Analysis of the terminal repeat binding abilities of mutant adeno-associated virus replication proteins.
The adeno-associated virus (AAV) Rep78 and Rep68 proteins play essential roles in viral DNA replication, trans activation of viral gene expression, and suppression of oncogene-mediated cellular transformation. By using an extensive set of linker insertion and deletion mutations in the replication gene, we mapped the regions of the Rep78 protein that mediate binding to the AAV origin of replication in vitro. Deletions that removed amino acid codons 25 to 62, 88 to 113, 125 to 256, and 346 to 400 abolished binding. Alterations in several other regions of the protein affected the binding affinity of the mutant proteins. All of the mutant proteins that support AAV DNA replication or p40 trans activation bound to the terminal repeat sequence, thus verifying the importance of binding for these functions. Several mutant rep genes that failed to suppress oncogene-mediated cellular transformation produced proteins that were capable of binding to the AAV terminal repeat sequences.
Yang
Q
Q
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008.
Trempe
J P
JP
D004252:DNA Mutational Analysis; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C068734:rep proteins, Adeno-associated virus 2