Adeno-associated viruses having nonsense mutations in the capsid genes: growth in mammalian cells containing an inducible amber suppressor.
When an adeno-associated virus (AAV) genome contained in a recombinant plasmid is transfected into adenovirus-infected cells, infectious AAV particles are efficiently generated. We previously described the construction of a conditional lethal mutant of AAV having an amber termination codon inserted in the rep gene. This mutant was propagated on a monkey kidney cell line (SupD12) having an inducible amber suppressor tRNAser. We now describe the construction and propagation of two additional conditional lethal mutants of AAV having amber codons affecting all three capsid proteins (AAV Capam) or only the VP1 capsid protein (AAV VP1am). Suppression of the amber mutations in the capsid proteins was demonstrated directly by immunoblot analysis. The efficiency of amber suppression on the SupD12 cell was about 6 to 10% for AAV VP1am and 4 to 5% for AAV Capam. The reversion frequency of either mutant was apparently less than 10(-5). On nonsuppressing cells AAV VP1am exhibited an Lip (Inf) phenotype, whereas AAV Capam exhibited a Cap phenotype.
Smuda
J W
JW
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Carter
B J
BJ
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D016153:Genes, Suppressor; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D015345:Oligonucleotide Probes; D012333:RNA, Messenger