Damaging the conical morphology of HIV-1 capsid by targeting the FG-binding pocket and disfavoring pentameric subunits needed for core closure

The HIV-1 capsid is an essential viral component, targeted by the long-acting antiretroviral Lenacapavir (LEN). LEN binds to the HIV-1 capsid protein (CA) at the phenylalanine-glycine (FG) binding pocket (FGBP), a site for multiple host-factor and antiviral interactions in CA hexamers (CA HEX ). Previously, we generated a chemical library to investigate the FGBP; ZW-1261, a lead compound, exhibits potent antiviral activity and strong inter-subunit interactions within CA HEX . Here, we report the molecular mechanism by which ZW-1261 affects the morphology and integrity of capsid lattice. ZW-1261 alone rapidly induces tubular CA assemblies; simultaneous addition of ZW-1261 with the assembly cofactor inositol hexaphosphate (IP6) forms morphologically distinct tubes. In mature virions, IP6 is required for the assembly of both CA HEX and CA pentamers (CA PENT ). Cryogenic-electron microscopy analysis of in vitro assembled capsid-like particles (CLPs) with IP6 suggests that ZW-1261 leads to the absence of CA PENT and damages the pre-formed conical lattice. To elucidate how this FGBP-targeting antiviral impacts CA PENT , we further solved structures of CA PENT -only icosahedral assemblies (T = 1), formed by reported mutations, that were treated with ZW-1261. We find that ZW-1261 binding in these constrained T = 1 assemblies converts CA PENT to a CA HEX -like conformation. Collectively, this suggests a mechanism by which addition of FGBP-binding inhibitor to native cores leads to the absence of CA PENT , impacting capsid closure and core integrity.