Preferential localization of the Epstein-Barr virus (EBV) oncoprotein LMP-1 to nuclei in human T cells: implications for its role in the development of EBV genome-positive T-cell lymphomas

Abstract

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) is thought to play a role in the EBV-induced B-cell transformation and immortalization. EBV has also been implicated in certain human T-cell lymphomas; however, the phenotypic effects of the expression of this oncoprotein in T cells are not known. To learn whether LMP-1 also induces phenotypic changes in T cells, we stably expressed it in human cell lines of T and B lineages and 25 LMP-1-expressing T-cell clones and 7 B-cell clones were examined. Our results show for the first time that, in sharp contrast to B cells, LMP-1 preferentially localizes to nuclei in T cells and does not induce the phenotypic changes in these cells that it induces in B cells, does not associate with TRAF proteins, and does not arrest the cell cycle in the G2/M phase. A computer-assisted analysis revealed that LMP-1 lacks the canonical nuclear localization signal. Our results suggest that this oncoprotein may not play the same role in the lymphomagenesis of T cells as it does in B cells.

FIG. 1.

Expression of LMP-1 in CEM and BJA-B cells. LMP-1 was detected by indirect immunofluorescence in permeabilized cells with S12 antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The upper panels show LMP-expressing BJA-B cells stained with S12 (A) or control (B) antibody. The lower panels show LMP-expressing CEM cells stained with S12 (C) or control (negative) (D) antibody. Note the localization of immunofluorescence in the nucleus in CEM cells (C) and at the membranes in BJA-B cells (A).

FIG. 2.

Different patterns of LMP-1 localization in Molt4 cells. LMP-expressing Molt4 cells were stained with S12 as described in the legend to Fig. 1. Shown here are three different patterns of LMP-1 localization in these cells: on the membrane (A), both on the membrane and in the nucleus (B), and in the nucleus (C). Panel D represents an LMP-expressing cell stained with a control (negative) antibody.

FIG. 3.

Detection of LMP-1 by Western blotting in CEM and BJA-B cells. (A) Whole-cell lysates (40 μg) from LMP-expressing and control vector-transfected cells were resolved by SDS-10% PAGE. After transfer onto nylon membranes, LMP-1 was detected by anti-LMP monoclonal antibody S12 followed by detection with alkaline phosphatase-conjugated anti-mouse antibodies 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium. Lanes: 1, BJA-B transfected with the control vector; 2 to 4, three different LMP-expressing BJA-B clones; 5, CEM transfected with the control vector; 6 to 8, three different LMP-expressing CEM clones; 9, an EBV-transformed B-cell line (LCL). The LMP-expressing BJA-B and CEM cells were fractionated into cytoplasmic and nuclear lysates, and each fraction was analyzed for LMP-1 expression by Western blotting. Panels B and C show LMP-1 expression in cytoplasmic and nuclear fractions, respectively. The lanes represent the clones indicated for panel A. The arrows point to LMP-1 bands.

FIG. 4.

Cell cycle profiles of LMP-expressing cells. After staining with PI, the cell cycle profile was analyzed from the DNA content by using a flow cytometer and Cellfit software. (A) The figure shows the relative number of cells in various phases of the cell cycle for control vector-transfected BJA-B (a), LMP-expressing BJA-B (b), control vector-transfected CEM (c), and LMP-expressing CEM (d) cells. Note the accumulation of cells in the G₂/M phase in LMP-expressing BJA-B cells (b). (B) Proliferation of pIgLMP-1 and control vector (IgNEO)-transfected cells. Cells (10⁵ per well) were cultured in triplicate for 24 h and pulsed with [³H]TdR for 16 h to assess proliferation. The asterisk indicates significant difference (P < 0.01).

FIG. 5.

(A) LMP-1 induced the TRAF1 protein (arrow) in BJA-B cells but not in CEM cells. Whole-cell lysates (40 μg) from LMP-expressing and control vector-transfected cells were resolved by SDS-10% PAGE. After transfer onto nylon membranes, TRAF1 was incubated with anti-TRAF1 antibody followed by detection with alkaline phosphatase-conjugated anti-rabbit antibody. The lanes represent different clones (the same as lanes 1 to 9 of Fig. 3). (B) LMP-1 (arrows) interacted with TRAF1 in BJA-B cells but not in CEM cells. TRAF1 was immunoprecipitated from cytoplasmic fractions (a) and whole-cell lysates (b), resolved by SDS-PAGE, and electroblotted onto nylon membranes. The membranes were probed for LMP-1 with LMP-specific monoclonal antibodies (CS1-4) and alkaline phosphatase-conjugated secondary antibodies. Lane 1 represents immunoprecipitation with normal rabbit serum. Lanes 2 to 10 in panel B correspond to lanes 1 to 9 in panel A, respectively.

FIG. 6.

Expression of bcl-2 (A), LFA-1 (B), and ICAM-1 (C). LMP expression induces bcl-2, LFA-1, and ICAM-1 in BJA-B cells but not in CEM cells. The expression of these proteins in different clones of LMP-expressing cells was determined by Western blotting performed on 40 μg of whole-cell lysates. The lanes represent the cell clones indicated in the legend to Fig. 3. Arrows from top to bottom indicate bcl-2, LFA-1, and ICAM-1, respectively.

FIG. 7.

LMP-1 expression induces a tyrosine-phosphorylated protein in B, but not in T, cells (arrow). Far Western blots were prepared from whole-cell lysates by using immunoprecipitated LMP-1. The blots were probed with anti-phosphotyrosine and alkaline phosphatase-conjugated anti-mouse IgG as described in the legend to Fig. 3. The lanes indicate the cell clones described in the legend to Fig. 3.