Rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535.
Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.
Han
Song-Iee
SI
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
Kawano
Masa-Aki
MA
Ishizu
Ken-Ichiro
K
Watanabe
Hajime
H
Hasegawa
Makoto
M
Kanesashi
Shin-Nosuke
SN
Kim
Yang-Su
YS
Nakanishi
Akira
A
Kataoka
Kohsuke
K
Handa
Hiroshi
H
D048948:14-3-3 Proteins; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007840:Latex; D009154:Mutation; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry*; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D048948:14-3-3 Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D007840:Latex; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D012694:Serine; D014446:Tyrosine 3-Monooxygenase