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. IMPORTANCEUpon 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.

Complementation for an essential ancillary non-structural protein function across parvovirus genera

“…This timing suggests that it may well be a response to replicating nuclear DNA. and its induction should result in changes in the way the infected cell responds to experimental stimuli, as recently reported, for example, for IL-6 expression in response to poly I-C stimulation (Mattei et al 2013), or Chk1 activation in response hydroxyurea treatment (Adeyemi and Pintel, 2014a). Overall, plausible explanations include failure to evade aspects of the induced DNA damage response (Adeyemi et al, 2010; Ruiz et al, 2011), failure to subvert the cell cycle (Adeyemi and Pintel, 2014b: Cotmore and Tattersall, 2013), or the induction of an innate antiviral response to replicating viral DNA, which NS2P, and by extension NP1, would be able to suppress.…”

Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3

“…The ATR-Chk1 pathway is important for the stabilization of stalled replication forks. Several DNA viruses, including herpesvirus, adenovirus, and parvovirus, can disable ATR signaling (27,45,46). During HSV-1 infection, viral replication proteins and ATR/ATRIP are recruited to viral replication forks and, with ATR-Chk1 pathway inhibition, may lead to replication fork collapse.…”

Protoparvovirus Interactions with the Cellular DNA Damage Response

“…During host cell infection, MVM induces a vigorous DDR in murine cells in which p53 is continually activated. Surprisingly, however, p21 WAF1/Cip1 (hereafter referred to as p21) and CHK1 (as discussed more fully below), major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, remain depleted or are inactivated, respectively [ 27 , 28 , 38 ]. Cycling cells typically reversibly down regulate p21 upon S-phase entry or in response to DNA double strand breaks, which is then restored to basal levels upon S-phase exit.…”

“…Depleted levels of p21 can be restored by treating infected cells with the proteasome inhibitor MG132, suggesting that p21 degradation during MVM infection is performed by the proteasomal machinery. Interestingly, however, whereas ATR activity modulates p21 degradation in response to various DDR-inducing agents (as described more fully below), the activation of ATR and its substrate CHK1 is reduced during MVM infection, consistent with the independence of p21 signaling from this pathway [ 28 ]. Similar to the case described above for cyclin B1 expression, while MVM infection leads to a loss of p21, the ectopic expression of NS1 results in increased p21 levels [ 39 ].…”

“…Unlike ATM, ATR is essential for cellular survival, and it has essential functions in the maintenance of genome integrity in response to replication stress in S- and G2-phases [ 3 , 58 ]. As mentioned above, ATR activation of CHK1, a major effector typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, is inhibited during MVM infection [ 28 ].…”