Structure of a cleavage-independent HIV Env recapitulates the glycoprotein architecture of the native cleaved trimer

Anita Sarkar,Jesper Pallesen,Robyn L Stanfield,Ian A Wilson,Anna-Janina Behrens,Natalia De Val,Shailendra Kumar Sharma,Max Crispin,Adriana Irimia,Sonu Kumar,Shridhar Bale,Devan C Diwanji,Andrew B Ward,Richard T Wyatt

doi:10.1038/s41467-018-04272-y

Abstract

Furin cleavage of the HIV envelope glycoprotein is an essential step for cell entry that enables formation of well-folded, native-like glycosylated trimers, releases constraints on the fusion peptide, and limits enzymatic processing of the N-glycan shield. Here, we show that a cleavage-independent, stabilized, soluble Env trimer mimic (BG505 NFL.664) exhibits a “closed-form”, native-like, prefusion conformation akin to furin-cleaved Env trimers. The crystal structure of BG505 NFL.664 at 3.39 Å resolution with two potent bNAbs also identifies the full epitopes of PGV19 and PGT122 that target the receptor binding site and N332 supersite, respectively. Quantitative site-specific analysis of the glycan shield reveals that native-like glycan processing is maintained despite furin-independent maturation in the secretory pathway. Thus, cleavage-independent NFL Env trimers exhibit quaternary protein and carbohydrate structures similar to the native viral spike that further validate their potential as vaccine immunogen candidates.

Highlights

Furin cleavage of the HIV envelope glycoprotein is an essential step for cell entry that enables formation of well-folded, native-like glycosylated trimers, releases constraints on the fusion peptide, and limits enzymatic processing of the N-glycan shield
The purified BG505 native flexibly linked (NFL).[664] trimer was incubated with 2× molar excess per binding site of Fabs PGT122 and PGV19 for stabilization, and further treated with EndoH glycosidase (New England Biolabs) that partially trims accessible glycans to the first GlcNAc moiety (Supplementary Fig. 1a-c)
The structure was determined by molecular replacement (MR) using BG505 SOSIP.[664] (PDB 4TVP) and unbound PGV19 Fab

Summary

Introduction

Furin cleavage of the HIV envelope glycoprotein is an essential step for cell entry that enables formation of well-folded, native-like glycosylated trimers, releases constraints on the fusion peptide, and limits enzymatic processing of the N-glycan shield. We show that a cleavage-independent, stabilized, soluble Env trimer mimic (BG505 NFL.664) exhibits a “closed-form”, native-like, prefusion conformation akin to furin-cleaved Env trimers. Cleavage-independent NFL Env trimers exhibit quaternary protein and carbohydrate structures similar to the native viral spike that further validate their potential as vaccine immunogen candidates. A soluble mimic of the furincleaved HIV Env, termed SOSIP, was designed to yield native-like trimers, and contained an engineered disulfide bond that covalently tethers gp[120] to gp[41] and an I559P mutation that favors the gp[41] prefusion state and enhances Env trimerization[4, 5]. The NFL design retains the trimerization-enhancing I559P mutation[5] from the SOSIP design, while dispensing with the disulfide bond[4] between gp[120] Ala[501] to gp[41] Thr60520

Methods

Results

Conclusion