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Avian reovirus p17 and ?A act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6.

ABSTRACT: Although we have shown that avian reovirus (ARV) p17-mediated inhibition of Akt leads to induction of autophagy, the precise mechanisms remain largely unknown. This study has identified a specific mechanism by which ARV coordinately regulates the degradation of ribosomal proteins by p17-mediated activation of E3 ligase MDM2 that targets ribosomal proteins and by ?A-mediated upregulation of proteasome PSMB6. In addition to downregulating ribosomal proteins, p17 reduces mTORC2 assembly and disrupts mTORC2-robosome association, both of which inactivate mTORC2 leading to inhibition of Akt phosphorylation at S473. Furthermore, we discovered that p17 binds to and inhibits the CDK2/cyclin A2 complex, further inhibiting phosphorylation of Akt S473. The negative effect of p17 on mTORC2 assembly and Akt phosphorylation at S473 is reversed in cells treated with insulin or overexpression of CDK2. The carboxyl terminus of p17 is necessary for interaction with CDK2 and for induction of autophagy. Furthermore, p17-mediated upregulation of LC3-II could be partially reversed by overexpression of CDK2. The present study provides mechanistic insights into cooperation between p17 and ?A proteins of ARV to negatively regulate Akt by downregulating complexes of mTORC2 and CDK2/cyclin A2 and upregulating PSMB6, which together induces autophagy and cell cycle arrest and benefits virus replication.

SUBMITTER: Huang WR

PROVIDER: S-EPMC5507987 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6.

Huang Wei-Ru WR Chi Pei-I PI Chiu Hung-Chuan HC Hsu Jue-Liang JL Nielsen Brent L BL Liao Tsai-Ling TL Liu Hung-Jen HJ

Scientific reports 20170712 1

Although we have shown that avian reovirus (ARV) p17-mediated inhibition of Akt leads to induction of autophagy, the precise mechanisms remain largely unknown. This study has identified a specific mechanism by which ARV coordinately regulates the degradation of ribosomal proteins by p17-mediated activation of E3 ligase MDM2 that targets ribosomal proteins and by σA-mediated upregulation of proteasome PSMB6. In addition to downregulating ribosomal proteins, p17 reduces mTORC2 assembly and disrupt ...[more]

PMID: 28701787

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Project description:Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.

Project description:UnlabelledThe Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo.ImportanceEBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.

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Dataset Information

Avian reovirus p17 and ?A act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6.

Publications

Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6.

Similar Datasets

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