Natural variations at the E433 site in Env-gp120 β20-β21 cooperate with V2 and gp41 mutations to modulate HIV-1 infectivity.

HIV-1 infection begins with Env-gp120 binding to the CD4 receptor and CD4-triggered Env conformational changes that enable viral entry. To identify key Env sites that modulate Env-CD4 interactions, we analyzed growth-enhanced adaptive mutants generated through the in vitro passage of a replication-feeble HIV-1 clone carrying a CCR5-tropic env from a clinical isolate CRF08_BC. Sequencing the four adapted clones tested revealed multiple Env mutations, including the recurrent substitution at the gp120 residue E433 within the β20-β21 hairpin in the C4 domain. By using chimeric proviral and virus mutant clones, we found that viral infectivity enhancement required specific combinations of Env mutations. In one adapted clone, a four-amino acid deletion in the V2 domain, E433K, and a two-nucleotide insertion in gp41 that results in C-terminal truncation of the cytoplasmic tail acted cooperatively: only the triple combination reproduced the high infectivity of the adapted virus. In another adapted clone, while substitutions D166N and N195K in the V2 domain were insufficient, adding the E433K mutation to the two significantly elevated viral infectivity. Across the point mutants, D166N decreased infectivity whereas N195K increased infectivity. E433K alone was largely silent but compensated the D166N defect and potentiated the N195K effect. Introducing a positive charge at the 433 site strongly enhanced viral infectivity: E433R increased infectivity by ~five-fold on its own and markedly enhanced infectivity with D166N+N195K, whereas E433G had little impact. Mutants bearing D166N+N195K and/or E433R displayed increased sensitivity to soluble CD4, consistent with enhanced CD4-triggered Env responsiveness. In contrast, a CCR5-antagonist time-of-addition entry assay showed similar progression to CCR5 engagement, suggesting improved entry yield rather than faster entry kinetics. Neutralization profiling suggested limited global trimer remodeling, with mutation-dependent changes in V1/V2- and V3-glycan-directed antibodies but little impact on CD4-binding-site antibodies. In silico gp120-CD4 analyses supported this view, implicating inter-protomer V2 contacts and a positive-charge effect at E433 at the CD4 interface. Taken all together, these results identify E433 in the β20-β21 hairpin in the C4 domain as an electrostatic tuning site that cooperates with V2 and gp41 changes to increase productive entry.