The hydrophobic pocket of cyclophilin is the binding site for the human immunodeficiency virus type 1 Gag polyprotein.

Completion of an early step in the human immunodeficiency virus type 1 (HIV-1) life cycle requires incorporation into virions of the cellular peptidyl-prolyl isomerase cyclophilin A (CyPA) by the Gag polyprotein. Elucidation of the biochemical role of CyPA would be aided by a detailed analysis of the genetic requirements for the formation of the Gag-CyPA complex; previous experiments have demonstrated the requirement for a critical proline and the immediately preceding glycine, located within the capsid domain of Gag, but nothing is known about the necessary CyPA residues. Cyclophilins possess a hydrophobic pocket where proline-containing peptide substrates and the immunosuppressive drug cyclosporine A bind. In this study, we engineered five CyPA mutations, each of which alters a residue that contributes to the hydrophobic pocket. Compared with the wild-type protein, all of the mutants drastically reduced CyPA binding to HIV-1 Gag and similarly inhibited CyPA incorporation into virions. In addition, we demonstrated that previously reported differences between the Gag-binding properties of CyPA and CyPB are due to adventitious association involving residues in the signal sequence of CyPB and that the core domain of CyPB interacts with Gag in a fashion which is indistinguishable from that of CyPA. These studies indicate that, as with other proline-containing peptides or cyclosporine A, HIV-1 Gag directly contacts residues in the hydrophobic pocket of CyPA.