THE LATE EXPRESSION FACTOR-8 AND FACTOR-9 AND POSSIBLY THE PHOSPHOPROTEIN P78 83 OF AUTOGRAPHA-CALIFORNICA MULTICAPSID NUCLEOPOLYHEDROVIRUSARE COMPONENTS OF THE VIRUS-INDUCED RNA-POLYMERASE/

Attempts were made to identify some of the subunits of the baculovirus -induced RNA polymerase following purification of its enzymatic activi ty by conventional chromatography. Polymerase activity was extracted f rom lysates of insect cells infected with Autographa californica multi capsid nucleopolyhedrovirus by polyethylenimine precipitation and subs equently purified by phosphocellulose, anion exchange, poly(A) Sepharo se affinity, and gel filtration chromatography. The presence of the po lymerase was monitored by its alpha-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with t he enzymatic activity were identified. Peptide-specific antibodies wer e generated against a variety of late-expression factors (LEFs) and th ese antibodies, along with antisera directed against several other bac ulovirus proteins, were used in an immunoblot analysis of the purified polymerase, The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus- induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associ ate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in t he mammalian transcription complex TFIIF, and the RAP94 polypeptide as sociated with vaccinia virus RNA polymerase. The p78/83 protein has pr eviously been shown by our laboratory to be a capsid protein, but it m ay also play some role with the RNA polymerase. These results represen t a first attempt to identify specific components of the RNA polymeras e associated with infections of insect cells by A. californica nucleop olyhedrovirus.