The Respiratory Syncytial Virus Polymerase Has Multiple RNA Synthesis Activities at the Promoter

Abstract

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection. Respiratory syncytial virus (RSV) is a major pathogen of infants with the potential to cause severe respiratory disease. RSV has an RNA genome and one approach to developing a drug against this virus is to gain a greater understanding of the mechanisms used by the viral polymerase to generate new RNA. In this study we developed a novel assay for examining how the RSV polymerase interacts with a specific promoter sequence at the end of an RNA template, and performed analysis of RSV RNA produced in infected cells to confirm the findings. Our experiments showed that the behavior of the polymerase on the promoter was surprisingly complex. We found that not only could the polymerase initiate synthesis of progeny genome RNA from an initiation site at the end of the template, but it could also generate another small RNA from a second initiation site. In addition, we showed that the polymerase could add additional RNA sequence to the template promoter, which affected its ability to initiate RNA synthesis. These findings extend our understanding of the functions of the promoter, and suggest a mechanism by which RNA synthesis from the promoter is regulated.