title: Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli.
abstract: The rep gene of the defective human parvovirus, adeno-associated virus, (AAV) mediates several trans-acting functions important to virus replication, transcription, and gene expression. At least four overlapping polypeptides are expressed from the rep gene. We have constructed a prokaryotic vector which expressed in Escherichia coli a region of AAV comprising 93% of the largest AAV rep protein. The protein expressed in E. coli, rep 78.93, was used to raise specific antibodies in rabbits. These antibodies were capable of detecting all four AAV rep proteins in human cells transfected with AAV-containing plasmids as well as new species of 47 and 35 kDa in molecular weight. These new rep proteins originate from the transcription promoter at map unit 19 in the AAV genome and may indicate use of alternate AUG codons or protein modification. The antibodies also recognized novel forms of the rep proteins expressed from mutant AAV genomes. Immunofluorescence analysis of AAV-infected human cells revealed that the rep proteins are localized primarily in the nucleus of the infected cell and have a distribution different from that of AAV capsid protein. These results demonstrate that antisera raised against an AAV rep protein synthesized in E. coli are capable of detecting wild-type AAV rep proteins in virus-infected mammalian cells. These specific antibodies should facilitate further characterization of the functionally pleiotropic viral rep proteins.
authors.affiliation: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
authors.forename: J P
authors.initials: JP
authors.lastname: Trempe
authors.forename: E
authors.initials: E
authors.lastname: Mendelson
authors.forename: B J
authors.initials: BJ
authors.lastname: Carter
mesh_terms: D000914:Antibodies, Viral / Q000276:immunology*; D002213:Capsid / Q000032:analysis; D002460:Cell Line; D004274:DNA, Recombinant; D000229:Dependovirus / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005814:Genes, Viral; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay; D010957:Plasmids; D014158:Transcription, Genetic; D014162:Transfection; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D004274:DNA, Recombinant; D007106:Immune Sera; D014764:Viral Proteins