Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size

Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovi rus-infected cells by cotransfecting a plasmid containing the recombinant A AV2 genome, which is generally comprised of the viral terminal repeats flan king a transgene, together with a second plasmid expressing the AAV 2 rep a nd cap genes. However, recombinant viruses generally replicate inefficientl y, often producing 100-fold fewer virus particles per cell than can be obta ined after transfection with a plasmid containing a wild-type AAV2 genome. We demonstrate that this defect is due, at least in part, to the presence o f a positive-acting cis element between nucleotides 194 and 1882 of AAV2. R ecombinant AAV2 genomes lacking this region accumulated 14-fold less double -stranded, monomer-length replicative-form DNA than did wild-type AAV2. In addition, we demonstrate that a minimum genome size of 3.5 kb is required f or efficient production of single-stranded viral DNA. Relatively small reco mbinant genomes (2,992 and 3,445 bp) accumulated three- to eightfold less s ingle-stranded DNA per monomer-length replicative-form DNA molecule than wi ld-type AAV2. In contrast, recombinant AAV2 with larger genomes (3,555 to 4 ,712 bp) accumulated similar amounts of single-stranded DNA per monomer-len gth replicative-form DNA compared to wild-type AAV2. Analysis of two recomb inant AAV2 genomes less than 3.5 kb in size indicated that they were defici ent in the production of the extended form of monomer-length replicative-fo rm DNA, which is thought to be the immediate precursor to single-stranded A AV2 DNA.