Mutational analyses of the intergenic dinucleotide and the transcriptional start sequence of vesicular stomatitis virus (VSV) define sequences required for efficient termination and initiation of VSV transcripts

Abstract

We have used dicistronic vesicular stomatitis virus (VSV) minigenomes to dissect the functional importance of the nontranscribed intergenic dinucleotide and the conserved transcription start sequence found at the beginning of all VSV genes. The minigenomes were generated entirely from cDNA and contained the G and M protein genes, flanked by the leader and trailer regions from the Indiana serotype of VSV. All mutations were made either within the nontranscribed M-G intergenic dinucleotide or within the transcription start sequence of the downstream G gene. Immunofluorescence microscopy and immunoprecipitation analysis of the mutated minigenomes indicated that the first three nucleotides of the transcriptional start sequence are the most critical for efficient VSV gene expression, whereas the nontranscribed, intergenic dinucleotide and the other conserved nucleotides found at the 5' mRNA start sequence can tolerate significant sequence variability without affecting G protein production. RNA analysis indicated that nucleotide changes in the transcriptional start sequence which resulted in reduced G protein expression correlated with the amount of transcript present. Therefore, this conserved sequence appears to be required for efficient transcript initiation following polyadenylation of the upstream mRNA. While the minimum sequence for efficient transcription (3'-UYGnn-5') is similar to that of other rhabdoviruses, it is not homologous to the start sites for viruses from the Paramyxoviridae or Filoviridae families. Using Northern blot analysis, we also found that some nucleotide changes in the nontranscribed intergenic region resulted in higher levels of read-through transcription. Therefore, the nontranscribed intergenic dinucleotide plays a role in transcript termination.