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 <i>in vitro</i> 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.