THE PA INFLUENZA-VIRUS POLYMERASE SUBUNIT IS A PHOSPHORYLATED PROTEIN

The induction of proteolysis by expression of the influenza virus PA p olymerase subunit is the only biochemical activity ascribed to this pr otein, In the course of studying viral protein synthesis by two-dimens ional gel electrophoresis, we observed the existence of several PA iso forms with different isoelectric points, These isoforms were also pres ent when the PA gene was singly expressed in three different expressio n systems, indicating that a cellular activity is responsible for its post-translational modification, In vivo labelling with [P-32]orthopho sphate, followed by two-dimensional gel electrophoresis, clearly demon strated the incorporation of phosphate into the PA molecule, Phosphose rine and phosphothreonine epitopes were present in PA, while phosphoty rosine residues were absent, as tested by immunoblotting with specific antibodies, These facts, as well as the presence of multiple consensu s sites for casein kinase II (CKII) phosphorylation, prompted us to te st the involvement of this kinase in PA covalent modification, PA prot ein purified by immunoprecipitation could be specifically labelled by the catalytic a subunit of human CKII, which was expressed and purifie d from bacteria. Collectively, these data demonstrate that the PA subu nit of the influenza virus RNA polymerase is a phosphoprotein.