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.
Braaten
D
D
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Ansari
H
H
Luban
J
J
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D017353:Gene Deletion; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019696:Peptidylprolyl Isomerase; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion; D019065:Virus Assembly
D002352:Carrier Proteins; D015683:Gene Products, gag; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase