The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C

The M1 protein of influenza A virus has multiple regulatory functions durin g the infectious cycle, which include mediation of nuclear export of viral ribonucleoproteins, inhibition of viral transcription and a crucial role in virus assembly and budding. The only known modification of the M1 protein is by phosphorylation through yet-to-be-identified kinases. We postulated t hat at least some of the M1 functions are exerted or regulated through inte ractions with cellular components. In a screen for such cellular mediators, the protein receptor of the activated C-kinase (RACK 1) was identified by its interaction with the viral M1 protein in the yeast two hybrid system. T he physical MI-RACK 1 interaction was confirmed in glutathione-S-transferas e-based coprecipitation assays for the diverged M1 proteins of avian, swine and human influenza A virus strains. This conservation suggests that the M I-RACK 1 interaction is of general importance during influenza A virus infe ctions. RACK 1 has previously been identified to specifically bind the acti vated form of protein kinase C (PKC) and is assumed to anchor the kinase at membranes in the vicinity of its substrates. Since the M1 protein becomes phosphorylated during influenza virus infection, we examined if PKC could c atalyze the phosphate transfer. We demonstrate that virion-derived and reco mbinant M1 protein can indeed be efficiently phosphorylated by purified PKC . Moreover, in cell extracts, we detected M1 phosphorylation activity that was strongly reduced in the presence of the PKC-specific inhibitor compound GF109203X. These data suggest that PKC is the main M1-phosphorylating acti vity in the cell. Since both, the M1 protein and PKC have been shown to int eract with RACK I, we suggest that the M1-RACK 1 interaction is involved in M1 phosphorylation. (C) 2000 Elsevier Science B.V. All rights reserved.