Visualization of the major late r strand transcription unit of adenovirus serotype 2

Abstract

Using chromatin spreading for electron microscopy, we have visualized the major late r strand transcription unit of adenovirus serotype 2 in a transcriptionally active state. Adenovirus 2 genomes were identified in the dispersed nuclear contents of infected HeLa cells by their discrete length (8 to 12 μm), relatively smooth deoxyribonucleoprotein configuration, and occurrence as both Y-form and linear form replicative intermediates. At 16 hours after infection, near the time of maximal synthesis of late messenger RNA, any one genome was likely to exhibit either several or no nascent transcripts. The percentage of adenovirus 2 genomes utilized as transcriptional templates appeared low and was not limited to non-replicating molecules. Analysis of individual transcribing viral genomes revealed an average of 3.9 transcripts per adenovirus 2 genome (ranging between 1 and 15; n = 51) and, in general, single gradients of fibril length. Linear regression analyses on 20 genomes with four or more transcripts yielded an average extrapolated promoter site at adenovirus map unit 16.6, very near the previously determined promoter for the late r strand transcription unit of 16.4. Both individual genomes and a constructed “composite genome” exhibited increasing gradients of fibril length from the promoter to the distal end of the genome, suggesting that only one large transcription unit was active, and that transcription was completed before discernible RNA cleavage events. Viral transcripts exhibited a high degree of ribonucleoprotein packaging and a secondary structure suggesting intramolecular interactions. The low transcript density on the viral genomes was similar to that seen on host heterogeneous nuclear RNA transcription units. A calculation is presented that indicates that the observed frequency of transcription is sufficient to account for late viral mRNA production.