Visualization of nascent RNA transcripts and simultaneous transcription and replication in viral nucleoprotein complexes from adenovirus 2-infected HeLa cells

Abstract

The study of potential regulatory events in gene expression in animal cells, such as RNA processing and template specificity for transcription or replication, was approached by visualization of these cellular processes in animal virus-infected cells. Viral nucleoprotein complexes were selectively extracted from purified HeLa cell nuclei 15 to 24 hours following infection with human adenovirus serotype 2. These viral nucleoprotein complexes contained replicative and transcriptional activities as judged by biochemical and structural analysis. Methods were developed that permitted electron microscopic visualization of adenovirus serotype 2 nucleoprotein complexes separated from the mass of cellular chromatin and containing measurable nascent RNA transcripts. Importantly, nascent RNA transcripts were observed on molecules that were intermediate in replication. Quantitation revealed that transcripts were present on replicating molecules with approximately the same frequency with which they were present on non-replicating molecules. This demonstrated that molecules of adenovirus serotype 2 DNA could serve simultaneously as templates for replication and transcription and that there was no obvious preference for replicating molecules as transcriptional templates.