Structural analysis of adeno-associated virus transduction circular intermediates

Recombinant adeno-associated virus (rAAV) has recently been demonstrated to form circular intermediates following transduction in muscle tissue and ce ll lines. Although restriction enzyme and Southern blot analysis has reveal ed a consistent monomer and multimer head-to-tail conformation, detailed st ructural sequence analysis has been lacking due to the high secondary struc ture of the ITR arrays. To gain further insight into potential mechanisms b y which AAV circular genomes are formed from linear single-stranded viral D NA, we have performed chemical sequencing of ITR arrays within seven circul ar intermediates independently isolated from primary fibroblasts and Hela c ells. Results from these studies demonstrated several types of circular int ermediates with mosaic ITR elements flanked by two D sequences. The most pr edominant form consisted of a structure similar to that of previously gener ated AAV double-D plasmids, with one complete ITR flanked by two D-region e lements. However, intermediately deleted ITR arrays with more than one comp lete ITR were also seen. Based on this structural information, we have prop osed a model for formation of AAV circular intermediates by recombination/l igation between ITR ends of panhandle single-stranded AAV genomes. (C) 1999 Academic Press.