Effect of inserting paramyxovirus simian virus 5 gene junctions at the HN/L gene junction: Analysis of accumulation of mRNAs transcribed from rescued viable viruses

Simian parainfluenza virus 5 (SV5) is a prototype of the Paramyxoviridae family of nonsegmented negative-sense RNA viruses. The single-stranded RNA genomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences flanked by gene-end (GE) and gene-start (GS) sequences. The viral RNA polymerase (vRNAP) complex is thought to enter the genome at its 3' end, and synthesis of mRNAs is thought to occur by a stop- start mechanism in a sequential and polar manner, with transcriptional attenuation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNAP. To investigate the functions of GE, IG, and GS sequences in transcription, we constructed plasmids containing cDNAs of the full-length SV5 genome in which the gene junction sequences (GE, IG, and GS sequences) located between the hemagglutinin-neuraminidase (HN) and the polymerase (L) genes were replaced with the counterpart sequences from other gene junctions. By using reverse genetics, we recovered viable viruses from each cDNA construct, although their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of HN to L mRNAs varied over a fourfold range. The alteration of the gene junction sequences also permitted examination of the hypothesized requirement for hexamer nucleotide position of the GS sites. The recovery of infectious viruses with transcription initiation sites that occurred at nucleotide positions 1, 2, 3, 5, and 6 of the hexamer suggest that the requirement is nonstringent.