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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

Knockdown of Tpr is beneficial for virus replication.(A)To determine the effect of Tpr on ARV replication, depletion of Tpr with shRNA was carried out in ARV-infected Vero cells. The downstream molecules (p53, PTEN and LC3) of Tpr were also depleted using the respective shRNAs. All data shown represent the mean± SD calculated from three independent experiments. (B) In the present study, the effects of PI3K inhibitor (LY294002, 10 uM), mTORC1 inhibitor (rapamycin; 5 uM), and inhibitor of autophagy (3MA) on ARV replication were examined. Data shown represent the mean± SD calculated from three independent experiments. (C) In the case of CDK 4, vero cells were infected with ARV at a MOI of 5 for 3 hours, followed by CDK 4 shRNA transfection for 18 hours. All data shown represent the mean± SD calculated from three independent experiments.
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pone.0133699.g010: Knockdown of Tpr is beneficial for virus replication.(A)To determine the effect of Tpr on ARV replication, depletion of Tpr with shRNA was carried out in ARV-infected Vero cells. The downstream molecules (p53, PTEN and LC3) of Tpr were also depleted using the respective shRNAs. All data shown represent the mean± SD calculated from three independent experiments. (B) In the present study, the effects of PI3K inhibitor (LY294002, 10 uM), mTORC1 inhibitor (rapamycin; 5 uM), and inhibitor of autophagy (3MA) on ARV replication were examined. Data shown represent the mean± SD calculated from three independent experiments. (C) In the case of CDK 4, vero cells were infected with ARV at a MOI of 5 for 3 hours, followed by CDK 4 shRNA transfection for 18 hours. All data shown represent the mean± SD calculated from three independent experiments.

Mentions: As previously demonstrated, p17 is a Tpr suppressor leading to activation of p53, p21, and PTEN. To further determine the impact of Tpr on virus replication, Tpr knockdown was performed. Importantly, Tpr depletion increased virus production (Fig 10A). Conversely, knockdown of p53, PTEN, and autophagy markers (LC3) diminished virus yield (Fig 10A). Moreover, we also investigated the effects of PI3K inhibitor (LY294002), mTORC1 inhibitor (rapamycin), and autophagy inhibitor (3MA) on ARV replication. As presented in Fig 10B, inhibition of PI3K and mTORC1 by inhibitors increased virus titers, but inhibition of autophagy by 3MA reduced virus yield. These results are in consistent with our previous study [39]. Aside from this, a decrease in CDK4 level was observed in p17-transfected cells (Fig 8B; S3B Fig), and so we therefore also investigated the effect of CDK 4 on virus replication. In this work, expression of CDK4 was silenced. After vero cells were infected with ARV for 3 hours, CDK 4 knockdown in vero cells increased virus yield (Fig 10C). Collectively, our findings in this study reveal that p17-modulated suppression of Tpr and subsequent activation of upstream signaling (p53, PTEN, and p21) induces cell cycle arrest and autophagosome formation, which in turn enhancing virus replication [37–39, 64].


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)

Knockdown of Tpr is beneficial for virus replication.(A)To determine the effect of Tpr on ARV replication, depletion of Tpr with shRNA was carried out in ARV-infected Vero cells. The downstream molecules (p53, PTEN and LC3) of Tpr were also depleted using the respective shRNAs. All data shown represent the mean± SD calculated from three independent experiments. (B) In the present study, the effects of PI3K inhibitor (LY294002, 10 uM), mTORC1 inhibitor (rapamycin; 5 uM), and inhibitor of autophagy (3MA) on ARV replication were examined. Data shown represent the mean± SD calculated from three independent experiments. (C) In the case of CDK 4, vero cells were infected with ARV at a MOI of 5 for 3 hours, followed by CDK 4 shRNA transfection for 18 hours. All data shown represent the mean± SD calculated from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133699.g010: Knockdown of Tpr is beneficial for virus replication.(A)To determine the effect of Tpr on ARV replication, depletion of Tpr with shRNA was carried out in ARV-infected Vero cells. The downstream molecules (p53, PTEN and LC3) of Tpr were also depleted using the respective shRNAs. All data shown represent the mean± SD calculated from three independent experiments. (B) In the present study, the effects of PI3K inhibitor (LY294002, 10 uM), mTORC1 inhibitor (rapamycin; 5 uM), and inhibitor of autophagy (3MA) on ARV replication were examined. Data shown represent the mean± SD calculated from three independent experiments. (C) In the case of CDK 4, vero cells were infected with ARV at a MOI of 5 for 3 hours, followed by CDK 4 shRNA transfection for 18 hours. All data shown represent the mean± SD calculated from three independent experiments.
Mentions: As previously demonstrated, p17 is a Tpr suppressor leading to activation of p53, p21, and PTEN. To further determine the impact of Tpr on virus replication, Tpr knockdown was performed. Importantly, Tpr depletion increased virus production (Fig 10A). Conversely, knockdown of p53, PTEN, and autophagy markers (LC3) diminished virus yield (Fig 10A). Moreover, we also investigated the effects of PI3K inhibitor (LY294002), mTORC1 inhibitor (rapamycin), and autophagy inhibitor (3MA) on ARV replication. As presented in Fig 10B, inhibition of PI3K and mTORC1 by inhibitors increased virus titers, but inhibition of autophagy by 3MA reduced virus yield. These results are in consistent with our previous study [39]. Aside from this, a decrease in CDK4 level was observed in p17-transfected cells (Fig 8B; S3B Fig), and so we therefore also investigated the effect of CDK 4 on virus replication. In this work, expression of CDK4 was silenced. After vero cells were infected with ARV for 3 hours, CDK 4 knockdown in vero cells increased virus yield (Fig 10C). Collectively, our findings in this study reveal that p17-modulated suppression of Tpr and subsequent activation of upstream signaling (p53, PTEN, and p21) induces cell cycle arrest and autophagosome formation, which in turn enhancing virus replication [37–39, 64].

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