Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Department of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, N20, W10, Kita-ku, Sapporo 001-0020, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2009; 285(2):1544-54. DOI: 10.1074/jbc.M109.064311
Source: PubMed


Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

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    • "In addition to their ability to induce entry into S-phase, many PyVs appear to block progression into mitosis at the G 2 →M checkpoint (Orba et al., 2010; Rohaly et al., 2010; Demetriou et al., 2012; Jiang et al., 2012; Li et al., 2013; Tsang et al., 2014). The exact events leading to G 2 arrest are undefined. "
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    ABSTRACT: Viruses are obligate intracellular parasites that subvert cellular metabolism and pathways to mediate their own replication-normally at the expense of the host cell. Polyomaviruses are a group of small DNA viruses, which have long been studied as a model for eukaryotic DNA replication. Polyomaviruses manipulate host replication proteins, as well as proteins involved in DNA maintenance and repair, to serve as essential cofactors for productive infection. Moreover, evidence suggests that polyomavirus infection poses a unique genotoxic threat to the host cell. In response to any source of DNA damage, cells must initiate an effective DNA damage response (DDR) to maintain genomic integrity, wherein two protein kinases, ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), are major regulators of DNA damage recognition and repair. Recent investigation suggests that these essential DDR proteins are required for productive polyomavirus infection. This review will focus on polyomaviruses and their interaction with ATMand ATR-mediated DNA damage responses and the effect of this interaction on host genomic stability.
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    • "Inspection of Fig. 3C (top line) provides additional evidence that in C33A cells, JCV T-ag is in the nuclei where it clusters in discrete foci. An analogous punctate distribution of T-ag in the nuclei of replication competent cells was seen in previous studies of SV40 DNA Zhao et al., 2008 and JCV replication ((Gasparovic et al., 2009; Orba et al., 2010; Shishido-Hara et al., 2008); reviewed in (Shishido-Hara, 2010)). Inspection of Fig. 3C (middle and bottom rows) establishes that a similar punctate distribution of JCV T-ag occurs in the Hs 683 and U87 cells. "
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    ABSTRACT: Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.
    Full-text · Article · Nov 2014 · Virology
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    • "For example, simian virus 40 large T antigen promoted entry into S phase (33), and cells infected with mouse polyomavirus or simian virus 40 accumulated in S and G2/M phases (34). More recently, JC virus was shown to proliferate progeny in G2-arrested host cells (35). The present study consistently demonstrates virus infection–associated cell cycle modulation in human brain tissues and is the first to show an association between virus-induced cell cycle changes and nuclear enlargement. "
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    ABSTRACT: In progressive multifocal leukoencephalopathy, JC virus-infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed "promyelocytic leukemia nuclear bodies" (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 μm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
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