The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice

Abstract

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway.

Importance: Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway.

FIG 1

Viral replication is insufficient for Chk1 activation during MVM infection. (A) Murine A9 cells were infected with MVM at an MOI of 10 for 24 h. Uninfected control cells were treated with 1 mM hydroxyurea (HU) for 12 h or 150 ng/ml of neocarzinostatin (NCS) for 1 h. Cells were harvested and processed for Western blotting using antibodies directed against the indicated proteins. (B) Human 324K cell lines were treated as mentioned above for A9 cells. Cells were harvested, and RIPA lysates were blotted using antibodies directed against the indicated proteins.

FIG 2

ATR-Chk1 pathway proteins are stable, and ATR/ATRIP is recruited to viral replication bodies. (A) Human 324K cells were infected with MVM at an MOI of 10 for the indicated time points (18 and 24 h). Control cells were treated with 1 mM hydroxyurea (HU) for 12 h. Whole-cell lysates were processed for Western blotting using antibodies directed against the indicated proteins. (B and C) Murine 324K cell lines were infected with MVM at an MOI of 10. Cells were preextracted with cytoskeleton buffer, fixed, and stained using antibodies directed against the indicated proteins. Nuclei were visualized by DAPI staining. Colocalization with NS1 in infected cells with detectable APAR bodies was around 60% (ATR) and 90% (ATRIP).

FIG 3

Rad9 does not efficiently associate with chromatin during MVM infection. (A) Human 324K cells were infected with MVM at an MOI of 10 for 24 h. Control cells were treated with 1 mM HU for 6 h. Cells were harvested and fractionated as described in Materials and Methods, and 2% SDS lysates of the chromatin fraction were assayed by Western blotting using antibodies directed against the indicated proteins. (B and C) Human 324K cells were infected with MVM at an MOI of 10 for 24 h. Control cells were treated with 1 mM hydroxyurea (HU) for 12 h. Cells were preextracted with cytoskeleton buffer, fixed, and stained using antibodies directed against the indicated proteins. Nuclei were stained with DAPI.

FIG 4

Stalling of MVM replication “forks” early in infection triggers Chk1 phosphorylation. (A) Murine A9 cells were parasynchronized by isoleucine deprivation and infected with MVM at an MOI of 10 at the time of release into complete medium. At 15 hpr, cells were treated with 3 mM HU or vehicle control for 3 h and harvested at 18 hpr. RIPA lysates were quantitated by Bradford assay, and equal protein amounts were loaded in wells and processed for Western blotting using antibodies directed against phospho-Chk1. “Control” is a nonspecific band that served as a control for loading. (B) Murine A9 cells were treated as described above for Fig. 3A. Cells were infected with wild-type MVM or UV-inactivated virus and processed by Western blotting using antibodies directed against the indicated proteins. (C) Murine A9 cells treated as described for Fig. 3A were infected with increasing MOIs of MVM (0.1, 1, and 10). Cells were processed for Western blotting using antibodies directed against the indicated proteins. (D) Murine A9 cells were parasynchronized by isoleucine deprivation and then released into medium containing aphidicolin for an additional 16 h to achieve a tight synchronization at the G₁/S border. Infection with MVM at an MOI of 10 was performed at the time of release into medium containing aphidicolin. Control cells are shown in lane 3 to demonstrate the absence of NS1 expression in aphidicolin-treated cells in lanes 1 and 2. These cells were released into aphidicolin-free medium after isoleucine block and harvested at 18 h to allow progression into S phase and expression of NS1. Cell lysates were processed for Western blotting using antibodies directed against the indicated proteins.

FIG 5

Full MVM infection prevents ATR-Chk1 phosphorylation in response to exogenous DNA-damaging agents. (A) Parasynchronized murine A9 cells were released into complete medium and infected with MVM at an MOI of 10 at the time of release. At 24 hpr, cells were treated with 3 mM HU or vehicle control for 3 h. RIPA lysates were processed for Western blotting using antibodies directed against the indicated proteins. (B) Parasynchronized murine A9 cells were released into complete medium and infected with MVM at an MOI of 10 at the time of release. At 24 hpr, cells were treated with 150 ng/ml of NCS for 1 h. Cells were harvested and processed for Western blotting using antibodies directed against the indicated proteins.

FIG 6

Activation status of ATR and its role in MVM DDR signaling. (A) Human 324K cells were transfected twice with control siRNA or siRNA directed against ATR as indicated. Sixteen hours after the second transfection, cells were either mock infected or infected with MVM at an MOI of 10. Twenty-four hours later, cells were processed for Western blotting using antibodies directed against the indicated proteins. “*” indicates a nonspecific band. (B) Human 324K cells were infected with MVM at an MOI of 10 as indicated. Eight hours after infection, cells were treated with 10 μM ATR inhibitor (ATRi) for 16 h. Cells were harvested 24 hpi and processed for Western blotting using antibodies against the indicated proteins. (C) Human 324K cells were infected with MVM at an MOI of 10 for the indicated time periods. Control cells were treated with HU as described above. Cells were fractionated as described above, and ATR was immunoprecipitated from the chromatin fraction. Western blotting analyses were performed using antibodies directed against the indicated proteins.