Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function

Abstract

Transfection of a pBR322-based, recombinant plasmid, pAV2, containing the entire adeno-associated virus (AAV) type 2 genome into human 293 cells in the presence of helper adenovirus resulted in rescue and replication of AAV to yield infectious particles. We constructed mutants of pAV2 containing deletions within the AAV sequence. We describe here the phenotypes of these AAV deletion mutants. The results can be summarized as follows. Mutants (cap-) with deletions between map positions 53 and 85 did not synthesize capsid antigen or progeny single-stranded DNA but accumulated normal levels of duplex replicating form DNA. Mutants (rep-) with deletions between map positions 17 and 36 failed to rescue or replicate any AAV DNA. The rep- mutants could be complemented for replicating form DNA synthesis by a cap- mutant. This clearly demonstrates an AAV-coded replication function which is different from the capsid antigen. Other mutants (inf-) with deletions in the region between map positions 40 and 52 synthesized abundant amounts of replicating form DNA and capsid antigen but gave a low yield of infectious particles. This suggests that there may be an additional region of AAV, perhaps within the intron, which is required for efficient particle assembly. This work shows that AAV is genetically complex and expresses at least three clearly different functions.