Defining HIV-1 Envelope N-glycan Microdomains Through Site-specific Heterogeneity Profiles.

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 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 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.