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

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. Overall design: Env from the R5-tropic HIV-1 BaL strain was encoded by a synthetic codon-optimized gene with a CD5 leader sequence. The Env sequence was diversified by overlap-extension PCR to generate three single-site saturation mutagenesis libraries spanning the full length of the gene. The libraries were expressed in human Expi293F cells (a HEK293 derivative) and sorted by FACS for binding to soluble CD4 (sCD4) and the broadly-neutralizing antibodies PG16 and VRC01. Enrichment ratios were calculated for all single amino acid substitutions by comparing the frequencies of sequence variants in the sorted cells (from RNA transcripts) with the naive libraries (plasmid DNA). Evolution experiments were duplicated.