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Replication of minute virus of mice in murine cells is facilitated by virally induced depletion of p21 - PubMed

Replication of minute virus of mice in murine cells is facilitated by virally induced depletion of p21

Richard O Adeyemi et al. J Virol. 2012 Aug.

Abstract

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G(2)/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK.

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Figures

Fig 1
Fig 1

(A) MVM infection results in acetylation of p53. A9 cells parasynchronized by isoleucine deprivation (3) were released into complete medium and at the same time mock infected or infected with MVM at a multiplicity of infection (MOI) of 10. Cells were harvested at the indicated time points after release into complete medium and lysed in modified radioimmunoprecipitation assay (RIPA) buffer (3), and the amounts of protein were quantitated by the Bradford assay, and equal amounts of protein were loaded in each well. Western blot analyses were performed using antibodies against NS1 (10), actin (catalog no. MA515739; Pierce), phosphorylated p53 S15 (catalog no. 9284S; Cell Signaling) (p53-P-S15), and p53 acetylated on Lys379 (catalog no. 2570S; Cell Signaling) (acetyl-p53). Lanes M0 and M33 contain mock-treated cells at the time of release and 33 h postrelease, respectively. (B) p53 acetylation is significantly higher in MVM-infected cells. A9 cells were infected with MVM at the indicated time points as described above for panel A. Control cells were mock treated or treated with 1 mM hydroxyurea (HU) or 100 nM doxorubicin (Doxo) for 24 h. Western blots were performed against total p53 (catalog no. sc-6243; Santa Cruz Biotechnology) (phosphorylated p53 migrates as a slower running band), and acetyl-p53. (C) MVM infection results in upregulation of Bax and Mdm2. Cells were mock infected or infected with MVM as described above for panel A. Western blotting was performed using antibodies directed against NS1, tubulin (catalog no. T4026; Sigma), p53-S15, Bax (catalog no. 2772; Cell Signaling), and Mdm2 (catalog no. MAB3776; Millipore).

Fig 2
Fig 2

(A) MVM infection results in loss of p21 in the presence of activated p53. A9 cells parasynchronized as described in the legend to Fig. 1 were mock infected or infected with MVM at an MOI of 10. Western blot analyses were performed using antibodies against NS1, tubulin, phosphorylated p53 S15 and p21 (catalog no. sc-271532; Santa Cruz Biotechnology). Lanes M1 and M42 contain mock-treated cells harvested at 1 h and 42 h after release from isoleucine block into complete medium, respectively. Treatment of A9 cells with hydroxyurea (HU)—which induces an ATR-mediated DNA damage response and blocks cells in S phase—also reduced levels of p21 (lane 8) as previously documented by others (14). (B) G2/M restoration of p21 levels does not occur in MVM-infected cells. Mock- and MVM-infected A9 cells were harvested at the indicated time points after release from isoleucine-deprived medium. Western blot analyses were performed as described above for panel A. (C) Agents that block cells in G2/M result in increased levels of p21. A9 cells were treated with doxorubicin at the indicated concentrations for 24 h or were not treated with doxorubicin (−). Cells were harvested and processed for Western blotting. The band labeled Control in the top blot is a cellular cross-reacting band to the p21 antibody, which was used as a loading control. (D) p21 loss is proteasome mediated. Mock- and MVM-infected A9 cells, parasynchronized as described in the legend to Fig. 1, were treated with the proteasome inhibitor MG132 or DMSO vehicle for 6 h at 26 hours postinfection (hpi). Cells were harvested at 32 hpi, and Western blotting was performed as described in the legend to Fig. 1.

Fig 3
Fig 3

(A) NS1 leads to stabilization of p21. Stable A9 cell lines that inducibly express NS1 under a doxycycline-responsive promoter (A9-NS1) were generated using the pINDUCER lentivirus system (22) as described in the text. Cells were treated with doxycycline (500 ng/ml) (+) or vehicle (−) for 24 and 40 h and harvested, and the levels of NS1, actin, p53 phosphorylated on serine 15, and p21 were assayed by Western blotting. (B) NS1 expression does not lead to degradation of p21 in the context of DNA damage. A9 cells that were induced to express NS1 were treated with doxorubicin or vehicle control as indicated at a final concentration of 150 nM. Shortly thereafter, doxycycline (500 ng/ml) or vehicle control was added for 24 h to induce NS1 expression. Cells were then harvested and assayed via Western blotting as described above for panel A. (C) MVM infection prevents p21 upregulation by doxorubicin treatment. Parasynchronized A9 cells were released into complete medium and at the same time mock infected or infected with MVM at an MOI of 10. At 18 h postrelease, doxorubicin was added to mock- and MVM-infected cells at a final concentration of 150 nM. Cells were harvested at the indicated time points and assayed via Western blotting as described above for panel A.

Fig 4
Fig 4

(A) Inhibition of Cdk2 activity by roscovitine treatment. A9 cells parasynchronized by isoleucine deprivation were harvested at the time of release (T0) and 24 h after release (T24) (lanes 2 and 3). In the sample shown in lane 3, roscovitine was added for 6 h at 18 h after release. Lysates were quantified, immunoprecipitated, and used for kinase reactions as described in the text. Samples were run on 12% SDS-polyacrylamide gels, dried, and used for autoradiography. (B) Roscovitine treatment affects MVM replication following S-phase entry. A9 cells parasynchronized by isoleucine deprivation were infected with MVM at the time of release at an MOI of 1. At 18 hpi, cells were treated with vehicle or roscovitine for 4, 8, or 12 h and harvested at 22, 26, or 30 hpi, respectively. Cell lysates were used for Southern blotting. Equivalent loading was confirmed by measurements using a nanodrop spectrophometer as well as via ethidium bromide staining of cellular DNA. The positions of monomers and dimers of the 5-kb double-stranded replicative form (RF) are shown to the right of the blot. A representative blot is shown. (C) Quantification of the replication. The monomer double-stranded replicative forms from the Southern blots (an example of which is shown in panel B above) were quantitated using phosphorimager analysis (10). Data points represent averages from three independent experiments. The error bars represent the standard deviations. At both 26 and 30 h, the values for the treated samples were significantly different from the values for the untreated samples, with P values of <0.05.

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