Adeno-associated Virus Integration Research Articles

Chromatin-like structure of adeno-associated virus DNA in infected cells.

Adeno-associated virus (AAV) is a single-stranded DNA virus which requires adenovirus as a helper for productive infection. We studied whether intracellular AAV DNA in KB cells was present in a chromatin-like structure by digesting infected cell nuclei with micrococcal nuclease. Virus DNA was detected by agarose gel electrophoresis followed by blotting and hybridization to nick-translated [32P]DNA probes. After coinfection with adenovirus, AAV DNA was present in nucleosome-like structures which were similar to cell nucleosomes and were double stranded as judged by insensitivity to S1 nuclease digestion. In the absence of adenovirus, intracellular AAV DNA also formed similar nucleosome-like structures which were also insensitive to S1 digestion and were formed in both the presence and absence of hydroxyurea. These latter structures probably formed on AAV duplexes created either by reassociation of infecting parental single-stranded DNA or by covalent integration into the cell genome rather than by de novo AAV DNA synthesis. These results have implications for the mechanism of AAV genome replication, transcription, and integration into the cell genome.