Fidelity of adenovirus RNA transcription in isolated HeLa cell nuclei

Abstract

An in vitro nuclear system from adenovirus type 2-infected cells was developed to study transcription of viral RNA. Nuclei isolated from adenovirus-infected HeLa cells late in the infectious cycle synthesized in vitro only RNA from the r-strand of adenovirus DNA. Around 15% of the virus-specific RNA in isolated nuclei was polyadenylated. Short pulse labeling of nascent RNA followbd by hybrization of size-fractionated RNA to specific restriction endonuclease fragments of the genome suggested that the origin(s) for transcription is located on the r-strand in the left 30% of the adenovirus 2 genome at late times in the infectious cycle. Pulse-chase experiments were used to estimate the elongation rate of adenovirus high-molecular-weight RNA in isolated nuclei. An elongation of a least six nucleotides per second was observed in vitro. Viral RNA synthesis in the vitro nuclei showed several similarities to the in vivo system late in the infectious cycle.