Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus

The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon s ystem. The 3' ends of the genome and antigenome, which, respectively, conta in the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) r egions, should each contain a promoter for RNA replication. The 3' genome e nd also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3'-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when j uxtaposed with a transcription gene-start (GS) signal, this sequence was al so able to direct transcription. It was also shown that the region of Le im mediately preceding the GS signal of the first gene could be deleted with e ither no effect or with a slight decrease in transcription initiation. Thus , the TrC is competent to direct transcription even though it does not do s o in nature, and the partial sequence identity it shares with the 3' end of the genome likely represents the important elements of a conserved promote r active in both replication and transcription. Increasing the length of th e introduced TrC sequence incrementally to 147 nt resulted in a fourfold in crease in replication and a nearly complete inhibition of transcription. Th ese two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but ra ther are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enh ancing cis-acting element. In contrast, the inhibitory effect on transcript ion was due solely to the altered spacing between the 3' end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor t he inhibition of transcription was due to increased base-pairing potential between the 3' and 5' ends, The relative strengths of the Le and TrC promot ers for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity be tween the RSV antigenomic and genomic promoters, but provided a further dis tinction between promoter requirements for transcription and replication.