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.