The role of the diverse gene junction sequences in modulating paramyxovirus transcription and virus growth

Paramyxoviruses use a sequential, stop-start mechanism of gene expression and the activities of the viral polymerase are modulated by cis-acting elements located between the tandemly-linked genes. The transcribing activities of the viral RNA dependent RNA polymerase include termination of mRNA synthesis at an upstream gene and reinitiation of mRNA synthesis at a downstream gene to produce two monocistronic 5' capped and 3' polyadenylated mRNAs. Paramyxoviruses can be divided into two classes based on their gene junction sequences: those viruses which have highly conserved gene junctions and those viruses which exhibit variability in gene junction sequence and size. Interestingly, each of these classes of gene junctions direct similar polymerase activity. Using simian virus 5 (SV5), a model paramyxovirus, with variable gene junction sequences, this thesis addresses the relationship between the variable gene junction sequences and the common polymerase activities they modulate. A reverse genetics system was established in which viral transcription could be reconstituted in vivo from cDNA-derived polymerase components. Using this system several features concerning the variable gene junctions were elucidated. First, it was determined that a single nucleotide in the M gene end region was responsible for directing elevated levels of M-F transcriptional readthrough. Additionally, it was determined that termination at the M gene end required cooperation between the short M gene end U tract and the first nucleotide of the M-F intergenic region. Even though the remainder of the M-F intergenic region was not necessary for termination or reinitiation of transcription, naturally occurring mutations in this region have never been detected. A recombinant SV5 which contained a non-viral M-F intergenic region grew at wild type levels and directed slightly elevated levels of transcription. Additionally, it was also determined in the SH-HN gene junction that proper downstream gene reinitiation requires a minimum distance of six nucleotides to separate the two genes. This spacing requirement is mediated by the overall length of the gene junction region. This thesis suggests that the diversity in sequence at the SV5 gene junctions reflects specific combinations of nucleotides which may differentially affect viral gene expression and provide additional levels of transcriptional control.