Mutation of a consensus purine nucleotide binding site in the adeno-associated virus rep gene generates a dominant negative phenotype for DNA replication.
Adeno-associated virus (AAV) contains a multifunctional nonstructural gene, rep, which is required for AAV DNA replication and has pleiotropic effects on positive and negative regulation of gene expression. All of the parvovirus nonstructural genes contain a region of highly conserved amino acid homology. Within this conserved region is the consensus sequence for a purine nucleotide binding site. We constructed a mutant AAV having a mutation in this site by converting lysine 340 to histidine. The resulting mutant AAV genome, pNTC23, overproduced the mutant Rep proteins, indicating that these proteins are autoregulated. Furthermore, the mutant gene was unable to replicate but was able to inhibit in trans wild-type AAV DNA replication. Thus, pNTC23 represents a dominant negative mutant of AAV. These results suggest that rep has separate functional domains important for DNA replication.
Chejanovsky
N
N
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Carter
B J
BJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011685:Purine Nucleotides / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D004279:DNA, Viral; D011685:Purine Nucleotides; D014764:Viral Proteins