Isolation of two forms of SV40 nucleoprotein containing RNA polymerase from infected monkey cells

Abstract

Abstract Triton extraction of SV40 infected monkey cells separates the viral transcription complex (VTC) into two fractions. Both forms of VTC are comprised of viral DNA associated with RNA polymerase which had initiated RNA synthesis prior to extraction. Approximately 20% of the total VTC can be solubilized by a variety of extraction conditions, while the remainder is pelleted together with the cellular chromatin or DNA. The proportion of VTC in the pellet fraction is not altered by extraction at a 10-fold lower cell concentration or under conditions which release most of the protein from chromatin and solubilize nearly all of the nonintegrated SV40 DNA. The predominant template for SV40 transcription at a late stage of infection is apparently associated with cellular DNA in vivo , or else it becomes selectively associated during the extraction procedure. Most of the Triton-soluble VTC, detected by endogenous RNA polymerase activity, sediments somewhat faster than the DNA-labeled SV40 chromatin, suggesting that a minor fraction of the DNA is engaged in transcription. Treatment of the VTC with Sarkosyl reduces its sedimentation rate from 50–55 S to 21–25 S and enhances the endogenous RNA polymerase activity by a factor of 3–5. It is thus likely that some cellular and/or viral proteins associated with SV40 DNA affect the rate of viral transcription in vivo .