Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells to Ras-mediated apoptosis

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.     

A rapid screen for putative mislocalization mutations by using a GAL4-hybrid protein.

A GAL4 one-hybrid system was used to develop an indirect assay for intracellular localization. The Kar1 protein, a component of the yeast spindle pole body (SPB), was shown to be targeted to the SPB by electron microscopic analysis, or indirect immunofluorescence methods. To assay localization of the Kar1p by measuring the reporter gene expression on solid media, we constructed Kar1-Gal4 hybrid proteins with or without the SPB localization domain. The long fusion Kar1(299)-Gal4 with the localization domain led to non-growth on SC-His + AT media, and quite a low level of the lacZ reporter gene expression. The short fusion Kar1(107)-Gal4 without the localization domain caused a full activation of the reporter genes, HIS3 and lacZ, indicating that the protein remains in the nucleoplasm. These results suggest that the Kar1(299)-Gal4p fusion protein, localized to the SPB, can be clearly differentiated with the mislocalized protein by assaying the reporter gene expression. By utilizing the Kar1(299)-Gal4 construct, we isolated ten spontaneous mutations that were defective in the Kar1-Gal4p localization. Four of these mutations were in the same complementation group. We propose, therefore, that the Gal4-hybrid localization assay can be utilized in cases where the target organelle or the structure is too small for microscopic analysis, or in the initial screening for mutations defective in localization.