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 the 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.
- Research Organization:
- National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD (USA)
- OSTI ID:
- 5822140
- Journal Information:
- J. Virol.; (United States), Journal Name: J. Virol.; (United States) Vol. 63:2; ISSN JOVIA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DISEASES
DNA REPLICATION
DRUGS
ELECTROPHORESIS
EVEN-ODD NUCLEI
INFECTIOUS DISEASES
ISOTOPES
LABELLING
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MAN
METHIONINE
MICROORGANISMS
NUCLEI
NUCLEIC ACID REPLICATION
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
SULFUR 35
SULFUR ISOTOPES
SYNTHESIS
VERTEBRATES
VIRAL DISEASES
VIRUSES