Viral Transport of DNA Damage That Mimics a Stalled Replication Fork

Abstract

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G 2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G 2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.The human adeno-associated virus type 2 (AAV2) can perturb cell cycle progression (51, 71) and mediate specific killing of p53-deficient cells (51). Cells with intact p53 activity were able to arrest with 4N DNA content, whereas cells without functional p53 were not able to sustain this arrest and died. This effect was shown to depend not on the viral capsid proteins or other virus-encoded proteins but on the presence of the viral DNA. The AAV2 particle contains a single-stranded DNA molecule of 4.7 kb flanked by identical inverted terminal repeats which form T-shaped hairpin structures (5). The inverted terminal repeats are thought to function as primers for viral DNA replication. The hairpin structures of AAV2 DNA together with its single strandedness were hypothesized (51) to induce DNA damage signaling after AAV2 infection. In the work presented here, we set out to test this hypothesis, to identify proteins that recognize AAV DNA, and to elucidate how these proteins then activate the pathway that leads to G 2 arrest.An appropriate cellular response to DNA damage is crucial for maintenance of normal cell fate. Ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia-and Rad3-related (ATR) proteins are the two major signaling kinases that respond to DNA damage in cells.

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

“…Although ATM/ATR kinase phosphorylates Chk2 at Thr-68 and p53 at Ser-15, the ATR kinase predominantly targets Chk1 at Ser-345, leading to increased Chk1 activity. Our results indicate that HBV infection preferentially activates ATR DNA damage response signaling, as is the case with human adeno-associated-virus type 2 [30][31][32][33][34][35][36] . As a latent virus, HBV abrogates checkpoint signaling controlled by ATR, to prevent triggering of signals for apoptosis in multiple ways.…”

The Role of DNA Repair Pathways in Adeno-Associated Virus Infection and Viral Genome Replication / Recombination / Integration

“…This strongly supports a model in which AAV-ITR is recognized as a stalled replication fork and triggers the checkpoint response via ATR kinase. The actual activation of the ATR pathway by AAV genomes has been confirmed by the demonstration that the ATR-downstream effector proteins; i.e., Chk1 and RPA, become phosphorylated in cells infected with wtAAV2 or UV-irradiated wtAAV2, both of which are devoid of the ability to replicate or express viral genes in the system used for the experiment (Fragkos et al, 2008;Ingemarsdotter et al, 2010;Jurvansuu et al, 2005Jurvansuu et al, , 2007. Interestingly, rAAV2 genome devoid of the 55-nt CARE within the p5 promoter does not evoke the ATR-mediated checkpoint signal, and it has been shown that co-existence of both ITR and CARE in an AAV genome is essential for the activation (Fragkos et al, 2008).…”

“…Direct evidence for the association of AAV genomes with DNA repair machinery has obtained in chromatin immunoprecipitation (ChIP) studies where AAV genomes and their associated cellular factors were crosslinked by formalin and precipitated together using antibodies specific to DNA repair proteins. To date, the MRN complex, Ku86, Rad52, RPA and DNA polymerase delta have been identified as factors bound to ss AAV genomes (Cervelli et al, 2008;Jurvansuu et al, 2005;Zentilin et al, 2001). In addition, immunofluorescence microscopy has revealed that the MRN complex, ATR, TopBP1, BLM, Brca1, Rad17, RPA, and Rad51 are recruited to the discrete nuclear foci where AAV genomes accumulate (Cervelli et al, 2008;Jurvansuu et al, 2005).…”

“…To date, the MRN complex, Ku86, Rad52, RPA and DNA polymerase delta have been identified as factors bound to ss AAV genomes (Cervelli et al, 2008;Jurvansuu et al, 2005;Zentilin et al, 2001). In addition, immunofluorescence microscopy has revealed that the MRN complex, ATR, TopBP1, BLM, Brca1, Rad17, RPA, and Rad51 are recruited to the discrete nuclear foci where AAV genomes accumulate (Cervelli et al, 2008;Jurvansuu et al, 2005). Table 2 summarizes the roles of DNA repair proteins in AAV infection/transduction, AAV genome selfcircularization, and AAV genome integration into the host genome.…”

Interactions Between AAV-2 and HSV-1: Implications for Hybrid Vector Design

“…It was therefore hypothesized that activation of RPA by Rep might be responsible for inhibition of HSV-1 replication. However, hyperphosphorylation of RPA induced by other means, such as camptothecin or infection with UV-inactivated AAV-2 [177,178], did not reduce HSV-1 replication, suggesting that the activation of RPA per se is not involved in the inhibitory effect of Rep on HSV-1 [153]. It therefore appears that the ability of Rep to induce DNA damage, rather than the cellular response to it, is responsible for the inhibitory effect on HSV-1.…”