title: Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes.
abstract: Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.
authors: Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
authors: J A
authors: JA
authors: Chiorini
authors: S
authors: S
authors: Afione
authors: R M
authors: RM
authors: Kotin
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012703:Serotyping; D013379:Substrate Specificity; D020079:Terminal Repeat Sequences; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2