Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells

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Abstract
A clone of human cells (Detroit 6) latently infected by adeno-associated virus (AAV) was characterized with regard to the status of the viral DNA. In early (9-10) and late (118) passages of the clone, AAV DNA was recombined with host DNA, at least in some cases as a head-to-tail tandem repeat, via the terminal sequences of the viral genome. It was not possible to distinguish between integration into chromosomal DNA and very large plasmids (> 20 .times. 106 MW) which contain both viral and cellular DNA sequences. Although evidence for some modifications of the viral sequence was obtained, most integrated sequences appeared to be intact. In some cases, sequences of undetermined origin separated adjacent copies of the viral genome. Free copies of the AAV genome were detectable in late passage cells, but not in early passage cells. The orientation of nucleotide sequences present in the free AAV DNA from late passage cells was indistinguishable from that of virion DNA. With 1 notable exception, the organization of the integrated AAV sequences as determined by restriction enzyme digestion remained constant with continued passage. Digestion with SmaI, which cleaves within the palindromic region of the terminal repetition in AAV DNA, produced reproducibly different patterns when early and late passage DNA were compared. Several models for rescue of free copies of the genome from the integrated DNA are possible; all involve the terminal repetition.