases the exposure of the CD4 binding site while also making the V1/V2 loop more flexible and increasing the accessibility of V3. In gp120 monomers (bottom), without tertiary and quaternary structural constrains, the V1/V2/V3 loops are relatively disordered. Since the N197 glycan does not occlude the CD4 binding site, its removal has no significant influence on the binding of CD4bs or V3-specific antibodies.