Synthesis of message and genome RNAs in vitro by Sendai virus-infected cell nucleocapsids

Abstract

Purified Sendai virus nucleocapsids isolated from infected cells were used to programme a transcription system in vitro to study virus-specific RNA synthesis. The RNA products were analysed for size by centrifugation before and after denaturation with formamide or glyoxal. The polarity of the products [message (+) or genome (-) strands] was analysed by RNA-RNA hybridization. The non-denatured RNA products sedimented in three groups: 7S to 22S single-stranded RNA transcripts and two partially ribonuclease-resistant complexes. One complex, representing 12% of the total product, sedimented at 26S to 36S. After denaturing the 26S to 36S complex to single-stranded molecules, about half of the RNAs sedimented at 25S to 54S and about half at 6S to 24S. The second complex, representing about 13% of the total RNA product, sedimented at 42S to 52S. After denaturing, about 10% of the single-stranded RNAs sedimented at 38S to 52S and about 90% sedimented at 6S to 19S. In hybridization studies, single-stranded RNAs that sedimented at less than 19S were predominantly of message sense (+ strand), whereas RNAs that sedimented at 25S to 54S were a mixture of genome and anti-genome type. These results show that transcription and replication activities in vitro were associated with Sendai virus nucleocapsids obtained from infected cells and that some of the reaction products approached genome size.