Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus.
An RNA polymerase-viral RNA complex was purified from influenza A/PR/8 virions by combination of cesium trifluoroacetate centrifugation and phosphocellulose column chromatography. Surface proteins were removed from the detergent-treated virions by the centrifugation. Starting from the M protein-free ribonucleoprotein (RNP) fraction, an RNA polymerase-RNA complex lacking NP protein was isolated by repeated chromatography on phosphocellulose columns. The isolated RNA polymerase-RNA complex, which is composed of PB1, PB2, PA and vRNA, cleaved capped poly(A) endonucleolytically at 10-12 nucleotides from the 5' end and incorporated GMP into the 3' end of the resulting capped fragments. In the presence of all four ribonucleotide triphosphate substrates, the cleaved fragments were elongated to polynucleotides in the absence of exogenous vRNA. The RNA synthesis was primed not only by capped polynucleotides but also dinucleotide ApG. These results indicate that the purified RNA polymerase-RNA complex is as active in viral mRNAs synthesis as native RNP and that NP protein is not required for the catalytic function.
D002500:Centrifugation, Isopycnic; D002848:Chromatography, DEAE-Cellulose; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D009980:Influenza A virus / Q000201:enzymology / Q000235:genetics; D011061:Poly A / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000302:isolation & purification; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000302:isolation & purification; D014771:Virion / Q000201:enzymology
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014758:Viral Core Proteins; D011061:Poly A; D012316:RNA Nucleotidyltransferases; D012324:RNA-Dependent RNA Polymerase