RNA sequences involved in transcriptional termination of respiratory syncytial virus.

RNA signals at the ends of the genes of respiratory syncytial (RS) virus direct polyadenylation and termination of viral transcription. These gene ends contain two conserved regions, a pentanucleotide and a tract of uridylate (U) residues, separated by an A/U-rich central region that is less well conserved. The U tract is thought to be the template for polyadenylation of viral mRNAs by reiterative transcription. The cis-acting requirements for termination were investigated by mutagenesis of the matrix (M) gene end (3'-UCAAUUAUUUUUU-5') in a dicistronic RNA replicon. Termination efficiencies were quantitated by intracellular metabolic labeling of monocistronic mRNAs and the dicistronic readthrough RNAs that result when termination fails to occur. All three regions of the gene end were necessary for termination. Mutation of each of the first 8 nucleotides of the M gene end to all other nucleotides showed that nucleotides 2 to 6 were important for termination and intolerant of change, whereas nucleotides 1 and 7 were tolerant of change. At position 8, A or U allowed termination, but G or C did not. Both the length and the position of the U tract were important for termination. U residues at positions 9 to 12 were necessary, while additional U residues at position 8, and especially position 13, enhanced termination efficiency. Altering the length of the central region abolished termination, suggesting that the position of the U tract with respect to the 3'-UCAAU-5' sequence was critical. The termination efficiencies of each of the 10 genes of RS virus are different. Since transcription is obligatorily sequential and termination of each gene is required for transcription of the next gene downstream, these differences may contribute to gene regulation. In agreement with our data, the naturally occurring gene ends of RS virus that terminate inefficiently have short U tracts or other sequence features that correlated with decreased termination when similar mutations were analyzed in RNA replicons.

Diverse gene junctions of respiratory syncytial virus modulate the efficiency of transcription termination and respond differently to M2-mediated antitermination.

The ability of the diverse gene junctions of respiratory syncytial (RS) virus to signal the termination of transcription was analyzed. Nine dicistronic subgenomic replicons of RS virus were constructed; each contained one of the RS virus gene junctions in its natural upstream and downstream sequence context. The RNA synthesis activities of these subgenomic replicons were analyzed in the absence and presence of the M2 protein, which we showed previously to function as a transcription antiterminator. Our data showed that the efficiency with which the polymerase terminated transcription was affected by the gene junction that it encountered. The M2 protein significantly decreased the efficiency of the termination of transcription, resulting in increased levels of readthrough transcription at all the gene junctions. The diverse gene junctions fell into three broad groups with respect to their ability to signal transcription termination. One group of gene junctions (NS1/NS2, NS2/N, M2/L, and L/trailer) showed inefficient termination in the absence or the presence of the M2 protein. A second group of gene junctions (N/P, P/M, M/SH, SH/G, and G/F) terminated transcription efficiently. The SH/G gene junction terminated transcription with the greatest efficiency and produced low levels of readthrough transcripts in the absence or the presence of the M2 protein, correlating with the absence of SH/G polycistronic transcripts in RS virus-infected cells. The F/M2 gene junction was particularly sensitive to the M2 protein: it efficiently signaled termination in the absence of the M2 protein but produced high levels of readthrough transcripts in the presence of the M2 protein. This result suggests that the M2 protein may regulate its own production by negative feedback. The data presented here show that the different gene junctions of RS virus do modulate RS virus transcription termination. The M2 protein reduced termination at all gene junctions. The magnitude of antitermination due to the M2 protein, however, varied at the different gene junctions. The data presented here indicate that the mechanism for the regulation of RS virus gene expression is more complex than was previously appreciated.