Limits...
Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

Huang WR, Chiu HC, Liao TL, Chuang KP, Shih WL, Liu HJ - PLoS ONE (2015)

Bottom Line: To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner.The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4.Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan.

ABSTRACT
Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

No MeSH data available.


Related in: MedlinePlus

p17 functions as a Tpr suppressor leading to p53 and p21 nuclear accumulation.(A) Both Tpr and p53 levels in mock control (Vero cell only), pcDNA3.1 (mock transfection)-, pcDNA3.1-p17-transfected, and ARV-infected cells were examined. Whole cell lysates and nuclear extracts were collected at the indicated time points for Western blot assays. (B) To examine whether the Tpr transcription was down-regulated by p17, the Tpr mRNA levels in ARV-infected and pcDNA3.1-p17-transfected Vero cells were compared with those in mock treated cells. In semi-quantitative RT-PCR amplification of the p17 and Tpr genes, Vero cells were transfected with pcDNA3.1-p17 or infected with ARV at an MOI of 10. The pcCNA3.1-p17 transfected or ARV-infected cells were collected at 24 hours postinfection (hpi), and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock transfection (vector only) and mock infection (cell alone) were included as negative controls. Graph shown represents the mean± SD calculated from three independent experiments. (C) To study whether the p17 mutant (1–118), which does not possess a NLS, can influence the levels of p-p53 and p-p21 in the nucleus, vero cells were transfected with the p17 mutant (1–118) plasmid (negative control) for 24 hours. (D) To study whether Tpr depletion affects p53, p21, and PTEN nuclear accumulation, nuclear extracts from ARV-infected and pcDNA3.1-p17-transfected Vero cells were collected for Western blot assays. Vero cells were transfected with Tpr shRNA for 6 hours before being infected with ARV at an MOI of 10 for 18 hours. In a parallel experiment, Vero cells were co-transfected with pcDNA3.1-p17 and Tpr shRNA plasmid for 18 hours. Nuclear extracts were collected for Western blot assays using the indicated antibodies. Either actin or histone H2A was used as loading controls. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panel A) or in mock controls (cell alone) (panels C and D). The levels of indicated proteins at 0 h or in the mock controls (cell alone) were considered 1-fold.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526660&req=5

pone.0133699.g003: p17 functions as a Tpr suppressor leading to p53 and p21 nuclear accumulation.(A) Both Tpr and p53 levels in mock control (Vero cell only), pcDNA3.1 (mock transfection)-, pcDNA3.1-p17-transfected, and ARV-infected cells were examined. Whole cell lysates and nuclear extracts were collected at the indicated time points for Western blot assays. (B) To examine whether the Tpr transcription was down-regulated by p17, the Tpr mRNA levels in ARV-infected and pcDNA3.1-p17-transfected Vero cells were compared with those in mock treated cells. In semi-quantitative RT-PCR amplification of the p17 and Tpr genes, Vero cells were transfected with pcDNA3.1-p17 or infected with ARV at an MOI of 10. The pcCNA3.1-p17 transfected or ARV-infected cells were collected at 24 hours postinfection (hpi), and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock transfection (vector only) and mock infection (cell alone) were included as negative controls. Graph shown represents the mean± SD calculated from three independent experiments. (C) To study whether the p17 mutant (1–118), which does not possess a NLS, can influence the levels of p-p53 and p-p21 in the nucleus, vero cells were transfected with the p17 mutant (1–118) plasmid (negative control) for 24 hours. (D) To study whether Tpr depletion affects p53, p21, and PTEN nuclear accumulation, nuclear extracts from ARV-infected and pcDNA3.1-p17-transfected Vero cells were collected for Western blot assays. Vero cells were transfected with Tpr shRNA for 6 hours before being infected with ARV at an MOI of 10 for 18 hours. In a parallel experiment, Vero cells were co-transfected with pcDNA3.1-p17 and Tpr shRNA plasmid for 18 hours. Nuclear extracts were collected for Western blot assays using the indicated antibodies. Either actin or histone H2A was used as loading controls. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panel A) or in mock controls (cell alone) (panels C and D). The levels of indicated proteins at 0 h or in the mock controls (cell alone) were considered 1-fold.

Mentions: Because of our interest in p17 modulating Tpr, we next wanted to examine whether ARV infection and p17 transfection influence the level of Tpr in both Vero and DF-1 cells. To rule out the possibility of over-expression artifacts, all assays were carried out in both ARV-infected and p17-transfected cells. Importantly, a marked decrease in Tpr was seen in both whole cell lysates and nuclear extracts in ARV-infected cells at 12 and 24 hours postinfection and in pcDNA3.1-p17- transfected cells in a time-dependent manner (Fig 3A; S1A Fig). As shown in the negative control (Fig 3B, lane 5), the levels of Tpr, p-p53, andp-p21 were not changed in p17 (1–118)-transfected cells. The above unexpected finding inspired us to further explore whether Tpr was transcriptionally regulated by p17. Thus, further analysis of the Tpr mRNA level was performed by semi-quantitative RT-PCR. Our results reveal that a corresponding decrease in the Tpr mRNA level was observed (Fig 3C; S1B Fig), suggesting that Tpr is transcriptionally downregulated by p17. The potential role of p17 in transcription of Tpr is supported by previous studies that p17 decreases cellular transcription to induce host cell shutoff [38, 39] and might activate or repress the transcription of specific genes due to its specific double-stranded DNA binding activity [36].


Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

Huang WR, Chiu HC, Liao TL, Chuang KP, Shih WL, Liu HJ - PLoS ONE (2015)

p17 functions as a Tpr suppressor leading to p53 and p21 nuclear accumulation.(A) Both Tpr and p53 levels in mock control (Vero cell only), pcDNA3.1 (mock transfection)-, pcDNA3.1-p17-transfected, and ARV-infected cells were examined. Whole cell lysates and nuclear extracts were collected at the indicated time points for Western blot assays. (B) To examine whether the Tpr transcription was down-regulated by p17, the Tpr mRNA levels in ARV-infected and pcDNA3.1-p17-transfected Vero cells were compared with those in mock treated cells. In semi-quantitative RT-PCR amplification of the p17 and Tpr genes, Vero cells were transfected with pcDNA3.1-p17 or infected with ARV at an MOI of 10. The pcCNA3.1-p17 transfected or ARV-infected cells were collected at 24 hours postinfection (hpi), and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock transfection (vector only) and mock infection (cell alone) were included as negative controls. Graph shown represents the mean± SD calculated from three independent experiments. (C) To study whether the p17 mutant (1–118), which does not possess a NLS, can influence the levels of p-p53 and p-p21 in the nucleus, vero cells were transfected with the p17 mutant (1–118) plasmid (negative control) for 24 hours. (D) To study whether Tpr depletion affects p53, p21, and PTEN nuclear accumulation, nuclear extracts from ARV-infected and pcDNA3.1-p17-transfected Vero cells were collected for Western blot assays. Vero cells were transfected with Tpr shRNA for 6 hours before being infected with ARV at an MOI of 10 for 18 hours. In a parallel experiment, Vero cells were co-transfected with pcDNA3.1-p17 and Tpr shRNA plasmid for 18 hours. Nuclear extracts were collected for Western blot assays using the indicated antibodies. Either actin or histone H2A was used as loading controls. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panel A) or in mock controls (cell alone) (panels C and D). The levels of indicated proteins at 0 h or in the mock controls (cell alone) were considered 1-fold.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526660&req=5

pone.0133699.g003: p17 functions as a Tpr suppressor leading to p53 and p21 nuclear accumulation.(A) Both Tpr and p53 levels in mock control (Vero cell only), pcDNA3.1 (mock transfection)-, pcDNA3.1-p17-transfected, and ARV-infected cells were examined. Whole cell lysates and nuclear extracts were collected at the indicated time points for Western blot assays. (B) To examine whether the Tpr transcription was down-regulated by p17, the Tpr mRNA levels in ARV-infected and pcDNA3.1-p17-transfected Vero cells were compared with those in mock treated cells. In semi-quantitative RT-PCR amplification of the p17 and Tpr genes, Vero cells were transfected with pcDNA3.1-p17 or infected with ARV at an MOI of 10. The pcCNA3.1-p17 transfected or ARV-infected cells were collected at 24 hours postinfection (hpi), and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock transfection (vector only) and mock infection (cell alone) were included as negative controls. Graph shown represents the mean± SD calculated from three independent experiments. (C) To study whether the p17 mutant (1–118), which does not possess a NLS, can influence the levels of p-p53 and p-p21 in the nucleus, vero cells were transfected with the p17 mutant (1–118) plasmid (negative control) for 24 hours. (D) To study whether Tpr depletion affects p53, p21, and PTEN nuclear accumulation, nuclear extracts from ARV-infected and pcDNA3.1-p17-transfected Vero cells were collected for Western blot assays. Vero cells were transfected with Tpr shRNA for 6 hours before being infected with ARV at an MOI of 10 for 18 hours. In a parallel experiment, Vero cells were co-transfected with pcDNA3.1-p17 and Tpr shRNA plasmid for 18 hours. Nuclear extracts were collected for Western blot assays using the indicated antibodies. Either actin or histone H2A was used as loading controls. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panel A) or in mock controls (cell alone) (panels C and D). The levels of indicated proteins at 0 h or in the mock controls (cell alone) were considered 1-fold.
Mentions: Because of our interest in p17 modulating Tpr, we next wanted to examine whether ARV infection and p17 transfection influence the level of Tpr in both Vero and DF-1 cells. To rule out the possibility of over-expression artifacts, all assays were carried out in both ARV-infected and p17-transfected cells. Importantly, a marked decrease in Tpr was seen in both whole cell lysates and nuclear extracts in ARV-infected cells at 12 and 24 hours postinfection and in pcDNA3.1-p17- transfected cells in a time-dependent manner (Fig 3A; S1A Fig). As shown in the negative control (Fig 3B, lane 5), the levels of Tpr, p-p53, andp-p21 were not changed in p17 (1–118)-transfected cells. The above unexpected finding inspired us to further explore whether Tpr was transcriptionally regulated by p17. Thus, further analysis of the Tpr mRNA level was performed by semi-quantitative RT-PCR. Our results reveal that a corresponding decrease in the Tpr mRNA level was observed (Fig 3C; S1B Fig), suggesting that Tpr is transcriptionally downregulated by p17. The potential role of p17 in transcription of Tpr is supported by previous studies that p17 decreases cellular transcription to induce host cell shutoff [38, 39] and might activate or repress the transcription of specific genes due to its specific double-stranded DNA binding activity [36].

Bottom Line: To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner.The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4.Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan.

ABSTRACT
Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

No MeSH data available.


Related in: MedlinePlus