Defining HIV-1 Envelope N-Glycan Microdomains through Site-Specific Heterogeneity Profiles

Abstract

The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV-transmitted/founder (T/F) and immune escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos National Laboratory HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune escape variants. IMPORTANCE The Env protein of HIV is highly glycosylated, and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered, resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

Impact of glycan positioning on HIV-1 Env glycan shield density, function, and antibody recognition

“…To further clarify the function of the N262 glycans, we produced recombinant trimeric gp120 WEAU variants using our previously published approach that added a foldon and V5 and His tags to the gp120 (Hargett et al, 2019, Raska et al, 2014, Raska et al, 2008, Raska et al, 2010. We confirmed that gp120 WEAU glycoproteins were exclusively produced as trimers ( Fig 5A).…”

“…The glycopeptides for each NGS were expressed as relative abundance of each individual N-glycopeptide at each site for each rgp120 variant ( Fig 6A). Three different types of heterogeneity were observed that categorized individual NGS as (i) predominantly high-mannose glycans, (ii) predominantly complex glycans, and (iii) a mixed population of both high-mannose and complex glycans ( Fig EV2B) (Hargett et al, 2019). N-glycan sitespecific heterogeneity profiles of the four WEAU rgp120 trimers are shown in Fig 6A (Behrens et al, 2017).…”

“…Given the effect of the N262 glycans on Env structure and functionality, we decided to decipher how the N262 glycans could globally affect the Env glycan shield. To begin to address this question, we performed monosaccharide compositional analysis by using gas-liquid chromatography to assess the global glycan composition of WEAU rgp120 trimer variants (Hargett et al, 2019, Raska et al, 2014, Raska et al, 2010. Surprisingly, the monosaccharide compositional analysis revealed that the presence of N262 not only affected function, but also controlled the overall glycan composition of the Env gp120 trimers.…”

“…To determine the specific nature of changes at the individual potential NGS, we used our validated LC-MS-based glycomics workflow (Hargett et al, 2019). Trimeric rgp120 preparations with N262 NGS (WEAU-d16-N264S and WEAU-d391) and without N262 potential NGS (WEAU-d16 and WEAU-d391-S264N) were normalized for total protein (Fig EV2A), digested with various combinations of proteases and glycosidases, and the subsequent (glyco)peptides were analyzed by LC-MS.…”

“…However, the impact of these potential NGS mutations was only studied regarding their immediate location, and escape from nAbs, but not with respect to global effects on the Env structure. Our recent analysis of all deposited HIV-1 sequences in the LANL database demonstrated that certain N-glycan microdomains have a limited number of potential NGS combinations (Hargett et al, 2019). These findings suggest that some glycan units have a distinct functional role in terms of position and glycan density to maintain the overall massive glycan shield (Hargett et al, 2019).…”