Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway

Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two proteins involved in nonhomologous end joining (NHEJ), DNAPKcs and ligase IV, exhibit differential effects on AAV site-specific integration. DNAPKcs is not required; its presence increases the frequency of junction formation indicative of site-specific integration, but seems to reduce the ratio of site-specific integration to random integration (i.e., the latter is even more enhanced). In contrast, site-specific integration is significantly reduced relative to random integration in cells deficient in ligase IV expression. Furthermore, we show that single-stranded AAV vectors are better substrates for site-specific integration than are self-complementary AAV vectors; the absence of DNAPKcs did not affect the targeted integration of these double-stranded AAV vectors. Together, these data suggest that NHEJ proteins participate in site-specific integration, and indicate a role for the single-stranded form of AAV DNA in targeted integration.