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-UCAAUUAU UUUUU-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.

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.

The genes of respiratory syncytial (RS) virus are transcribed sequentially by the viral RNA polymerase from a single 3'-proximal promoter. Polyadenylation and termination are directed by a sequence at the end of each gene, after which the polymerase crosses an intergenic region and reinitiates at the start sequence of the next gene. The 10 viral genes have different gene end sequences and different termination efficiencies, which allow for regulation of gene expression, since termination of each gene is required for initiation of the downstream gene. RNA sequences within the previously characterized 13 nucleotide gene end, including a conserved sequence 3'-UCAAU-5' and a tract of U residues, are important for termination. In this study, two additional sequence elements outside of the 13 nucleotide gene end were found to modulate termination efficiency: the A residue upstream of the 3'-UCAAU-5' sequence, and the first nucleotide of the intergenic region when it follows a U(4) tract.

Human respiratory syncytial virus (HRSV) has a single-stranded, negative-sense RNA genome with 10 genes encoding 11 proteins. Sequences at the beginning of the HRSV genes are highly conserved; however, the gene end sequences vary around a semiconserved consensus sequence, and the nontranscribed intergenic regions vary in both length and sequence. The regions at the junctions between HRSV genes (the gene end sequence of an upstream gene, intergenic region, and the gene start sequence of a downstream gene) contain elements required for efficient termination of the upstream gene and transcription of the downstream gene. Previous studies have examined variation in the HRSV coding sequences, but none have systematically analyzed the noncoding transcriptional control regions for variability. We determined the gene start and gene end sequences of each of the 10 HRSV genes from 14 clinical isolates for variations from the sequence of the prototype A2 strain. No changes were found in any of the gene start sequences. Eight of the 10 gene end sequences, however, contained variations. Several of these, a U(4)-tract instead of a U(6)- or U(5)-tract at the M and SH gene ends, respectively, (U(4)A) and an A-to-G change at position four in the G gene end (A4G), were predicted to affect termination and were examined for their effects on transcription. The changes were found to inhibit transcriptional termination, resulting in increased polycistronic readthrough and correspondingly reduced initiation of the downstream monocistronic mRNA. Viruses with the A4G variant G gene end sequence produced less F protein than those with A2-like G gene end sequences. Examination of additional G gene end sequences available in GenBank revealed that the observed A4G variation was restricted to one phylogenetic lineage of HRSV. All viruses examined within this lineage possessed this variant G gene end sequence. The data presented show that the gene end sequences of naturally occurring HRSV clinical isolates vary from those of the prototypic A2 strain and that certain of these changes inhibit efficient transcriptional termination and downstream gene expression.