Journal of Biological Chemistry
Volume 293, Issue 32, 10 August 2018, Pages 12542-12562
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Microbiology
Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK–cyclin complexes and enhancement of p53 and cyclin H interaction

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The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK–cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK–cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53–cyclin H interaction. p17 binds to CDK–cyclin except for CDK1–cyclin B1 and CDK7–cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1–cyclin B1, but it is required for other CDK–cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127. Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.

cancer
cell cycle
cyclin
mutagenesis
cyclin-dependent kinase 7 (CDK7)
avian reovirus
CDK inhibitor
CDK–cyclin complexes
cyclin-dependent kinase (CDK)
p17

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This work was supported by Taichung Veterans General Hospital Grants TCVGH-NCHU1057603 and TCVGH-NCHU1067601, National Chung Hsing University Grants TCVGH-NCHU1057603 and TCVGH-NCHU1067601, Ministry of Science and Technology of Taiwan Grants MOST 106-2313-B-005-054-MY3 and 106-2622-B-005-009-CC2, and the iEGG and Animal Biotechnology Center from the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. The authors declare that they have no conflicts of interest with the contents of this article.

This article contains Tables S1–S4 and Figs. S1–S11.

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The abbreviations used are:

    CDK

    cyclin-dependent kinase

    ARV

    avian reovirus

    CAK

    CDK-activating kinase

    IP

    immunoprecipitation

    DMEM

    Dulbecco's modified Eagle's medium

    FBS

    fetal bovine serum

    GST

    glutathione S-transferase

    MOI

    multiplicity of infection

    p-

    phosphorylated

    PMSF

    phenylmethylsulfonyl fluoride

    hpi

    hours postinfection

    MEM

    minimum essential medium.