Structural principles controlling HIV envelope glycosylation

Abstract

The heavily glycosylated, trimeric HIV-1 envelope (Env) protein is the sole viral protein exposed on the HIV-1 virion surface and is thus a main focus of antibody-mediated vaccine development. Dense glycosylation at the outer domain of Env constrains normal enzymatic processing, stalling the glycans at immature oligomannose-type structures. Furthermore, native trimerization imposes additional steric constraints, which generate an extensive 'trimer-induced mannose patch'. Importantly, the immature glycans present a highly conserved feature of the virus that is targeted by broadly neutralizing antibodies. Quantitative mass spectrometry of glycopeptides together with structures of the trimeric viral-spike define the steric principles controlling processing and provide a detailed map of the glycan shield.

Figure 1. Envelope cleavage induces a trimer-associated mannose patch

(A) The conformational and oligomeric state of recombinant or natural Env glycoprotein has significant impact on glycan processing. The Env proprotein precursor is synthesized in the endoplasmic reticulum and glycosylated with oligomannose-type glycans at Asn-X-Ser/Thr (X is any amino acid except proline) sequons. Trafficking through the secretory pathway exposes the glycoprotein to α-mannosidases and other glycan processing enzymes, which generally lead to the conversion of oligomannose-type glycans (green) to complex-type glycans (pink). However, intrinsic structural features of Env determine how well individual glycans can be accessed and processed by these enzymes. The outer domain of monomeric gp120 is characterised by a dense cluster of glycans that limits accessibility of ER and Golgi α-mannosidases and thus leads to the emergence of an intrinsic mannose patch. Cleaved Env engages a compact quaternary structure that further constrains glycan processing and induces the formation of an additional trimer-associated mannose population. Uncleaved, non-native Env pseudotrimers, however, have an open and irregular structure (inset EM image in B) that allows easier access to processing glycans and hence leads to an elevation in complex-type glycans. (B) Glycan profiles of released and fluorescently labelled N-glycans from three soluble, recombinant clade A BG505 constructs (monomeric gp120, pseudotrimeric uncleaved BG505.SEKS and trimeric cleaved BG505 SOSIP.664) generated by hydrophilic interaction chromatography-ultra performance liquid chromatography (HILIC-UPLC) [29].

Figure 2. Fine structure of the intrinsic mannose patch of recombinant gp120

(A) Glycan crowding in the outer domain of recombinant monomeric gp120 (BG505 strain) leads to an intrinsic mannose patch (IMP) consisting of two main oligomannose-microclusters, centered around N295 and N392 (see magnifications). Model of the glycosylated gp120 monomer is derived from the one previously described elsewhere [27]. (B) Bar graphs of the percentage of conservation of the 24 N-glycosylation sites on BG505 gp120 [11]. (C) Site-specific glycosylation profiles of IMP gp120 glycans, categorized as follows: oligomannose series (M4 to M9; Man₄GlcNAc₂ to Man₉GlcNAc₂) in green; hybrid- and complex-type glycans in pink (grouped by the number of their branching antennae and by the presence or absence of a core fucose) [27,29].

Figure 3. The trimer-associated mannose patch

Models of the glycosylated BG505 SOSIP.664 trimer are derived from the one previously described elsewhere [27]. (A) The glycans are colored according to their oligomannose content [29]. (B) Heat map of the effect of native-like trimerization on the percentage point of oligomannose-type glycans of recombinant gp120 [29]. (C) Schematic of the localization of the intrinsic and the trimer-associated mannose patches on Env.

Figure 4. Enzyme resistance and antibody recognition of the HIV-1 glycan shield

(A) ER α1,2-mannosidase I (PDB ID 5KIJ) [52] (cyan) was modelled to bind the Man₉GlcNAc₂ N332 glycan (green) on a fully glycosylated model of a previously described model of BG505 SOSIP.664 [27]. The surrounding glycans that sterically clash with enzyme recognition of N332 are highlighted in red. (B) The Fab variable region of broadly neutralizing antibody PGT128 (PDB ID 5ACO) (orange) targets the N301 glycan and also interacts and depends on additional nearby glycans [53]. Green: oligomannose-type glycans; pink: complex-type glycans. Removal of the N137 glycan has been shown