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

ABSTRACT 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.

  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Virology 11/2014; s 468–470:113–125. DOI:10.1016/j.virol.2014.07.042 · 3.28 Impact Factor
  • Source
    • "Interaction with p53 also prevents apoptosis induced by checkpoint activation when cells aberrantly enter S phase [10]. Additionally, large T antigen can promote viral replication in G2-arrested cells by inducing DNA damage response pathways, and this function was related to binding of cellular DNA by TAg [9]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: John Cunningham virus (JCV) is a member of the Polyomaviridae family. It was first isolated from the brain of a patient with Hodgkin disease in 1971, and since then the etiological agent of the progressive multifocal leukoencephalopathy (PML) was considered. Until the human immunodeficiency virus (HIV) pandemic, PML was rare: in fact HIV-induced immunodeficiency is the most common predisposing factor accounting for 85% of all instances of PML. This data led to intense research on JCV infection and resulted in better understanding of epidemiology and clinic-pathologic spectrum. Recently, cases of PML have been observed after the introduction of monoclonal antibodies, such as natalizumab, rituximab, efalizumab, and infliximab, in the treatment of autoimmune disease, underlining the important role of host immunity in PML pathogenesis. In this review current understanding of the JCV infection and the new findings relating to the pathogenesis of PML has been comprehensively revised, focusing our attention on the interaction between the cellular and viral molecular pathways implicated in the JCV infection and the modulating role of host immune surveillance in the viral reactivation from a latent state.
    Clinical and Developmental Immunology 04/2013; 2013:839719. DOI:10.1155/2013/839719 · 2.93 Impact Factor
  • Source
    • "In addition to their influence upon the G1/S phase transition of the cell cycle, polyomaviruses promote cell cycle arrest at the G2/M phase (Davy and Doorbar, 2007). JCV TAg induces G2 arrest as a result of the activation of the ATM and ATR checkpoint pathways, thereby promoting viral replication in permissive cells (Orba et al. 2010). βTrCP1 is a key player in the S and G2 DNA damage checkpoint pathways, and it promotes cell cycle arrest following DNA damage by attenuating CDK1 activity through degradation of Cdc25A. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lytic infection and transformation of cultured cells by JC virus (JCV) require five tumor proteins, which interact with factors regulating critical cellular processes. We demonstrate that JCV large T antigen (TAg) binds the F-box proteins β-transducin-repeat containing protein-1 and 2 (βTrCP1/2). These interactions involve a phosphodegron (DpSGX(2-4)pS) found in βTrCP substrates. TAg stability is unaltered, suggesting TAg is a pseudo-substrate. βTrCP and TAg co-localize in the cytoplasm, and a functional SCF complex is required. We examined whether TAg influences the levels of β-catenin, a βTrCP substrate. We were unable to demonstrate that TAg elevates β-catenin as previously reported, and a mutant TAg unable to bind βTrCP also had no detectable effect on β-catenin stability. Results presented in this study link JCV TAg to the cellular degradation complex, SCF(βTrCP1/2). Proteasomal degradation is essential for proper regulation of cellular functions, and interference with proteasomal pathways highlights possible JCV pathogenic and oncogenic mechanisms.
    Virology 02/2011; 410(1):119-28. DOI:10.1016/j.virol.2010.10.038 · 3.28 Impact Factor
Show more