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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 induces cytoplasmic accumulation of E2F4/5. (A and B) E2F4 (A) or HA-tagged E2F5 (B) was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 4) or GFP-tagged CRM1 (3) in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and HA-tagged E2F5 was determined by immunostaining with anti-HA antibody. Cells were treated with 2.5 ng/ml of LMB (4). Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for E2F4/5 expression. (A and B) Arrows indicate cells expressing E2F alone (1) or cells expressing both E2F and GFP-tagged LMP1 (2 and 4) or cells expressing both E2F and GFP-tagged CRM1 (3). (C) Activation of the human Rb gene promoter by E2F4 or E2F5 was blocked by coexpression of LMP1 in SVts8 cells. Expression plasmids encoding the indicated proteins were introduced into SVts8 cells along with 0.7 μg of Rb gene promoter–luciferase construct and with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. For all panels, error bars indicate SD.
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fig4: LMP1 induces cytoplasmic accumulation of E2F4/5. (A and B) E2F4 (A) or HA-tagged E2F5 (B) was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 4) or GFP-tagged CRM1 (3) in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and HA-tagged E2F5 was determined by immunostaining with anti-HA antibody. Cells were treated with 2.5 ng/ml of LMB (4). Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for E2F4/5 expression. (A and B) Arrows indicate cells expressing E2F alone (1) or cells expressing both E2F and GFP-tagged LMP1 (2 and 4) or cells expressing both E2F and GFP-tagged CRM1 (3). (C) Activation of the human Rb gene promoter by E2F4 or E2F5 was blocked by coexpression of LMP1 in SVts8 cells. Expression plasmids encoding the indicated proteins were introduced into SVts8 cells along with 0.7 μg of Rb gene promoter–luciferase construct and with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. For all panels, error bars indicate SD.

Mentions: Recent reports suggest that E2F4/5 mainly act as “repressor” E2Fs, which have opposing functions against “the activator” E2Fs, E2F1–3 (Trimarchi and Lees, 2002). Mouse embryonic fibroblasts (MEFs) lacking both repressor E2Fs, E2F4/5, grow normally but are insensitive to a p16INK4a-induced G1 arrest (Gaubatz et al., 2000). Moreover, enforced nuclear export of E2F4/5 by overexpression of CRM1 prevents the ability of p16INK4a to induce a G1 arrest in U2OS cells (Gaubatz et al., 2001). This evidence strongly suggests that E2F4/5 are essential downstream mediators of the p16INK4a-induced growth arrest pathway (Gaubatz et al., 2000). Therefore, we tested if LMP1 has any effect on the subcellular localization of E2F4 and/or E2F5, which have previously been shown to be regulated by CRM1-dependent nuclear export machinery (Gaubatz et al., 2001; Trimarchi and Lees, 2002). Although 50–60% of cells expressed transfected E2F4/5 in both cytoplasm and nucleus under normal proliferating conditions, coexpression of LMP1 significantly abolished the nuclear localization of E2F4/5 in Svts8 cells (Fig. 4, A and B, 1 and 2). As reported previously (Gaubatz et al., 2001), similar effects were seen by ectopic expression of CRM-1 (Fig. 4, A and B, 3). This cytoplasmic accumulation was blocked by the addition of LMB (Fig. 4, A and B, 4), indicating that LMP1 also promotes intracellular redistribution of E2F4/E2F5 from the nucleus to the cytoplasm in a CRM1-dependent manner. To obtain further proof of the inactivation of E2F4/5 by LMP1, we monitored the transcriptional activity of E2F4/5 using the human Rb gene promoter, which is known to be a target of E2F4 (Ren et al., 2002). Although E2F4/E2F5 induced Rb gene promoter activity, this was abolished when LMP1 was coexpressed, suggesting that the transcriptional activity of E2F4/5 is indeed blocked by LMP1 (Fig. 4 C, 5–8).


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 induces cytoplasmic accumulation of E2F4/5. (A and B) E2F4 (A) or HA-tagged E2F5 (B) was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 4) or GFP-tagged CRM1 (3) in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and HA-tagged E2F5 was determined by immunostaining with anti-HA antibody. Cells were treated with 2.5 ng/ml of LMB (4). Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for E2F4/5 expression. (A and B) Arrows indicate cells expressing E2F alone (1) or cells expressing both E2F and GFP-tagged LMP1 (2 and 4) or cells expressing both E2F and GFP-tagged CRM1 (3). (C) Activation of the human Rb gene promoter by E2F4 or E2F5 was blocked by coexpression of LMP1 in SVts8 cells. Expression plasmids encoding the indicated proteins were introduced into SVts8 cells along with 0.7 μg of Rb gene promoter–luciferase construct and with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. For all panels, error bars indicate SD.
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Related In: Results  -  Collection

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fig4: LMP1 induces cytoplasmic accumulation of E2F4/5. (A and B) E2F4 (A) or HA-tagged E2F5 (B) was expressed alone (1) or coexpressed with GFP-tagged LMP1 (2 and 4) or GFP-tagged CRM1 (3) in SVts8 cells. E2F4 was detected by immunostaining with anti-E2F4 antibody and HA-tagged E2F5 was determined by immunostaining with anti-HA antibody. Cells were treated with 2.5 ng/ml of LMB (4). Histograms on the right side of the micrographs indicate the percentage of nuclei that were positive (N+) or negative (N−) for E2F4/5 expression. (A and B) Arrows indicate cells expressing E2F alone (1) or cells expressing both E2F and GFP-tagged LMP1 (2 and 4) or cells expressing both E2F and GFP-tagged CRM1 (3). (C) Activation of the human Rb gene promoter by E2F4 or E2F5 was blocked by coexpression of LMP1 in SVts8 cells. Expression plasmids encoding the indicated proteins were introduced into SVts8 cells along with 0.7 μg of Rb gene promoter–luciferase construct and with 0.2 μg of MMLV-lacZ plasmid. Luciferase activities were normalized by lac-Z activities. For all panels, error bars indicate SD.
Mentions: Recent reports suggest that E2F4/5 mainly act as “repressor” E2Fs, which have opposing functions against “the activator” E2Fs, E2F1–3 (Trimarchi and Lees, 2002). Mouse embryonic fibroblasts (MEFs) lacking both repressor E2Fs, E2F4/5, grow normally but are insensitive to a p16INK4a-induced G1 arrest (Gaubatz et al., 2000). Moreover, enforced nuclear export of E2F4/5 by overexpression of CRM1 prevents the ability of p16INK4a to induce a G1 arrest in U2OS cells (Gaubatz et al., 2001). This evidence strongly suggests that E2F4/5 are essential downstream mediators of the p16INK4a-induced growth arrest pathway (Gaubatz et al., 2000). Therefore, we tested if LMP1 has any effect on the subcellular localization of E2F4 and/or E2F5, which have previously been shown to be regulated by CRM1-dependent nuclear export machinery (Gaubatz et al., 2001; Trimarchi and Lees, 2002). Although 50–60% of cells expressed transfected E2F4/5 in both cytoplasm and nucleus under normal proliferating conditions, coexpression of LMP1 significantly abolished the nuclear localization of E2F4/5 in Svts8 cells (Fig. 4, A and B, 1 and 2). As reported previously (Gaubatz et al., 2001), similar effects were seen by ectopic expression of CRM-1 (Fig. 4, A and B, 3). This cytoplasmic accumulation was blocked by the addition of LMB (Fig. 4, A and B, 4), indicating that LMP1 also promotes intracellular redistribution of E2F4/E2F5 from the nucleus to the cytoplasm in a CRM1-dependent manner. To obtain further proof of the inactivation of E2F4/5 by LMP1, we monitored the transcriptional activity of E2F4/5 using the human Rb gene promoter, which is known to be a target of E2F4 (Ren et al., 2002). Although E2F4/E2F5 induced Rb gene promoter activity, this was abolished when LMP1 was coexpressed, suggesting that the transcriptional activity of E2F4/5 is indeed blocked by LMP1 (Fig. 4 C, 5–8).

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