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Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

ABSTRACT: Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

SUBMITTER: Manocha E

PROVIDER: S-EPMC7911508 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

Manocha Ekta E Bugatti Antonella A Belleri Mirella M Zani Alberto A Marsico Stefania S Caccuri Francesca F Presta Marco M Caruso Arnaldo A

Cells 20210128 2

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to s ...[more]

PMID: 33525607

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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:In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and μNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

Dataset Information

Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

Publications

Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

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