Visualization of the HIV-1 Env glycan shield across scales

Significance The HIV-1 Env “glycan shield” masks the surface of the protein from immune recognition, yet intrinsic heterogeneity defies a typical structure–function description. Using an integrated approach of cryo-EM, computational modeling, and mass spectrometry, we visualized the glycan shield structure in a new light. Our approach facilitated development of cryo-EM analysis methods and allowed for validation of models against experiment. Comparison of Env expressed in different cell lines revealed how subtle differences in composition impact glycan shield structure and affect the accessibility of epitopes on the surface, providing insights for vaccine design. Finally, time-resolved cryo-EM experiments uncovered how highly connected glycan clusters help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion. 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.