Multifunctional roles of the respiratory syncytial virus transcription and genome replication machinery
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Respiratory syncytial virus (RSV) is the leading cause of pediatric lower respiratory tract infection and a major cause of hospitalization. The RSV genome is a single strand negative-sense RNA and encodes a large RNA-dependent RNA polymerase (L). L is responsible for transcribing viral mRNAs and replicating viral genomes within inclusion bodies. The RSV L protein contains six regions conserved in other viruses (CRI-VI). CRV contains the enzymatic residues necessary for cap addition and forms part of a lobe surrounding the polymerization active site. Analysis of CRV with amino acid substitutions identified variants defective in transcription or replication. The inhibition of transcription was due to the inability of variants to co-transcriptionally cap mRNAs. The failure to produce replicative products was due to an inability of the variants to efficiently elongate the RNA within the promoter region. A small molecule that targets CRV also inhibited transcription and replication. In this case, the polymerase elongated an RNA generated from the promoter region past the first transcription start site. Thus, in addition to its role in capping, CRV modulates polymerase elongation properties to enable proper transcription and replication. The RSV M2-1 protein is an RNA binding protein that functions as an RSV transcription elongation factor. To understand at what point M2-1 becomes associated with the transcribing polymerase, we mapped points of M2-1:RNA interactions using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). M2-1 was found to contact nucleotides along each viral mRNA, indicating that M2-1 functions as a component of the polymerase, transiently associating with mRNA as it is synthesized. Additionally, we observed that there are two pools of M2-1. At early times in infection, both pools localize with the polymerase in cytoplasmic inclusions, but at later times, one has a diffuse cytoplasmic distribution while the other remains in inclusions. We showed that M2-1 binds specific cellular mRNAs as well as viral mRNAs. We conclude that in addition to its previously described role in transcription elongation, a subpopulation of M2-1 has an additional role involving cellular RNA interactions. Thus, the studies presented here highlight the multifunctional roles for the RSV transcription and replication machinery.