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 p olyhedrosis virus-infected Spodoptera frugiperda cells was separated f rom the three host nuclear RNA polymerases by DEAE-Sephadex chromatogr aphy and then purified through two more steps: heparin-agarose chromat ography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayed in vitro for the abilit y 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 amount s of nonspecific late initiation occur, but specific late initiation w as still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound i n a complex. After the glycerol gradient ultracentrifugation step, SDS -PAGE showed fewer than 10 prominent polypeptides remaining in the act ive fractions, which suggests a high degree of purity of the transcrip tion machinery. (C) 1996 Academic Press, Inc.