Identification of a Single Amino Acid Change in the Human Respiratory Syncytial Virus L Protein That Affects Transcriptional Termination

Infectious Human respiratory syncytial virus (HRSV) with an aberrant RNA synthesis pattern was recovered from a cDNA clone. The virus displayed increased levels of polycistronic readthrough mRNAs resulting from failure of the polymerase to terminate transcription efficiently at the gene ends. An asparagine (N) to aspartic acid (D) change at amino acid 1049 in the large (L) polymerase protein was found to be responsible for the readthrough phenotype. Virus encoding N at position 1049 displayed an RNA synthesis pattern indistinguishable from the A2 strain of HRSV. We compared the transcriptional activities of polymerases that encoded an N or D at position 1049 (L1049N or L1049D) by using dicistronic subgenomic replicons and found that the alteration of amino acid 1049 specifically affected transcriptional termination but had no effect on genome replication. L1049N recognized and terminated transcription at each of the naturally occurring gene junctions with differing efficiencies but at significantly higher efficiency than L1049D. The abilities of the polymerases to recognize the cis-acting gene end signals required for termination were compared by examining the effect of single nucleotide changes at positions 1 to 8 of the M gene end and were found to be similar. This work identifies a single amino acid position in the polymerase that specifically affects the ability of the polymerase to terminate transcription, but which does not affect genome replication or interaction with the M2-1 protein. This work shows the critical importance of the previously defined cis-acting signals for termination, confirms differential termination at the varied gene junctions, and shows that the polymerase responds to the cis-acting sequences similarly, whether it has N or D at position 1049. Human respiratory syncytial virus (HRSV) is a nonsegmented negative-strand (NNS) RNA virus belonging to the Paramyxoviridae family in the order Mononegavirales. The HRSV genome contains 10 genes, in the order 3-NS1-NS2-N-P-M-SHG-F-M2-L-5, encoding at least 11 proteins (10, 11). The genes are flanked by noncoding leader (Le) and trailer (Tr) sequences at the 3 and 5 ends of the genome, respectively. The virally encoded RNA-dependent RNA polymerase (RdRp), containing the large (L) protein and the phosphoprotein (P), carries out genome replication and transcription (17, 47). Genome replication additionally requires the synthesis of nucleocapsid (N) protein for encapsidation of progeny genome RNA, which is essential for recognition by the RdRp (17, 47). In addition to the L and P proteins, efficient transcription of HRSV requires a further viral component, the M2-1 protein. Produced from the first open reading frame of the M2 gene, the M2-1 protein is a transcription factor that decreases intragenic and intergenic termination, allowing synthesis of fulllength mRNAs (9, 15, 18, 20). Available evidence supports a stop-start model of transcription for the Mononegavirales (5, 14, 24) in which the RdRp enters the genome at a single 3 site and transcribes each gene in an obligatorily sequential manner from 3 to 5, producing capped and polyadenylated mRNAs (13). The RdRp must terminate synthesis of each upstream mRNA to initiate syn