Reconstitution studies detect a single polymerase entry site on the vesicular stomatitis virus genome

Abstract

To identify the initial steps of vesicular stomatitis virus transcription, we reconstituted purified nucleocapsid template with solubilized transcriptase and characterized the in vitro products of de novo transcription. In the absence of UTP and GTP, only leader gene products were synthesized; mRNA oligonucleotides were detected only after transcription of full-length leader was permitted. These data suggest that vesicular stomatitis virus polymerase does not enter the genome independently at each gene, but each polymerase begins transcription at the 3′ end of the genome, and reaches internal genes only by sequentially transcribing the 3′ preceding sequences. These results are consistent with the conclusion that the observed sequential transcription of vesicular stomatitis virus mRNAs is due to obligatory entrance of all polymerases at the leader gene, and suggest that the transcriptase and replicase may recognize the same promoter.