Involvement of human CRM1 (exportin 1) in the export and multimerization of the Rex protein of human T-cell leukemia virus type 1

Abstract

We investigated the role of human CRM1 (hCRM1) (exportin 1) in the function of Rex protein encoded by human T-cell leukemia virus type 1. hCRM1 promoted the export of Rex protein from the nucleus to the cytoplasm. A Rex protein with a mutation in the activation domain, RexM90, lost both the ability to bind to hCRM1 and the ability to multimerize. The overexpression of hCRM1 complemented the functional defects of RexM64, which contains a mutation in the multimerization domain of Rex. A dominant-negative mutant of Rex which sequesters cofactors of Rex abrogated multimerization as well as the activity of the wild-type Rex protein. These two functions were simultaneously restored by the overexpression of hCRM1. Taken together, these results suggest that hCRM1 plays important roles in the multimerization and export of Rex protein.

FIG. 1

Subcellular localization of wild-type and mutant Rex proteins. At 24 h after transfection, cells transfected with 0.1 μg of pSRαRex (A and D), pSRαRexM64 (B and E), or pSRαRexM90 (C and F) in combination with 0.5 μg of pSRαhCRM1 (D, E, and F) or 0.5 μg of pSRα296 instead of pSRαhCRM1 in order to adjust the total amounts of the plasmids (A, B, and C) were subjected to immunofluorescence microscopy. Magnification, ×1,720.

FIG. 2

Overexpression of hCRM1 restores Rex activity inhibited by TAgRexM64. HeLa cells were transfected with 0.05 μg of pSRαRex, 0.5 μg of pTU50RXRE as a reporter plasmid, and various amounts of pSRαhCRM1 with (•) or without (▪) 0.2 μg of pSRαTAgRexM64. pCDM-β-gal (0.1 μg) was included in all samples as an internal control. At 48 h after transfection, the cells were harvested, the amount of Env and the activity of β-galactosidase were quantified, and the ratios of Env to β-galactosidase were calculated. The ratio of the amounts of Env and β-galactosidase obtained in the sample in which TAgRexM64 and pSRαhCRM1 were omitted was arbitrarily set at 1.

FIG. 3

Interaction of hCRM1 with Rex, RexM64, and RexM90 in the nuclei of mammalian cells. A mammalian version of the two-hybrid assay with transient expression by transfection into HeLa cells was performed. The hCRM1 protein was expressed as a GAL4 fusion, and the other proteins were expressed as VP16 fusions. pCDM-β-gal (0.1 μg) was included in all samples as an internal control. At 24 h after transfection, the cells were harvested, and the amount of CAT and the activity of β-galactosidase were quantified. The amount of CAT and the activity of β-galactosidase obtained after transfection of Rex-VP and GAL-hCRM1 were 370 pg and 2.5 × 10⁻³ U, respectively, and the ratio (CAT/β-galactosidase) was arbitrarily set at 1. GAL4 represents the plasmid expressing only the GAL4 region.

FIG. 4

Activities of Rex, RexM64, and RexM90. Cells were transfected with 0.5 μg of pTU50RXRE and increasing quantities of pSRαRex (•), pSRαRexM64 (▪), or pSRαRexM90 (▴). The culture medium was harvested at 48 h posttransfection, the amount of Env protein produced was assayed, and the cell lysates were used for the quantification of β-galactosidase expression levels. The Env/β-galactosidase ratio for each sample was divided by that for the sample without Rex and expressed as fold activation.

FIG. 5

Analysis of in vivo multimerization of the wild-type and mutant Rex proteins. HeLa cells were cotransfected with the expression plasmids for GAL and VP16 fusion proteins as indicated. The cells were harvested 24 h after transfection, and the amount of CAT and the activity of β-galactosidase were quantified. The amount of CAT and the activity of β-galactosidase resulting from the coexpression of GAL-Rex and Rex-VP were 270 pg and 4.8 × 10⁻³ U, respectively, and the ratio was assigned a value of 1.

FIG. 6

Western blot analysis of GAL-Rex and Rex-VP fusion protein expression in HeLa cells. The results for the control (lane 1) and for RexM90-VP (lane 2), RexM64-VP (lane 3), Rex-VP (lane 4), GAL-RexM90 (lane 5), GAL-RexM64 (lane 6), and GAL-Rex (lane 7) are shown.

FIG. 7

Inhibitory effect of TAgRexM64 and TAgRexM6490 on Rex-Rex multimerization. HeLa cell cultures were transfected with pGAL-Rex and pRex-VP in combination with various amounts of pSRαTAgRexM64 (▪) or with pSRαTAgRexM6490 (•). At 24 h posttransfection, the cells were harvested, and the amount of CAT and the activity of β-galactosidase were quantified. The amount of CAT and the activity of β-galactosidase obtained with transfection of pRex-VP and pGAL-Rex without any TAgRex expression plasmid were 230 pg and 4.5 × 10⁻³ U, respectively, and the ratio was arbitrarily set at 1.

FIG. 8

Overexpression of hCRM1 restores Rex-Rex multimerization inhibited by TAgRexM64. HeLa cell cultures were transfected with pGAL-Rex, pRex-VP, and 0.2 μg of pSRαTAgRexM64 in combination with various amounts of pSRαhCRM1. The amount of CAT and the activity of β-galactosidase obtained with transfection of pRex-VP and pGAL-Rex without pSRαTAgRexM64 were 380 pg and 6.8 × 10⁻³ U, respectively, and the ratio was arbitrarily set at 1.