Recombinant Adeno-Associated Viral Vectors Are Deficient in Provoking a DNA Damage Response

Abstract

Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.

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).…”

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).…”

“…Among the viral components, large Rep proteins, the cis-acting replication element (CARE) within the p5 promoter (Fragkos et al, 2008;Francois et al, 2005;Nony et al, 2001;Tullis & Shenk, 2000), AAV-ITR, and the unusual single-stranded nature of the viral genome are particularly important in AAV-evoked DDR and interaction with DNA repair machinery. These elements could potentially activate DDR without AAV viral genome replication.…”