title: Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase.
abstract: Respiratory syncytial virus RNA dependent RNA polymerase (RdRp) initiates RNA synthesis from the leader (le) and trailer-complement (trc) promoters. The RdRp can also add nucleotides to the 3' end of the trc promoter by back-priming, but there is no evidence this occurs at the le promoter in infected cells. We examined how environmental factors and RNA sequence affect de novo RNA synthesis versus back-priming using an in vitro assay. We found that replacing Mg(2+) with Mn(2+) in the reaction buffer increased de novo initiation relative to back-priming, and different lengths of trc sequence were required for the two activities. Experiments with le RNA showed that back-priming occurred with this sequence in vitro, but less efficiently than with trc RNA. These findings indicate that during infection, the RdRp is governed between de novo RNA synthesis and back-priming by RNA sequence and environment, including a factor missing from the in vitro assay.
authors.affiliation: Department of Microbiology, Boston University Medical Campus, 72 East Concord Street, Boston, MA 02118, USA. Electronic address: slnoton@bu.edu.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Noton
authors.affiliation: Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, ic2 Building, Liverpool L3 5RF, UK. Electronic address: w.aljabr@liverpool.ac.uk.
authors.forename: Waleed
authors.initials: W
authors.lastname: Aljabr
authors.affiliation: Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, ic2 Building, Liverpool L3 5RF, UK; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, ic2 Building, Liverpool L3 5RF, UK. Electronic address: julian.hiscox@liverpool.ac.uk.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Hiscox
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK. Electronic address: d.a.matthews@bristol.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Matthews
authors.affiliation: Department of Microbiology, Boston University Medical Campus, 72 East Concord Street, Boston, MA 02118, USA. Electronic address: rfearns@bu.edu.
authors.forename: Rachel
authors.initials: R
authors.lastname: Fearns
mesh_terms: D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000201:enzymology*; D014158:Transcription, Genetic
chemical_list: D002413:Cations, Divalent; D003067:Coenzymes; D012367:RNA, Viral; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase; D008274:Magnesium
keywords: Back-priming; Mononegavirales; Paramyxovirus; RNA dependent RNA polymerase; RNA synthesis; Respiratory syncytial virus