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

Simian parainfluenza virus 5 (SV5) is a prototype of the Paramyxoviridae fa mily of nonsegmented negative-sense RNA viruses. The single-stranded RNA ge nomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences banked by gene-end(GE) and gene-start (GS) seq uences. The viral RNA polymerase (vRNAP) complex is thought to enter the ge nome at its 3' end, and synthesis of mRNAs is thought to occur by a stop-st art mechanism in a sequential and polar manner, with transcriptional attenu ation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNA P, To investigate the functions of GE, IG, and GS sequences in transcriptio n, we constructed plasmids containing cDNAs of the full-length SV5 genome i n which the gene junction sequences (GE, IG, and GS sequences) located betw een 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, al though their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of BN to L mRNAs varied over a fourfold range. The alteration of the gene junction se quences also permitted examination of the hypothesized requirement for hexa mer 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 nonstringe nt.