Nucleotide Sequence and Secondary Structure of VSV Leader RNA and Homologous DNA Involved in Inhibition of DNA-dependent Transcription

We have analyzed the nucleotide sequences and secondary structure required for the transcriptional inhibitory activity of the plus-strand leader RNA of vesicular stomatitis virus (VSV) in a reconstituted HeLa cell transcription system using the adenovirus-2 late promoter (LP) and virus-associated (VA) genes as templates. The New Jersey serotype (VSVNJ) leader and the leader of the Indiana serotype (VSVInd) both contain cleavage sites for the double-strand-specific ribonuclease V1, and these sites are consistent with the presence of a predicted AU-rich stem-loop structure. Studies in which the secondary structure was perturbed with the intercalating agent proflavin suggested that a stem-loop structure enhances the efficiency of transcription inhibition in the VSVNJ leader. Experiments using leader RNA fragments, a VSVInd cDNA derived from the 3' end of the genome, and synthetic oligodeoxynucleotide homologous to regions of the VSV leader indicated that the AU(AT)-rich center region of the VSV leader molecule is sufficient to inhibit DNA-dependent transcription directed by both polymerase II and III, but flanking nucleotide sequences are important for more efficient inhibition of transcription.