Interaction between the DNA repair machinery and the adeno-associated virus (AAV) : vector transduction and site-specific integration

Adeno-associated virus type 2 (AAV-2) is a nonpathogenic, replication defective parvovirus containing a single-stranded DNA genome of 4.7 kb. Despite the increasing utilization of recombinant vectors derived from this virus (rAAV) in gene transfer applications, several aspects of the biology of both the wild type virus and of its vectors remain poorly understood. Here we developed a technique to visualize the conversion of rAAV vector genomes from single-stranded (ss) to double-stranded (ds) DNA in real time. We report that rAAV DNA accumulates into discrete foci inside the nucleus. These rAAV foci are defined in number, increase in size over time after transduction, and their presence correlates with the efficiency of cell transduction. These structures overlap with, or lie in close proximity to, the foci in which proteins of the MRN (Mrell-Rad50-Nbsl) complex and Mdcl accumulate after DNA damage. Silencing of Nbsl or Mdcl by RNA interference markedly increases the formation of rAAV foci, the extent of rAAV transduction, and AAV Rep dependent site-specific integration at chromosome 19q.l3.3-qter, so called AAVS1 locus. The adenovirus E4orf6 and ElB55k proteins-mediated degradation of the MRN complex also correlated with high levels of rAAV transduction and foci formation. Taken together, these observations indicate that the MRN complex plays an inhibitory role at the level of rAAV ss to double-stranded DNA (dsDNA) genome conversion, vector transduction, and site-specific integration. On the other hand, similar experiments using siRNAs against histone H2AX, Rad52, and DNA-PKcs indicated that these factors are required for effective rAAV transduction and site-specific integration