Bridging HIV neutralization sensitivities and Env dynamic phenotypes across genetically diverse variants

Abstract

The envelope glycoprotein (Env) is the sole target for neutralizing antibodies against HIV and the most rapidly evolving, variable part of the virus. High-resolution structures of Env trimers captured in the prefusion, closed conformation have revealed a high degree of structural similarity across diverse isolates. Biophysical data, however, indicate that Env is highly dynamic, and the level of dynamics and conformational sampling are believed to vary dramatically between HIV isolates. Dynamic differences likely influence neutralization sensitivity, receptor activation, and overall trimer stability. The primary objective of this dissertation is to understand how sequence diversity impacts dynamics, and better understand how differences in dynamics impact antibody recognition. After briefly introducing the HIV Env fusion protein, chapter 1 will review one of the primary biophysical techniques I use throughout this dissertation to characterize dynamics-- hydrogen-deuterium exchange mass spectrometry (HDX-MS) -- and highlight recent advances pertaining to its use in structural biology. In chapter 2 I use HDX-MS to map local dynamics across five native-like Env trimers from diverse isolates selected for their high yields, stability, and ability to be purified to a high degree of purity. We demonstrate that significant differences in epitope structural ordering are observed across most sites targeted by broadly neutralizing antibodies (bnAbs). We also observe isolate-dependent conformational switching that occurs over a broad range of timescales. Lastly, we report that hyper-stabilizing mutations that dampen dynamics in some isolates have little effect in others. In chapter 3 I show that variation in structural ordering in the V1/V2 apex of Env across a widely used panel of HIV-1 isolates is a major source of isolate-specific antigen variation that results in a marked effect on antibody association rates. With help from collaborators, we also report cryo-EM reconstructions of the apex-targeting PGT145 bnAb bound to two divergent Env that exhibit dramatically different degrees of structural ordering throughout much of the trimer structure. Parallel HDX-MS experiments demonstrate that the PGT145 bnAb has an exquisitely focused binding footprint at the apex and is unaffected by local and global structural fluctuations throughout the trimers. In chapter 4 we extend our characterization of Env dynamic phenotypes across trimers derived from isolates in a global panel that is representative of the vast genetic diversity of HIV-1. These data highlight the impact of extreme sequence diversity on dynamic phenotype, elucidates a structure-dynamic link between local dynamics and neutralization phenotype, and provides a mechanistic explanation for IgG recognition of Env trimers.