title: Adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin.
abstract: The adeno-associated virus (AAV) rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of the rep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the carboxyl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin.
authors.affiliation: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: R A
authors.initials: RA
authors.lastname: Owens
authors.forename: J P
authors.initials: JP
authors.lastname: Trempe
authors.forename: N
authors.initials: N
authors.lastname: Chejanovsky
authors.forename: B J
authors.initials: BJ
authors.lastname: Carter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D004261:DNA Replication; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics*
chemical_list: D000906:Antibodies; D014764:Viral Proteins