FIG. 1.

Specific interaction between hCrm1 and functional leucine-rich NESs detected in the mammalian two-hybrid assay. (A) The hCrm1 protein was expressed fused to the GAL4 DNA binding domain, while the indicated retroviral proteins were expressed as VP16 transcription activation domain (AD) fusions. COS cell cultures (in 100-mm-diameter dishes) were transfected by the DEAE-dextran procedure, using 1 μg of the pG6(−31)HIVLTRΔTAR reporter plasmid, 0.5 μg of the pGAL4-Crm1 expression plasmid, 1 μg of the relevant VP16 fusion protein expression plasmid, and 1 μg of the pBC12/CMV/βgal internal control plasmid. Transfected cells were harvested at ∼48 h posttransfection and analyzed for CAT and β-Gal expression levels as previously described (4, 18). (B) As for panel A except that COS cell cultures (on 35-mm-diameter dishes) were transfected by the calcium phosphate procedure, using 2 μg of GAL4-Crm1, 2 μg of each VP16 fusion expression plasmid, 0.5 μg of the pG6(−31)HIVLTRΔTAR indicator, and 0.1 μg of pBC12/CMV/βgal. The indicated mutants of the Rev NES (75-LPPLERLTLD-84) have been described elsewhere (31) and are as follows: M10, Leu 78 and Glu 79 to Asp and Leu, respectively; M19, Pro 77 to Asp; M21, Leu 81 and Thr 82 to Asp and Leu; M22, Leu 83 and Asp 84 to Asp and Leu; M27, Leu 78 to Ala; M29, Leu 83 to Ala; M32, Leu 78, Leu 81, and Leu 83 all to Ala. The only active Rev mutant is M19. These data are representative of three separate transfection experiments.