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 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 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 make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) 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 HIV-1 capsid that is lost upon viral disassembly.
Author Summary The early steps of HIV-1 infection are poorly understood, in part because of the difficulty in obtaining high-resolution information on encapsidated virus and its interaction with host cofactors. This, in turn, has made it difficult to design effective anti-capsid (CA) drugs. In our present study, we have used stabilized hexamers of HIV-1 CA to obtain complexed crystal structures with two cellular cofactors that are important for HIV-1 infection. These structures and accompanying virology reveal an essential interface in the capsid of HIV-1 that is lost upon viral uncoating. This interface is used to recruit both the nuclear targeting cofactor CPSF6 and NUP153, a nuclear pore component that facilitates nuclear entry. The high-resolution information provided by these structures reveals that the interface is degenerate and CA mutations can be made that selectively perturb sensitivity to each cofactor. This interface is also competed by two antiviral drugs, PF74 and BI-2, whose different mechanisms of action are not fully understood. We show that PF74, but not BI-2, binds across monomers within multimerized capsid affecting an inter-hexamer interface that is crucial for maintaining intact virions and that the addition of saturating concentrations of PF74 causes an irreversible block to viral reverse transcription.