Investigation of cis -acting sequences involved in control of vesicular stomatitis virus *transcription

Vesicular stomatitis virus (VSV) is the prototype member of the family Rhabdoviridae in the order Mononegavirales, the nonsegmented, negative-strand (NNS) RNA viruses. The VSV genomic architecture is among the simplest of the NNS viruses, containing five genes separated by conserved gene junctions. Transcription of the VSV mRNAs begins from a single 3' polymerase entry site and proceeds sequentially, 3' to 5', to generate five discrete mRNAs. Gene expression for VSV is controlled at the level of mRNA transcription by the position of a gene relative to the 3' transcriptional promoter and by cis-acting sequences present at the genomic termini and the gene junctions.;The gene junctions of VSV contain sequences that direct the viral polymerase to polyadenylate and terminate the upstream mRNA and to initiate and 5' cap the downstream mRNA. These polymerase activities are controlled, respectively, by a gene end sequence and a gene start sequence, which are separated by a nontranscribed intergenic region. Upstream transcriptional termination at the gene end sequence is absolutely required before downstream initiation can occur at the gene start sequence. Because of the sequential nature of transcription for VSV and the requirement for mRNA termination to occur before initiation, it was not previously possible to test for any role that the gene end sequence may have had in conjunction with the gene start sequence to signal initiation of the downstream mRNA.;In this study, we have separated the cis-acting signals present at the gene junctions of VSV and investigated what role the gene end sequence and intergenic region had in downstream transcription. We found that a region of the gene end sequence was involved in signaling efficient downstream transcription, independent of its function in signaling upstream mRNA termination. We also determined that the length of the intergenic region inversely correlated with the abundance of the downstream mRNA. Finally, we engineered similar mutations into a gene junction of the infectious cDNA of VSV and used selective pressure to test for the importance of the sequences upstream of the gene start sequence in transcription of the downstream mRNA.