Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: Characterization of Viral High Molecular Weight Nuclear RNA

Abstract

SUMMARY High mol. wt. RNA (HMW-RNA) extracted from nuclei of HEp-2 cells 5 h post infection with herpes simplex 1 virus has been shown to have the following characteristics: (i) the amount of HMW-RNA hybridizing to viral DNA fixed on filters increased with input multiplicity as well as following heat denaturation prior to hybridization; (ii) purified self-annealed HMW-RNA was enriched for viral RNA sequences which hybridized to viral DNA following denaturation; (iii) hybridization of excess unlabelled HMW-RNA which labelled viral DNA fragments followed by isopycnic centrifuging in Cs2SO4 led to the partitioning of a fraction of DNA with HMW-RNA. This DNA self annealed at a faster rate than the parental DNA population from which it was derived indicating that the HMW-RNA contained transcripts derived from symmetric transcription of a fraction of viral DNA; (iv) excess unlabelled, heat-denatured, HMW viral RNA drove 50% of viral DNA into DNA-RNA hybrid in hybridization tests with trace amounts of labelled viral DNA. Analysis of the kinetics of hybridization indicated that HMW-RNA consisted of 2 classes arising from 24 to 26% of viral DNA and differing 5000-fold in molar concentration. Since HMW-RNA contains symmetric viral transcripts which self anneal during the hybridization this is probably a minimal estimate of the amount of viral DNA represented in HMW-RNA.