Hyper-phosphorylation of the adeno-associated virus Rep78 protein inhibits terminal repeat binding and helicase activity.
The replication (Rep) proteins of adeno-associated virus (AAV) play prominent roles in regulation of viral DNA replication, RNA transcription, assembly of an infectious virion and establishment of the provirus. We have previously demonstrated that all four Rep proteins are phosphorylated on serine residues [Virology 23 (1997) 332-336]. Reversible phosphorylation may provide a mechanism for regulating Rep protein function. To test this hypothesis, we used the phosphatase inhibitor okadaic acid (OA) to obtain hyper-phosphorylated Rep proteins. OA treatment of AAV- and adenovirus (Ad)-infected cells and baculovirus-infected insect cells at a concentration of 100 nM resulted in a significant increase in Rep protein phosphorylation. This concentration suggests that protein phosphatase 2A (PP2A) is one of the enzymes involved in regulation of Rep phosphorylation. The increased phosphorylation occurred primarily on serine residues with a detectable amount of phosphate on threonine. Hyper-phosphorylation of Rep78 resulted in reduced binding to the AAV origin of DNA replication. Hyper-phosphorylated Rep78 also had diminished helicase activity. These results suggest that regulated phosphorylation of Rep78 plays a role in controlling Rep functions in the virus replication cycle.
Narasimhan
Diwahar
D
Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA.
Collaco
Roy
R
Kalman-Maltese
Vivian
V
Trempe
James P
JP
D002460:Cell Line; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D019319:Okadaic Acid / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020079:Terminal Repeat Sequences; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D019319:Okadaic Acid; D004265:DNA Helicases