EFFECT OF MUTATIONS IN THE GENE-START AND GENE-END SEQUENCE MOTIFS ONTRANSCRIPTION OF MONOCISTRONIC AND DICISTRONIC MINIGENOMES OF RESPIRATORY SYNCYTIAL VIRUS

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) mo tifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequenti al transcription were analyzed with cDNA-encoded RNA analogs (minigeno mes) of nonsegmented negative-sense RSV genomic RNA, Two minigenomes w ere used, The monocistronic RSV-CAT minigenome consists of the chloram phenicol acetyltransferase (CAT) translational open reading frame (ORF ) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA, The dicistronic RSV-CAT-LU C minigenome is a derivative of RSV-CAT into which the ORF for lucifer ase (LUG), bordered by GS and GE motifs, was inserted downstream of th e CAT gene with an intergenic region positioned between the two genes, Each minigenome, was synthesized in vitro and transfected into RSV-in fected cells, where it was replicated and transcribed to yield the pre dicted polyadenylated subgenomic mRNA(s), The only RSV sequences requi red for efficient transcription and RNA replication were the 44-nucleo tide 3' leader region, the last 40 nucleotides of the 5' trailer regio n, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable trans cription signals which could be attached to foreign sequences to direc t their transcription into subgenomic mRNAs, Removal of the GS motif g reatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream positi on, Ablation of the promoter-proximal GS signal was not associated wit h increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV- CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUG mRNA. The latter result showed that a dow nstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal, This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry, R emoval of both the GS and GE signals of the upstream CAT gene in RSV-C AT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant, Remarkably, whe reas both genes were silent when the CAT GS and GE signals were both a bsent, restoration of the CAT CE signal alone restored a significant l evel (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequent ial transcription that,vas independent of the promoter-proximal GS sig nal and appeared to involve readthrough from the leader region.