Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes

Adeno-associated virus (AAV) replication depends on two viral components fo r replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct vir us compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce rec ombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro re plication assays indicated that the block occurs at the level of replicatio n instead of at viral assembly. AAV2 and AAV5 Rep binding activities demons trate similar affinities for either an AAV2 or AAV5 ITR; however, compariso n of terminal resolution site (TRS) endonuclease activities showed a differ ence in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a ty pe 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficient ly. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG T GGC) which is absent from the ITRs of other AAV serotypes. Comparison of th e TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage sit e but little homology upstream.