Phosphorylation of specific serine residues within the acidic domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro.
The phosphorylated state of the vesicular stomatitis virus phosphoprotein (P), an essential component of the virion-associated RNA polymerase complex, has been shown to be important for the transcriptional activity of the complex. Recent studies indicate that phosphorylation within the acidic domain of the P protein by cellular casein kinase II is necessary for its activity. In an attempt to identify the exact location of the cell kinase-mediated phosphorylation, we altered specific serine and threonine residues within the acidic domain of the New Jersey serotype of P protein by site-directed mutagenesis. The altered P proteins were then tested to determine what effect these mutations had on the phosphorylated state of the protein in vivo as well as its transcriptional activity in vitro. We report that serine residues 59 and 61 within the acidic domain of the P protein must be phosphorylated for it to be functionally active in a reconstituted transcription assay. These results demonstrate the importance of site-specific phosphorylation in the transcriptional activity of a negative-strand RNA viral phosphoprotein and the crucial role played by a cell protein kinase in this process.
Takacs
A M
AM
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5178.
Barik
S
S
Das
T
T
Banerjee
A K
AK
D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D047388:Casein Kinases; D002460:Cell Line; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D018116:Vesiculovirus / Q000235:genetics / Q000378:metabolism*; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D009841:Oligonucleotides; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D012694:Serine; D011494:Protein Kinases; D047388:Casein Kinases