host cofactors and pharmacologic ligands share an essential interface in hiv1 capsid that is lost upon disassembly the hiv1 capsid is involved in all infectious steps from reverse transcription to integration site selection and is the target of multiple host cell and pharmacologic ligands however structural studies have been limited to capsid monomers ca and the mechanistic basis for how these ligands influence infection is not well understood here we show that a multisubunit 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 bi2 each ligand is anchored via a shared phenylalanineglycine fg motif to a pocket within the nterminal domain of one monomer and all but bi2 also make essential interactions across the nterminal domain cterminal domain ntdctd interface to a second monomer dissociation of hexamer into ca monomers prevents high affinity interaction with cpsf6 and pf74 and abolishes binding to nup153 the second interface is conformationally dynamic but binding of nup153 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 multiple ligands share an overlapping interface in hiv1 capsid that is lost upon viral disassembly