Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

Abstract

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNAdependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6,40,47,70,75,77,87,93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 het...

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

“…S10). The pan-γH2AX staining has been reported to occur in human fibroblast cells subjected to Adeno-associated virus (58,60), Chlamydia (33), and UV and ionizing radiation (30,61,62). Recently, such nuclear-wide γH2AX has been shown to occur in highly DNAdamaged cells and is mediated by ATM kinase (30).…”

SiDNA and Other Tools for the Indirect Induction of DNA Damage Responses

“…ATR may be the principal kinase phosphorylating H2AX in these conditions, although ATM and DNA-PK also seem to be involved (Nichols et al, 2009). Pan-nuclear H2AX phosphorylation has also been observed in cells infected with the adeno-associated virus (AAV) (Collaco et al, 2009;Fragkos et al, 2008;Schwartz et al, 2009). The AAV genome, like that of Ad, consists of an ssDNA molecule with ITRs at both ends, resulting in the formation of double-hairpin structures (Brown, 2010).…”

“…AAV replication in the presence of minimal Ad helper proteins induces a robust DDR-like response. This response is independent of the MRN complex and seems to be mediated principally by DNA-PKcs and, to a lesser extent, by ATM (Collaco et al, 2009;Schwartz et al, 2009). The response involves the accumulation of DNA-PK in compartments in which viral replication is occurring, and the pan-nuclear phosphorylation, not only of H2AX, but also of Smc1 and ATM (Schwartz et al, 2009).…”

“…This response is independent of the MRN complex and seems to be mediated principally by DNA-PKcs and, to a lesser extent, by ATM (Collaco et al, 2009;Schwartz et al, 2009). The response involves the accumulation of DNA-PK in compartments in which viral replication is occurring, and the pan-nuclear phosphorylation, not only of H2AX, but also of Smc1 and ATM (Schwartz et al, 2009). In another study, the phosphorylation of RPA, Nbs1 and Chk1/2 was observed, but the phosphorylation pattern was not investigated (Collaco et al, 2009).…”

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

“…The adenoviral components required in the lytic infection are E1a, E1b55k, E4orf6, DBP, and VAI. A series of adenovirus/AAV co-infection studies has provided significant insights into how DDR and DNA repair machinery play roles in AAV genome replication in the presence of adenovirus helper functions (Collaco et al, 2009;Schwartz et al, 2009). It has been shown that adenoviral E1b55k/E4orf6 degrades the MRN complex via the ubiquitin-proteasome pathway and E4orf3 mislocalizes the MRN complex to aggresome, abrogating the MRN function in triggering the ATM and ATR pathways (Figure 2a) (Collaco et al, 2009).…”