Characterization of the 5' termini of purified nascent simian virus 40 late transcripts

Abstract

The primary transcripts of simian virus 40 are extensively processed in the nuclei of infected monkey cells before they are transported to the cytoplasm as mature mRNAs. To investigate the early steps in this process, in particular, to determine which events occur on nascent chains before the termination of transcription, we have developed a procedure for the purification of nascent viral transcripts. This technique involves the in vitro incorporation of mercurated residues into the growing 3' ends of pre-initiated nascent chains, allowing their specific purification by sulfhydrylcellulose affinity chromatography. We further selected viral specific transcripts by hybridization to simian virus 40 DNA-cellulose. We describe here our analysis of the 5' termini of purified nascent simian virus 40 transcripts. This analysis revealed various cap structures, providing direct evidence that primary viral transcripts are capped before chain completion. The various cap structures exhibited a full range of methylation states. Completely unmethylated GpppA cap cores were identified, as well as caps methylated at the penultimate position only. The presence of GpppAm and GpppmAm caps indicates that, in BSC-1 cells, the penultimate nucleotide can be methylated before 7-methyl-G formation. Furthermore, the proportions of the various intermediates suggest that, in contrast to the viral capping enzymes of vaccinia virus and reovirus, the cellular enzymes methylate in the following order: GpppA leads to GpppAm leads to GpppmAm leads to 7mGpppmAm. In addition to capped ends, we also detected some unprocessed pppA ends. To our knowledge, this is the first time uncapped termini have been identified on RNAs known to be polymerase II products.