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, 105 (14), 5489-94

EBV-encoded EBNA-6 Binds and Targets MRS18-2 to the Nucleus, Resulting in the Disruption of pRb-E2F1 Complexes

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EBV-encoded EBNA-6 Binds and Targets MRS18-2 to the Nucleus, Resulting in the Disruption of pRb-E2F1 Complexes

Elena Kashuba et al. Proc Natl Acad Sci U S A.

Abstract

Epstein-Barr virus (EBV), like other DNA tumor viruses, induces an S-phase in the natural host cell, the human B lymphocyte. This is linked with blast transformation. It is believed that the EBV-encoded nuclear antigen 6 (EBNA-6) is involved in the regulation of cell cycle entry. However, the possible mechanism of this regulation is not approached. In our current study, we found that EBNA-6 binds to a MRPS18-2 protein, and targets it to the nucleus. We found that MRPS18-2 binds to both hypo- and hyperphosphorylated forms of Rb protein specifically. This binding targets the small pocket of pRb, which is a site of interaction with E2F1. The MRPS18-2 competes with the binding of E2F1 to pRb, thereby raising the level of free E2F1. Our experimental data suggest that EBNA-6 may play a major role in the entry of EBV infected B cells into the S phase by binding to and raising the level of nuclear MRPS18-2, protein. This would inhibit pRb binding to E2F1 competitively and lift the block preventing S-phase entry.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
EBNA-6 binds S18-2 in vitro. (A) GST-S18-2 precipitates EBNA-6 from freshly EBV-infected B cells in the GST pulldown assay. The membrane was probed with anti-EBNA-6 antibody. Lane 1: GST-S18-2 precipitated EBNA-6 from freshly EBV-infected B cells (72 h, 5 × 106 cells); lane 2: GST-S18-2 precipitation from MUTUI cell lysates; lane 3: GST precipitation from freshly infected B cells (as in lane 1); lane 4: GST precipitation from MUTUI cell lysate; lane 5: lysate of 0.5 × 106 freshly infected B cells; lane 6: lysate of 0.5 × 106 MUTUI cells. (B) SPR responses at the stage of protein interaction. The stepwise changes are due to solution replacement and correspond to changes in refractive index. EBNA-6 from LCL cell lysate binds to anti-EBNA-6 on the surface (first curve). GST-S18-2 binds to surface EBNA-6 (second curve). The binding of protein A to the chip surface and the anti-EBNA-6 antibody to protein A is not shown.
Fig. 2.
Fig. 2.
EBNA-6 targets S18-2 to the nucleus of the transfected cells. (a–e) MCF7 cells transfected with GFP-S18-2 (green signal). Mitochondria are stained with MitoFluor in red (see a, c, and d) and DNA in blue (a and e). Notice that GFP-S18-2 (green signal in a, b, and d) shows predominantly cytoplasmic distribution. (f–j) MCF7 cells that expressed GFP-S18-2 for 24 h were infected with recombinant vaccinia virus that expressed EBNA-6 (stained in blue; see f, h, and i). Mitochondria are stained in red (see f and j). Notice the translocation of GFP-S18-2 (green signal in f, g, and i) to the nucleus and colocalization with EBNA-6 (see overlapping blue and green signals in layers f and i). MitoFluor red was used to visualize mitochondria. They remain in cytoplasm (see red signal in f and j). (k–o) MCF7 cells that expressed MT-S18-2 constitutively (green signal) were transfected with pBabe-EBNA-6 (red signal). Cells after 24 h were stained with anti-EBNA-6 mouse monoclonal antibody, followed with horse anti-rabbit TR conjugated antibody. After blocking with mouse serum, we stained MT-S18-2 with FITC-conjugated anti-c-myc monoclonal antibody. Predominantly cytoplasmic MT-S18-2 (green signal in k, l, and n) translocated to the nucleus in the presence of EBNA-6. Moreover, the green S18-2 signal followed the red EBNA-6 pattern in the EBNA-6-expressing cell (k and n). (p–t) MCF7 cells that expressed MT-S18-2 constitutively were infected with EBNA-6-expressing recombinant vaccinia virus. Images were captured by using a confocal microscope. EBNA-6 was stained with anti-EBNA-6 mouse monoclonal antibody and TR-conjugated secondary horse anti-mouse antibody (red signal in p, r, and s). After blocking with total mouse serum, MT-S18-2 was stained with anti-c-myc mouse FITC-conjugated antibody (green signal in p, q, and s). Notice the predominantly cytoplasmic expression of MT-S18-2 in cells where EBNA-6 was absent (type I cell). Observe the change in S18-2 distribution (types II and III cells) when EBNA-6 was expressed. Moreover, EBNA-6 and S18-2 showed a high degree of colocalization (types II and III cells, see p and s).
Fig. 3.
Fig. 3.
Increased level of nuclear S18-2 in the EBNA-6-expressing cells; Western blot of the nuclear extracts and cytoplasmic fractions of RAJI and RAJI-EBNA-6 cells. (Left) S18-2 expression in RAJI and RAJI-EBNA-6 cells. Ten micrograms of total protein in nuclear extracts and cytoplasmic fractions was loaded on the gel. The membrane was probed with rabbit serum (76-2) raised against the S18-2 peptide. Notice the increase of nuclear S18-2 signal in RAJI-EBNA-6 cells. (Right) EBNA-6 expression. The mouse monoclonal ani-EBNA-6 antibody was used to monitor EBNA-6 in the nuclear extract and the cytoplasmic fraction of RAJI-EBNA-6 and RAJI cells.
Fig. 4.
Fig. 4.
EBNA-6 and S18-2 are components of the one protein complex in vivo. For immunoprecipitations, 5 μg of antibodies was coupled to the CN-Br Sepharose, and such supports were used to capture the corresponding proteins from the Nonidet P-40 LCL cell lysates. After extensive washing, the protein complexes were eluted by heat and separated on the polyacrylamide gel. Beads not coupled to antibodies were used as a negative control. Ten micrograms of LCL lysate was used as a control for protein expression. (Left) EBNA-6 from LCL was precipitated by anti-S18-2 antibody (with two clones, 75-5 and 76-2). The EBNA-6 antibody was used to probe the membrane. (Right) Precipitation of S18-2 from LCL by the anti-EBNA-6 mouse antibody. Clone 76-2 was used as a probe for Western blot analysis. Notice that beads not coupled to antibodies did not give any signal.
Fig. 5.
Fig. 5.
S18-2 binds EBNA-b and pRb in vitro specifically. (A) GST-S18-2 precipitates pRb regardless of EBNA-6 presence in GST pulldown assay. The membranes were probed with anti-pRb (Upper) and anti-ppRb (Lower) antibodies. Lane 1: lysate of 0.4 × 106 MUTUI cells; lane 2: lysate of 0.4 × 106 MUTUIII cells; lane 3: GST precipitation from MUTUI cell lysate (10 × 106 cells); lane 4: GST precipitation from MUTUIII cell lysate (10 × 106 cells); lane 5: GST-S18-2 precipitated pRb and ppRb from the MUTUI cell lysates (10 × 106 cells); lane 6: GST-S18-2 precipitated pRb and ppRb from MUTUIII cell lysates (10 × 106 cells). (B) GST-S18-2 precipitates pRb specifically in GST pulldown assay. The membranes were probed with anti-p130, anti-pRb, and anti-E2F1 antibodies. Lane 1: MUTUIII cell lysate; lane 2: GST beads; lane 3: GST-S18-2 precipitated pRb, but not p130 neither E2F1. (C) GST-S18-2 precipitates EBNA-6 and pRb simultaneously in GST pulldown assay. Membranes were probed with anti-EBNA-6 and pRb antibody. Lanes 1, 3, and 5: RAJI cell lysate, lanes 2, 4, and 6: RAJI-EBNA-6 cell lysate. Lanes 1 and 2: cell lysates (5% of input); lanes 3 and 4: GST beads; lanes 5 and 6: GST-S18-2 beads.
Fig. 6.
Fig. 6.
S18-2 expression decreases pRb levels. (A) MCF7 cells, transfected with GFP-S18-2 (green). Phosphorylated pRb (ppRb) is stained in red, and DNA is stained in blue. Notice the red signal decrease in the GFP-S18-2-expressing cells (a and c). The arrow indicates a transfected cell. (B) MCF7 cells, transfected with GFP-S18-2 (green). The total pRb protein is stained in red (see the red signal in a and c), and DNA is in blue. Notice that the pRb signal is essentially decreased in GFP-S18-2-expressing cells (red signal in a and c). The arrows indicate the transfected cells.
Fig. 7.
Fig. 7.
S18-2 expression decreases pRb levels. (A) Western blot showing protein levels in the nucleus and cytoplasm of the RAJI-EBNA-6 cells compared with RAJI. Notice the increase in pRb and E2F1 levels when EBNA-6 is expressed. (B) Immunoprecipitations from RAJI and RAJI-EBNA-6 cell lysates. (Left) Precipitations with anti-E2F1 antibody. Notice the decrease in the amount of pRb, bound to E2F1. (Right) Precipitations with anti-pRb antibody. The amount of pRb in complex with E2F1 protein is lower in RAJI-EBNA-6 cells. (C) Levels of total E2F1 and pRb proteins and in complex with each other. Lanes 1 and 2: amount of E2F1 in the RAJI and RAJI-EBNA-6 cell lysate (as a ratio to actin); lanes 3 and 4: amount of E2F1, precipitated with anti-pRb antibody from cell lysates; lanes 5 and 6: amount of total pRb in the cell lysates; lanes 7 and 8: 10-fold magnified amount of pRb, precipitated with anti-E2F1. (D) EMSA of DNA oligo, containing the two binding sites for E2F1. Lane 1: labeled DNA probe; lane 2: DNA probe with RAJI nuclear extract; lane 3: DNA probe with RAJI-EBNA-6 nuclear extract; lane 4: as lane 2 and anti-E2F1 mouse monoclonal antibody; lane 5: as lane 3 and anti-E2F1 antibody. Notice that almost all DNA is bound to E2F1 in RAJI-EBNA-6 cells compared with RAJI cells (lane 3 compared with lane 2). Observe that treatment with anti-E2F1 antibody leads to disappearance of DNA shift (lanes 4 and 5).
Fig. 8.
Fig. 8.
Functional consequences of the binding between S18-2 and EBNA-6: S18-2 bound to EBNA-6 translocates to the nucleus. This may facilitate the binding of S18-2 to pRb. As long as it is bound to S18-2, pRb may not influence the level of free E2F1. This may promote the entry of the EBV infected B cell into the cell cycle.

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