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Title: Adeno-associated virus rep protein synthesis during productive infection

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of themore » Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.« less
Authors:
; ;
Publication Date:
OSTI Identifier:
5822140
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Virol.; (United States); Journal Volume: 63:2
Research Org:
National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD (USA)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PROTEINS; BIOSYNTHESIS; VIRUSES; DNA REPLICATION; ANIMAL CELLS; ELECTROPHORESIS; LABELLING; MAN; METHIONINE; PHOSPHORUS 32; SULFUR 35; VIRAL DISEASES; AMINO ACIDS; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBOXYLIC ACIDS; DAYS LIVING RADIOISOTOPES; DISEASES; DRUGS; EVEN-ODD NUCLEI; INFECTIOUS DISEASES; ISOTOPES; LIGHT NUCLEI; LIPOTROPIC FACTORS; MAMMALS; MICROORGANISMS; NUCLEI; NUCLEIC ACID REPLICATION; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PARASITES; PHOSPHORUS ISOTOPES; PRIMATES; RADIOISOTOPES; SULFUR ISOTOPES; SYNTHESIS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques