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 cellula r peptidyl-prolyl isomerase cyclophilin A (CyPA) by the Gag polyprotei n. Elucidation of the biochemical role of CyPA would be aided by a det ailed analysis of the genetic requirements for the formation of the Ga g-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 neces sary CyPA residues. Cyclophilins possess a hydrophobic pocket where pr oline-containing peptide substrates and the immunosuppressive drug cyc losporine A bind. In this study, we engineered five CyPA mutations, ea ch of which alters a residue that contributes to the hydrophobic pocke t. Compared with the wild-type protein, all of the mutants drastically reduced CyPA binding to HIV-1 Gag and similarly inhibited CyPA incorp oration into virions. In addition, we demonstrated that previously rep orted differences between the Gag-binding properties of CyPA and CyPB are due to adventitious association involving residues in the signal s equence 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 cyclospor ine A, HIV-1 Gag directly contacts residues in the hydrophobic pocket of CyPA.