THE STRUCTURALLY DIVERSE INTERGENIC REGIONS OF RESPIRATORY SYNCYTIAL VIRUS DO NOT MODULATE SEQUENTIAL TRANSCRIPTION BY A DICISTRONIC MINIGENOME

The first nine genes of respiratory syncytial virus (RSV), a nonsegmen ted negative-strand RNA virus, are separated by intergenic regions whi ch range in size from 1 to 52 nucleotides for strain A2 and lack obvio us consensus elements except that each ends in an A (genome sense). Th eir significance for gene expression was investigated by using RSV-CAT -LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV ge nomic RNA in which the viral genes were replaced by a negative-sense c opy of the translational open reading frame (ORF) encoding chloramphen icol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUG) as the downstream gene, These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (G-E) transc ription signals and separated by the naturally occurring G/F intergeni c region, The RSV-GAT-LUC minigenome was synthesized in vitro and tran sfected into RSV-infected cells, and synthesis of the CAT and LUC mRNA s was monitored by enzyme assay and Northern (RNA) blot hybridization, Surprisingly, substitution of each of the other naturally occurring R SV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs, Substitution of a nonnatural 10 -nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurr ing intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRN As would hybridize equally with a CAT-specific probe and their relativ e molar amounts could be determined. The level of expression of the do wnstream gene was 0.30 to 0.36 that of the upstream one. This determin ed the magnitude of RSV transcriptional polarity across a gene pair an d confirmed that this value was very similar among the various interge nic regions. Minigenome transcription also yielded a CAT-LUG readthrou gh mRNA at a level 0.10 to 0.13 that of the LUC mRNA, In summary, the structurally diverse RSV intergenic regions do not appear to play a ro le in modulating RSV gene expression.