Journal of General Virology, Vol 79, 471-478, Copyright © 1998 by Society for General Microbiology

ARTICLES

The PA influenza virus polymerase subunit is a phosphorylated protein

JJ Sanz-Ezquerro, J Fernandez Santaren, T Sierra, T Aragon, J Ortega, J Ortin, GL Smith and A Nieto
Centro Nacional de Biotecnologia (CSIC), Cantoblanco, Madrid, Spain.

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two- dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

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