Sequential synthesis of small capped RNA transcripts in vitro by vesicular stomatitis virus.

Using purified viral or intracellular transcriptive complexes (RNP cores) of vesicular stomatitis virus (VSV), we have identified several small RNA species, ranging in size from 12 to 47 nucleotides in length that are synthesized in vitro by the genomic RNA. One group of small RNA transcripts is composed of three species that are capped at their 5' termini. Two of the capped species are from the start of the N gene and one is from the start of the NS gene. Unlike the previously described 5' triphosphated small RNAs, the templates encoding these small capped RNAs had uv target sizes greater than their respective lengths. In addition, these RNAs appeared sequentially during synchronized in vitro transcription reactions. Thus, these results provide evidence that sequences representing the 5'-capped termini of N and NS mRNAs are synthesized concomitantly with their respective mRNAs rather than simultaneously at the onset of transcription as proposed for the multiple entry, start-stop model (D. Testa, P. K. Chanda, and A. K. Banerjee, 1980, Cell 21, pp. 267-275). Together with the inability of the internally initiated 5'-triphosphated RNAs to be chased into mRNA (R. A. Lazzarini, I. Chien, F. Yang, and J. D. Keene, 1982, J. Gen. Virol. 58, 429-441), these results support a single entry model of VSV mRNA transcription.