The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus

Abstract

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5–6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1–10 units/μg RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0·02 μg/μg RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)25, suggesting that the poly(A) sequence on the 3′ end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [3H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.