ROLE OF THE TERMINAL REPEAT GAGC TRIMER, THE MAJOR REP78 BINDING-SITE, IN ADENOASSOCIATED VIRUS-DNA REPLICATION

The adeno-associated virus (AAV) terminal repeats (TR) are cis require d, and the AAV encoded Rep78 protein is trans required, for AAV DNA re plication. The Rep78 protein recognizes and interacts with at least th ree regions within the TR DNA. The major binding site, with the highes t affinity for Rep78 binding, is within the TR stem (nt 36-16) and inc ludes the 'core' GAGC trimer (GAGC(3) nt 33-22; Fig. 2) sequence. In t his study mutations mere made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, p repackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two ins ide GAGC motifs are mutated, with only motif no. 1 left intact (see Fi g, 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no, 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (3 4%) DNA replication. Finally, if only the middle motif no. 2 was mutat ed, with motifs no. 1 and 3 left intact (mutC), the resulting DNA repl ication for both ds and ss forms was essentially wild type (80% that o f mild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important o f the three motifs for both ds and ss DNA replication.