Purification of a Virus-Induced RNA Polymerase fromAutographa californicaNuclear Polyhedrosis Virus-Infected Spodoptera Frugiperda Cells That Accurately Initiates Late and Very Late Transcriptionin Vitro

Abstract

AbstractThe virus-induced RNA polymerase fromAutographa californicanuclear polyhedrosis virus-infectedSpodoptera frugiperdacells was separated from the three host nuclear RNA polymerases by DEAE–Sephadex chromatography and then purified through two more steps: heparin–agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayedin vitrofor the ability to perform accurate initiation of transcription on a late (p6.9) and a very late (polyhedrin) template using primer extension analysis. In each case, the ability to accurately initiate transcription of these genes coincided with the virus-induced polymerase activity. Only after the glycerol gradient ultracentrifugation step did significant amounts of nonspecific late initiation occur, but specific late initiation was still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound in a complex. After the glycerol gradient ultracentrifugation step, SDS–PAGE showed fewer than 10 prominent polypeptides remaining in the active fractions, which suggests a high degree of purity of the transcription machinery