ACTIVE-SITE RESIDUES OF CYCLOPHILIN-A ARE CRUCIAL FOR ITS INCORPORATION INTO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VIRIONS

Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosoli c receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not int eract with CyPA, The incorporation of CyPA into HIV-1 virions is media ted by a specific interaction with a proline containing, solvent-expos ed loop in the capsid (CA) domain of the Gag polyprotein, CsA, which d isrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-sit e mutants of human CyPA, Expression vectors for epitope-tagged wild-ty pe and mutant CyPA were transfected into COS-7 cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagg ed CyPA into HIV-1 virions. One CyPA mutant with a substantially decre ased sensitivity to CsA was incorporated with wild-type efficiency, de monstrating that the requirements for binding to CsA and to HIV-1 CA a re not identical. The remaining sis CyPA mutants were incorporated wit h markedly reduced efficiency, providing in vivo evidence that HIV-1 C A interacts with the active site of CyPA.