Host Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost upon Disassembly

The HIV-1 capsid is involved in all infectious c us steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligancls. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection well understood. Here we show that multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also al interactions across he N-terminal domain: C-terminal domain (NTD:CTD) interface to a second niononle Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding t NUP153. The second interface is conformationally dynamic ' but binding of NLIP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that ul e ligands share an overlapping. interface in HIV-1 capsid that is lost upon viral disassembly.