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Mutational tolerance of HIV-1 Env in the CD4, VRC01 and PG16 bound states

ABSTRACT: HIV-1 infection begins with binding of the viral envelope glycoprotein Env to the host receptor CD4, triggering a series of conformational changes that lead to fusion of the virus and cell membranes. Env, a trimer of gp120 and gp41 subunits, occupies a ‘closed’ conformation with contacts between gp120 subunits at the apex, and transitions through an ‘open’ conformation with the gp120 subunits spread apart following CD4 binding. Using deep mutational scanning, sequence-fitness landscapes were mapped for full-length Env from the clade B BaL strain interacting with CD4, and broadly neutralizing antibodies VRC01 and PG16, which preferentially bind closed Env. Contacting residues are conserved for CD4 binding, and glycosylation at N262 is critical for accessing the high-affinity CD4-bound state. By comparison, VRC01 binding is resistant to most single amino acid substitutions, an ideal quality in a broadly neutralizing antibody. Also in contrast to CD4 interaction, Env interfacial residues are under tight selection for PG16 binding to maintain a closed conformation. Screening for mutations that enhanced PG16 binding, we identified several important sites, in particular neutralization of the electropositive apical cavity that we hypothesize promotes trimer opening by electrostatic repulsion. Mutations were combined to generate Quaternary Epitope Stabilized (QES) mutants with enhanced presentation of the PG16 epitope, and the mutations were partially transferable to other HIV-1 strains. These mutational analyses offer insight into Env conformational stabilization that may assist immunogen design.

ORGANISM(S): Human immunodeficiency virus 1

PROVIDER: GSE102276 | GEO | 2019/02/03

REPOSITORIES: GEO

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Project description:Impact of Common Modifications on the Antigenic Profile and Glycosylation of Membrane-Expressed HIV-1 Envelope Glycoprotein Recent HIV-1 vaccine development has centered on near native soluble envelope glycoprotein (Env) trimers. These trimers are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These same stabilizing mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their precise effects in this context are unclear. To address this question, we investigated the effects of Env mutations expressed on virus-like particle (VLP) in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 (NFL) neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed processing post-expression, resulting in increased trimer stability. Gp41 N-helix mutations I559P and NT1-5 both imparted lateral trimer stability, but concomitantly reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ donor plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common effect of mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. We observed a minor species of high mannose glycan only gp160 in particle preparations. This was unaffected by any mutations and instead bypasses normal folding and glycan maturation processes. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and increased the proportion of high mannose gp160. Remarkably, NAbs were unable to bind to full-length Env trimers. Overall, our findings suggest caution in leveraging mutations to ensure they impart valuable membrane trimer phenotypes for vaccine use. The files enclosed are labelled according to the enzyme used in the digest. T = Trypsin, C = Chymotrypsin, A = Alpha-lytic protease

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Mutational tolerance of HIV-1 Env in the CD4, VRC01 and PG16 bound states

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