Avian reovirus p17 and σA 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 σA-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 σA 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.

Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17

“…Cellular proteins hnRNP A1 and lamin A/C coimmunoprecipatate with p17. As shown previously, we identified several cellular proteins, such as nucleoporin Tpr, vimentin, and lamin A/C, that interact with the ARV p17 protein (27,28,30). To explore whether and how cellular proteins are involved in mediating nucleocytoplasmic shut-Chiu et al FIG 1 p17 interacts with hnRNP A1.…”

“…A monopartite-type functional NLS near the C terminus of p17 is necessary for nuclear import (26) and interacts with nucleoporin Tpr localized within the nuclear basket of the NPC (27), which suppresses Tpr, thereby activating p53, PTEN, and p21 (27). Our team suggested that the avian reovirus (ARV) p17 protein performs specific duties in both the nucleus and the cytoplasm, as in mediating the Tpr/p53/PTEN/Akt/mTORC1 and mTORC2/Akt pathways (27,28), cell cycle (29)(30)(31), autophagy (28,32), and cellular translation (27,33). To date, the precise mechanisms of the involvement of cellular proteins in mediating nucleocytoplasmic shuttling of p17 remain unknown.…”

Avian reovirus σA-modulated suppression of lactate dehydrogenase and upregulation of glutaminolysis and the mTOC1/eIF4E/HIF-1α pathway to enhance glycolysis and the TCA cycle for virus replication

“…A previous study revealed that two arginine residues (Arg155 and Arg273) of σA are critical for nucleolus import and double-stranded RNA binding (Guardado-Calvo et al, 2008). Recently, we demonstrate that the ARV σA and p17 proteins function cooperatively to suppress Akt by inhibition of mTORC2 and CDK2 and activation of proteasome PSMB6 (Huang et al, 2017). …”

“…The constructs pCI-neo-σA and pcDNA 3.1-p17 were used as described previously (Huang et al, 2017). For the construction of the pCI-neo-LDHA plasmid, the LDHA-encoded gene was amplified by polymerase chain reaction (PCR), followed by digestion with NheI and EcoRI and ligation into the pCI-neo vector that was predigested with the same restriction endonucleases (Promega).…”

“…Vero cells were infected with ARV at an MOI of 5 followed by Co-IP with a σA monoclonal antibody as described previously (Huang et al, 2017). Briefly, noninfected and 3-hr ARV-infected cells were lysed and co-immunoprecipitated with an σA monoclonal antibody (2 μg).…”