Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro

The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells 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 assayed in vitro for 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. (C) 1996 Academic Press, Inc.