Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants

Abstract

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate singlestranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VP1, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.

The adeno‐associated virus Rep78 major regulatory protein forms multimeric complexes and the domain for this activity is contained within the carboxy‐half of the molecule

“…This study directly demonstrates that AAV Rep78 forms homo-multimers by both West-Western and chemical crosslinking analysis. We believe that this self recognition is to be expected due to Rep78's multiple roles in transcriptional regulation [7-15] and DNA replication [3,4]. Rep78's interaction with DNA has been studied extensively [6,16,17,[28][29][30][31][32], but its potential protein-protein interactions have not.…”

“…Adeno-associated virus (AAV) is a helper dependent human parvovirus, which requires that the cell be co-infected with an adeno-or herpes virus to allow for AAV productive infection [1,2]. The Rep78 nuclear protein, encoded by AAV, plays important roles in AAV DNA replication [3,4], AAV chromosomal DNA integration into human chromosome 19 [5,6], the irans-regulation of AAV gene expression [7][8][9], and the /raniregulation of various heterologous genes [10][11][12][13][14][15]. Consistent with Rep78's many biological roles it has many defined biochemical activities.…”

The Role of the Adeno-Associated Virus Capsid in Gene Transfer

“…These systems have advanced the structural studies of the AAV capsid as a result of the large amount of empty capsids or virus-like particles (VLPs) that can be produced. Studies of AAV assembly have demonstrated that VP3 alone is sufficient to form VLPs [26], but VP1 is required for infectivity [27]. Subsequently, it was shown that the unique N-terminus of VP1 has phospholipase A2 activity and contains a nuclear localization signal (NLS) [28].…”

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.…”