A NOVEL TERMINAL RESOLUTION-LIKE SITE IN THE ADENOASSOCIATED VIRUS TYPE-2 GENOME

The adeno-associated virus 2 (AAV) contains a single-stranded DNA geno me of which the terminal 145 nucleotides are palindromic and form T-sh aped hairpin structures, These inverted terminal repeats (ITRs) play a n important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep), However, the Rep proteins al so interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome f rom latently infected cells or from recombinant AAV plasmids, To disti nguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genome s that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus t ype 2-infected human KB cells, as expected. However, excision and abun dant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome, T his site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RES. Furthermore, deletion of this RB S abolished the rescue and replication of the vector sequences, These studies suggest that the Rep-mediated cleavage at the RES during viral DNA replication may, in part, account for the generation of the AAV d efective interfering particles.