Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication.
The adeno-associated virus (AAV) rep gene is transcribed from two promoters, p5 and p19, which code for two over-lapping families of rep proteins. The proteins coded by p5 transcripts contain an amino-terminal domain not present in the proteins coded by p19 transcripts. The rep gene is required for AAV DNA replication and also mediates pleiotropic effects in positive and negative regulation of expression of genes driven by either AAV or heterologous promoters. All three functions require rep proteins coded by p5 transcripts. The functions of the rep proteins coded by the p19 transcripts could not be independently discerned since the coding region for these proteins and the p19 promoter are embedded within the coding region of the p5 transcript unit. We describe here an AAV mutant in which the putative AUG initiation codon in the p19 transcripts was altered and which did not express p19-coded rep proteins. This mutant exhibited normal AAV duplex RF DNA replication but was deficient in accumulation of AAV single-stranded progeny DNA and infectious AAV particles. This mutant defines a novel phenotype for a rep gene mutation and suggests a role for the rep proteins in the generation or accumulation of the viral SS DNA. Moreover this mutant distinguishes two different functions of the rep protein(s) in accumulation of the RF molecules and accumulation or processing of the SS DNA molecules, respectively.
Chejanovsky
N
N
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Carter
B J
BJ
D001483:Base Sequence; D002461:Cell Line, Transformed; D003062:Codon / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D005814:Genes, Viral; D005816:Genetic Complementation Test; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication / Q000235:genetics*
D003062:Codon; D004279:DNA, Viral; D009841:Oligonucleotides; D012333:RNA, Messenger