Purification and Molecular Structure of RNA Polymerase from Influenza Virus A/PRS1

Abstract

The RNA–dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA–NP–P proteins) complexes were isolasgted by glycerol gradient centrifugation of detergent–treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins–viral RNA) complexes were further dissociated into P proteins and virasl RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunobloting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250, 000 Da. Upon addition of the template vRNA, the RNA–free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co–precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA–dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA–free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.