Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

The third variable (V3) loop on the HIV-1 Env glycoprotein is required for viral entry. Here, the authors applied DARPin technology to produce broadly neutralizing inhibitors targeting a region of V3 that becomes accessible after binding to the CD4 receptor. The third variable (V3) loop on the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein trimer is indispensable for virus cell entry. Conformational masking of V3 within the trimer allows efficient neutralization via V3 only by rare, broadly neutralizing glycan-dependent antibodies targeting the closed prefusion trimer but not by abundant antibodies that access the V3 crown on open trimers after CD4 attachment. Here, we report on a distinct category of V3-specific inhibitors based on designed ankyrin repeat protein (DARPin) technology that reinstitute the CD4-bound state as a key neutralization target with up to >90% breadth. Broadly neutralizing DARPins (bnDs) bound V3 solely on open envelope and recognized a four-turn amphipathic & alpha;-helix in the carboxy-terminal half of V3 (amino acids 314-324), which we termed '& alpha;V3C'. The bnD contact surface on & alpha;V3C was as conserved as the CD4 binding site. Molecular dynamics and escape mutation analyses underscored the functional relevance of & alpha;V3C, highlighting the potential of & alpha;V3C-based inhibitors and, more generally, of postattachment inhibition of HIV-1.