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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 intracellular redistribution of endogenous Ets2 and E2F4 proteins. (A) LMP1-inducible cell line was established using SVts8 cells and Ecdyson inducible system (lanes 1 and 2). LMP1 expression was induced by the incubation with 0.5 μg/ml of Ecdyson homologue, Ponasteron A for 2 d, and levels were compared with that of EBV-positive (lane 4) and -negative (lane 3) human B cells. MEK was used here as a loading control. (B) LMP1-inducible cell line described in A, was treated with (lanes 2 and 4) or without (lanes 1 and 3) 0.5 μg/ml of Ponasteron A for 2 d. Next, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (C) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding LMP1 (lanes 2 and 4) or control vector (lanes 1 and 3). 4 d after infection, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (D) Both cytoplasmic and nuclear fractions were prepared from BL41 cells (EBV negative) and BL41 + B95 cells (EBV positive). The levels of endogenous E2F4 were analyzed by Western blotting. α-Tubulin and Sp1 were used here as cytoplasmic or nuclei marker, respectively. LMP1 expression was confirmed by anti-LMP1 antibody.
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fig5: LMP1 induces intracellular redistribution of endogenous Ets2 and E2F4 proteins. (A) LMP1-inducible cell line was established using SVts8 cells and Ecdyson inducible system (lanes 1 and 2). LMP1 expression was induced by the incubation with 0.5 μg/ml of Ecdyson homologue, Ponasteron A for 2 d, and levels were compared with that of EBV-positive (lane 4) and -negative (lane 3) human B cells. MEK was used here as a loading control. (B) LMP1-inducible cell line described in A, was treated with (lanes 2 and 4) or without (lanes 1 and 3) 0.5 μg/ml of Ponasteron A for 2 d. Next, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (C) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding LMP1 (lanes 2 and 4) or control vector (lanes 1 and 3). 4 d after infection, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (D) Both cytoplasmic and nuclear fractions were prepared from BL41 cells (EBV negative) and BL41 + B95 cells (EBV positive). The levels of endogenous E2F4 were analyzed by Western blotting. α-Tubulin and Sp1 were used here as cytoplasmic or nuclei marker, respectively. LMP1 expression was confirmed by anti-LMP1 antibody.

Mentions: To examine whether this is also the case for the endogenous proteins, we have established an LMP1-inducible cell line using an Ecdyson-inducible vector. The level of LMP1 induced in this cell line was similar to the levels expressing in EBV-positive human B cells (Fig. 5 A, lanes 2 and 4), suggesting that the levels of LMP1 in this cell line is likely to be a physiological level. Because the levels of both endogenous Ets2 and endogenous E2F4 were under detectable level by immunofluorescence in this cell line, we examined the levels of both proteins in isolated nuclear and cytoplasmic fractions in the presence or absence of LMP1 expression by immunoblotting. As shown in Fig. 5 B, the levels of endogenous Ets2 and E2F4 in the nuclear fraction were significantly reduced in cells expressing LMP1 (Fig. 5 B, lanes 3 and 4). Moreover, similar results were obtained using TIG-3 cells expressing LMP1 using a retroviral vector (Fig. 5 C, lanes 3 and 4). Although we were unable to examine endogenous E2F5 due to lack of an antibody, these results strongly suggest that LMP1 induces intracellular redistribution of endogenous Ets2 and E2F4/5 from the nucleus to the cytoplasm in human fibroblasts. To test whether this is also the case under the physiological condition of EBV infection, we examined the subcellular localization of endogenous E2F4 in Burkitt lymphoma cells that are positive or negative for EBV infection. As shown in Fig. 5 D, significant levels of E2F4 were observed in both nuclear and cytoplasmic fractions of EBV-negative Burkitt lymphoma cell line, BL41 cells. However, we were unable to detect E2F4 in the nuclear fraction of the BL41 + B95 cells, which are experimentally infected with EBV (Fig. 5 D, lane 4). Ets2 levels were under detectable levels in these cell lines (unpublished data). These results further support the idea that LMP1 affects the intracellular location of Ets2 and E2F4/5 under the physiological condition.


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 intracellular redistribution of endogenous Ets2 and E2F4 proteins. (A) LMP1-inducible cell line was established using SVts8 cells and Ecdyson inducible system (lanes 1 and 2). LMP1 expression was induced by the incubation with 0.5 μg/ml of Ecdyson homologue, Ponasteron A for 2 d, and levels were compared with that of EBV-positive (lane 4) and -negative (lane 3) human B cells. MEK was used here as a loading control. (B) LMP1-inducible cell line described in A, was treated with (lanes 2 and 4) or without (lanes 1 and 3) 0.5 μg/ml of Ponasteron A for 2 d. Next, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (C) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding LMP1 (lanes 2 and 4) or control vector (lanes 1 and 3). 4 d after infection, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (D) Both cytoplasmic and nuclear fractions were prepared from BL41 cells (EBV negative) and BL41 + B95 cells (EBV positive). The levels of endogenous E2F4 were analyzed by Western blotting. α-Tubulin and Sp1 were used here as cytoplasmic or nuclei marker, respectively. LMP1 expression was confirmed by anti-LMP1 antibody.
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Related In: Results  -  Collection

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fig5: LMP1 induces intracellular redistribution of endogenous Ets2 and E2F4 proteins. (A) LMP1-inducible cell line was established using SVts8 cells and Ecdyson inducible system (lanes 1 and 2). LMP1 expression was induced by the incubation with 0.5 μg/ml of Ecdyson homologue, Ponasteron A for 2 d, and levels were compared with that of EBV-positive (lane 4) and -negative (lane 3) human B cells. MEK was used here as a loading control. (B) LMP1-inducible cell line described in A, was treated with (lanes 2 and 4) or without (lanes 1 and 3) 0.5 μg/ml of Ponasteron A for 2 d. Next, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (C) Early passage (38 PDLs) HDFs expressing ecotropic receptor were infected with a retrovirus encoding LMP1 (lanes 2 and 4) or control vector (lanes 1 and 3). 4 d after infection, both cytoplasmic (lanes 1 and 2) and nuclear (lanes 3 and 4) fractions were prepared and the levels of endogenous E2F4 and Ets2 were analyzed. α-Tubulin and LaminA/C were used here as cytoplasmic or nuclei marker, respectively. (D) Both cytoplasmic and nuclear fractions were prepared from BL41 cells (EBV negative) and BL41 + B95 cells (EBV positive). The levels of endogenous E2F4 were analyzed by Western blotting. α-Tubulin and Sp1 were used here as cytoplasmic or nuclei marker, respectively. LMP1 expression was confirmed by anti-LMP1 antibody.
Mentions: To examine whether this is also the case for the endogenous proteins, we have established an LMP1-inducible cell line using an Ecdyson-inducible vector. The level of LMP1 induced in this cell line was similar to the levels expressing in EBV-positive human B cells (Fig. 5 A, lanes 2 and 4), suggesting that the levels of LMP1 in this cell line is likely to be a physiological level. Because the levels of both endogenous Ets2 and endogenous E2F4 were under detectable level by immunofluorescence in this cell line, we examined the levels of both proteins in isolated nuclear and cytoplasmic fractions in the presence or absence of LMP1 expression by immunoblotting. As shown in Fig. 5 B, the levels of endogenous Ets2 and E2F4 in the nuclear fraction were significantly reduced in cells expressing LMP1 (Fig. 5 B, lanes 3 and 4). Moreover, similar results were obtained using TIG-3 cells expressing LMP1 using a retroviral vector (Fig. 5 C, lanes 3 and 4). Although we were unable to examine endogenous E2F5 due to lack of an antibody, these results strongly suggest that LMP1 induces intracellular redistribution of endogenous Ets2 and E2F4/5 from the nucleus to the cytoplasm in human fibroblasts. To test whether this is also the case under the physiological condition of EBV infection, we examined the subcellular localization of endogenous E2F4 in Burkitt lymphoma cells that are positive or negative for EBV infection. As shown in Fig. 5 D, significant levels of E2F4 were observed in both nuclear and cytoplasmic fractions of EBV-negative Burkitt lymphoma cell line, BL41 cells. However, we were unable to detect E2F4 in the nuclear fraction of the BL41 + B95 cells, which are experimentally infected with EBV (Fig. 5 D, lane 4). Ets2 levels were under detectable levels in these cell lines (unpublished data). These results further support the idea that LMP1 affects the intracellular location of Ets2 and E2F4/5 under the physiological condition.

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