Abstract
The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.
Highlights
The dense array of N-linked glycans on the HIV type 1 (HIV-1) envelope glycoprotein (Env), known as the “glycan shield,” is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure–function analysis
We found that dynamics give rise to a network of interglycan interactions that drive the formation of higherorder structure within the glycan shield
Using Env expressed in three common cell lines, we show how differences in glycan composition and occupancy can be detected by cryo-EM and result in changes to glycan shield structure and dynamics that affect the accessibility of epitopes on the surface
Summary
The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the “glycan shield,” is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure–function analysis. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. One crystallography study addressing glycan shield structure was recently published and the data show evidence for highly stabilized glycans engaging in a wide range of interglycan contacts [6], which is at odds with the cryo-EM results.
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