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.

The Use of Heterologous Promoters for Adeno‐Associated Virus (AAV) Protein Expression in AAV Vector Production

“…Analysis of the factors which affect AAV vector production will provide insights into the development of more efficient rAAV production systems. Since Rep78/68, whose expression is driven from the p5 promoter, are the only AAV gene products required for replication and regulate both p19 and p40 promoters (21,28,34), we constructed various AAV helper plasmids in which the p5 promoter was replaced with heterologous promoters (EF, CMV, SV40, B l9p6 and CAG) to modulate the expression of AAV proteins, and examined the relation between the expression patterns of AAV proteins and the efficiency of rAAVLacZ production using these helper plasmids.…”

“…The AAV rep gene is transcribed from p5 and p19 promoters, and the AAV cap gene is transcribed from p40 promoter. Transcription from p5 promoter generates spliced and unspliced transcripts which encode Rep68 and Rep78, respectively (34). Rep78/68 are required to regulate transcription from all three promoters and to replicate the viral DNA (9,34).…”

“…Transcription from p5 promoter generates spliced and unspliced transcripts which encode Rep68 and Rep78, respectively (34). Rep78/68 are required to regulate transcription from all three promoters and to replicate the viral DNA (9,34). In the absence of a helper virus, Rep78/68 repress both p5 and p19 transcription (18).…”

“…Since Rep78/68 proteins are required for replication from an AAV origin within the ITR and transactivate both p19 and p40 promoters during productive infection in the presence of adenovirus (21,28,34), a change in the expression level of Rep78/68 would affect the expression of other AAV proteins and thereby alter the efficiency of AAV vector production. In this study, we constructed various helper plasmids in which p5 promoter was replaced with heterologous promoters [human polypeptide chain elongation factor 1-oc (EF) (24), cytomegalovirus immediate-early (CMV-LE) (36), simian virus 40 early (SV40), B19 parvovirus p6 (B 19p6) (27) and chicken P-actin promoter plus cytomegalovirus enhancer (CAG) (25) promoters] to examine the effect of different amounts of Rep78/68 on the expression of Rep52/40 and Cap, rAAV DNA replication and the efficiency of AAV vector production.…”

Efficient Production of Adeno‐associated Virus Vectors Using Split‐type Helper Plasmids

“…Rep78/68 are required to regulate transcription from all three promoters and to replicate the viral DNA. 10,11) Furthermore, Rep78/68 are also involved in the preferential integration of AAV genomes into the AAVS1 locus. 12) Spliced and unspliced transcripts from the p19 promoter encode Rep40 and Rep52, respectively.…”