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Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

Ohtani N, Brennan P, Gaubatz S, Sanij E, Hertzog P, Wolvetang E, Ghysdael J, Rowe M, Hara E - J. Cell Biol. (2003)

Bottom Line: Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression.As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway.These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.

ABSTRACT
The p16INK4a-RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

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LMP1 promotes cytoplasmic accumulation of Ets2. (A) Flag-tagged Ets2 was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 3) or Id1 (4) in SVts8 cells, as indicated. Flag-tagged Ets2 was detected by immunostaining with an anti-Flag antibody. Expression of Id1 was confirmed by immunostaining with polyclonal anti-Id1 antibody. Cells were treated with 2.5 ng/ml of LMB (3). The histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Ets2 expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2 and 3) or cells expressing both Flag-tagged Ets2 and Id (4). (B) Flag-tagged JunB (1) or p27Kip1 (3) was expressed alone or coexpressed with GFP-tagged LMP1 (2 and 4) in SVts8 cells, as indicated. Flag-tagged JunB and p27Kip1 were detected by immunostaining with an anti-Flag antibody. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged protein alone (1 and 3) or cells expressing both Flag-tagged protein and GFP-tagged–LMP1 (2 and 4). (C) Flag-tagged Ets2 expression vector (pRc/RSV; Invitrogen) was cotransfected with an Ecdyson-inducible vector encoding GFP-tagged LMP1 (pIND; Invitrogen) into early passage (38 PDLs) TIG-3 cells. Expression of GFP-tagged LMP1 was induced by addition of 0.5 μg/ml of Ecdyson homologue, Muristeron A (panel 2). Flag-tagged Ets2 was detected with an anti-Flag antibody 2 d after addition of Muristeron A. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2). For all panels, error bars indicate SD.
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fig2: LMP1 promotes cytoplasmic accumulation of Ets2. (A) Flag-tagged Ets2 was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 3) or Id1 (4) in SVts8 cells, as indicated. Flag-tagged Ets2 was detected by immunostaining with an anti-Flag antibody. Expression of Id1 was confirmed by immunostaining with polyclonal anti-Id1 antibody. Cells were treated with 2.5 ng/ml of LMB (3). The histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Ets2 expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2 and 3) or cells expressing both Flag-tagged Ets2 and Id (4). (B) Flag-tagged JunB (1) or p27Kip1 (3) was expressed alone or coexpressed with GFP-tagged LMP1 (2 and 4) in SVts8 cells, as indicated. Flag-tagged JunB and p27Kip1 were detected by immunostaining with an anti-Flag antibody. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged protein alone (1 and 3) or cells expressing both Flag-tagged protein and GFP-tagged–LMP1 (2 and 4). (C) Flag-tagged Ets2 expression vector (pRc/RSV; Invitrogen) was cotransfected with an Ecdyson-inducible vector encoding GFP-tagged LMP1 (pIND; Invitrogen) into early passage (38 PDLs) TIG-3 cells. Expression of GFP-tagged LMP1 was induced by addition of 0.5 μg/ml of Ecdyson homologue, Muristeron A (panel 2). Flag-tagged Ets2 was detected with an anti-Flag antibody 2 d after addition of Muristeron A. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2). For all panels, error bars indicate SD.

Mentions: Because the LMP1 protein localizes to the cytoplasm (Eliopoulos and Young, 2001), we next asked how LMP1 blocks Ets2 binding to the p16INK4a promoter. We examined the subcellular localization of ectopically expressed Ets2 protein in the presence or absence of LMP1 in the human fibroblast cell line, SVts8 cells. In most of the cells, flag-tagged Ets2 is expressed either in the nucleus or in the nucleus and cytoplasm (Fig. 2 A, 1). Coexpression with GFP-tagged LMP1, however, resulted in the accumulation of Flag-tagged Ets2 in the cytoplasm (Fig. 2 A, 2). Similar results were obtained using nontagged Ets2 and nontagged LMP1 expression vectors (unpublished data). This effect was blocked by treatment with leptomycin B (LMB; Fornerod et al., 1997; Stade et al., 1997; Kudo et al., 1998), a specific inhibitor of CRM1-dependent nuclear export (Fig. 2 A, 3). In contrast, overexpression of GFP-tagged CRM1 resulted in the accumulation of Ets2 in the cytoplasm (unpublished data), suggesting that these effects were mediated through a CRM1-dependent nuclear export mechanism. These effects were specific to Ets2, because LMP1 expression did not have any significant impact on the subcellular localization of other transcription factor, such as JunB (Fig. 2 B, 1 and 2), Elk1, or p53 (not depicted). Moreover, LMP1 failed to promote nuclear export of p27Kip1, which is known as a nuclear shuttling protein (Tomoda et al., 1999; Rodier et al., 2001; Ishida et al., 2002; Fig. 2 B, 3 and 4). Furthermore, another p16INK4a repressor, Id1 (Lyden et al., 1999; Ohtani et al., 2001), did not have any impact on the subcellular localization of Ets2 (Fig. 2 A, 4). To confirm that these effects were not due to transfection artifacts, nor limited to this cell line, the subcellular localization of Ets2 was examined using an Ecdyson-inducible vector encoding GFP-tagged LMP1 in the TIG-3 strain of primary HDFs. Although nuclear staining of Ets2 was predominantly observed in the absence of the Ecdysone analogue, Muristeron A (Fig. 2 C, 1), cytoplasmic staining of Ets2 was predominantly observed in the significant percentage of the cells when the expression of GFP-tagged LMP1 was induced by the addition of the Muristeron A (Fig. 2 C, 2). These results strongly suggest that LMP1 represses p16INK4a expression through, at least partly, blocking the nuclear localization of Ets2 transcription factor.


Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

Ohtani N, Brennan P, Gaubatz S, Sanij E, Hertzog P, Wolvetang E, Ghysdael J, Rowe M, Hara E - J. Cell Biol. (2003)

LMP1 promotes cytoplasmic accumulation of Ets2. (A) Flag-tagged Ets2 was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 3) or Id1 (4) in SVts8 cells, as indicated. Flag-tagged Ets2 was detected by immunostaining with an anti-Flag antibody. Expression of Id1 was confirmed by immunostaining with polyclonal anti-Id1 antibody. Cells were treated with 2.5 ng/ml of LMB (3). The histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Ets2 expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2 and 3) or cells expressing both Flag-tagged Ets2 and Id (4). (B) Flag-tagged JunB (1) or p27Kip1 (3) was expressed alone or coexpressed with GFP-tagged LMP1 (2 and 4) in SVts8 cells, as indicated. Flag-tagged JunB and p27Kip1 were detected by immunostaining with an anti-Flag antibody. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged protein alone (1 and 3) or cells expressing both Flag-tagged protein and GFP-tagged–LMP1 (2 and 4). (C) Flag-tagged Ets2 expression vector (pRc/RSV; Invitrogen) was cotransfected with an Ecdyson-inducible vector encoding GFP-tagged LMP1 (pIND; Invitrogen) into early passage (38 PDLs) TIG-3 cells. Expression of GFP-tagged LMP1 was induced by addition of 0.5 μg/ml of Ecdyson homologue, Muristeron A (panel 2). Flag-tagged Ets2 was detected with an anti-Flag antibody 2 d after addition of Muristeron A. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2). For all panels, error bars indicate SD.
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fig2: LMP1 promotes cytoplasmic accumulation of Ets2. (A) Flag-tagged Ets2 was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 3) or Id1 (4) in SVts8 cells, as indicated. Flag-tagged Ets2 was detected by immunostaining with an anti-Flag antibody. Expression of Id1 was confirmed by immunostaining with polyclonal anti-Id1 antibody. Cells were treated with 2.5 ng/ml of LMB (3). The histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Ets2 expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2 and 3) or cells expressing both Flag-tagged Ets2 and Id (4). (B) Flag-tagged JunB (1) or p27Kip1 (3) was expressed alone or coexpressed with GFP-tagged LMP1 (2 and 4) in SVts8 cells, as indicated. Flag-tagged JunB and p27Kip1 were detected by immunostaining with an anti-Flag antibody. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged protein alone (1 and 3) or cells expressing both Flag-tagged protein and GFP-tagged–LMP1 (2 and 4). (C) Flag-tagged Ets2 expression vector (pRc/RSV; Invitrogen) was cotransfected with an Ecdyson-inducible vector encoding GFP-tagged LMP1 (pIND; Invitrogen) into early passage (38 PDLs) TIG-3 cells. Expression of GFP-tagged LMP1 was induced by addition of 0.5 μg/ml of Ecdyson homologue, Muristeron A (panel 2). Flag-tagged Ets2 was detected with an anti-Flag antibody 2 d after addition of Muristeron A. Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for Flag-tagged protein expression. Arrows indicate cells expressing Flag-tagged Ets2 alone (1) or cells expressing both Flag-tagged Ets2 and GFP-tagged LMP1 (2). For all panels, error bars indicate SD.
Mentions: Because the LMP1 protein localizes to the cytoplasm (Eliopoulos and Young, 2001), we next asked how LMP1 blocks Ets2 binding to the p16INK4a promoter. We examined the subcellular localization of ectopically expressed Ets2 protein in the presence or absence of LMP1 in the human fibroblast cell line, SVts8 cells. In most of the cells, flag-tagged Ets2 is expressed either in the nucleus or in the nucleus and cytoplasm (Fig. 2 A, 1). Coexpression with GFP-tagged LMP1, however, resulted in the accumulation of Flag-tagged Ets2 in the cytoplasm (Fig. 2 A, 2). Similar results were obtained using nontagged Ets2 and nontagged LMP1 expression vectors (unpublished data). This effect was blocked by treatment with leptomycin B (LMB; Fornerod et al., 1997; Stade et al., 1997; Kudo et al., 1998), a specific inhibitor of CRM1-dependent nuclear export (Fig. 2 A, 3). In contrast, overexpression of GFP-tagged CRM1 resulted in the accumulation of Ets2 in the cytoplasm (unpublished data), suggesting that these effects were mediated through a CRM1-dependent nuclear export mechanism. These effects were specific to Ets2, because LMP1 expression did not have any significant impact on the subcellular localization of other transcription factor, such as JunB (Fig. 2 B, 1 and 2), Elk1, or p53 (not depicted). Moreover, LMP1 failed to promote nuclear export of p27Kip1, which is known as a nuclear shuttling protein (Tomoda et al., 1999; Rodier et al., 2001; Ishida et al., 2002; Fig. 2 B, 3 and 4). Furthermore, another p16INK4a repressor, Id1 (Lyden et al., 1999; Ohtani et al., 2001), did not have any impact on the subcellular localization of Ets2 (Fig. 2 A, 4). To confirm that these effects were not due to transfection artifacts, nor limited to this cell line, the subcellular localization of Ets2 was examined using an Ecdyson-inducible vector encoding GFP-tagged LMP1 in the TIG-3 strain of primary HDFs. Although nuclear staining of Ets2 was predominantly observed in the absence of the Ecdysone analogue, Muristeron A (Fig. 2 C, 1), cytoplasmic staining of Ets2 was predominantly observed in the significant percentage of the cells when the expression of GFP-tagged LMP1 was induced by the addition of the Muristeron A (Fig. 2 C, 2). These results strongly suggest that LMP1 represses p16INK4a expression through, at least partly, blocking the nuclear localization of Ets2 transcription factor.

Bottom Line: Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression.As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway.These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.

ABSTRACT
The p16INK4a-RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

Show MeSH
Related in: MedlinePlus