McFadden, W. M.; Kirby, K. A.; Lorson, Z. C.; Wang, L.; Highland, C. M.; Harvey, S. R.; Brancato, S.; Emanuelli Castaner, A.; Du, H.; Wysocki, V. H.; Wang, Z.; Dick, R. A.; Sarafianos, S. G.

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 (CAHEX). 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 CAHEX. 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 CAHEX and CA pentamers (CAPENT). Cryogenic-electron microscopy analysis of in vitro assembled capsid-like particles (CLPs) with IP6 suggests that ZW-1261 leads to the absence of CAPENT and damages the pre-formed conical lattice. To elucidate how this FGBP-targeting antiviral impacts CAPENT, we further solved structures of CAPENT-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 CAPENT to a CAHEX-like conformation. Collectively, this suggests a mechanism by which addition of FGBP-binding inhibitor to native cores leads to the absence of CAPENT, impacting capsid closure and core integrity.