RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8.
Ribonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in two fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol, and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into two major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P proteins complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only three P proteins (P1, P2, and P3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P proteins complex was dependent on either ApG or capped RNA primers, and required four ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the three P proteins was separated from RNA by glycerol gradient centrifugation after ribonuclease treatment or cesium chloride equilibrium centrifugation.
D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D009693:Nucleic Acid Hybridization; D009975:Orthomyxoviridae / Q000032:analysis*; D012367:RNA, Viral / Q000302:isolation & purification*; D014764:Viral Proteins / Q000302:isolation & purification
D012367:RNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases