title: An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.
abstract: Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the 'promoter'). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.
authors.affiliation: Grenoble Outstation, European Molecular Biology Laboratory, Grenoble 38042, France.|Unit of Virus-Host Cell Interactions, EMBL-UGA-CNRS, Grenoble 38042, France.
authors.forename: Stefan
authors.initials: S
authors.lastname: Reich
authors.affiliation: Unit of Virus-Host Cell Interactions, EMBL-UGA-CNRS, Grenoble 38042, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Guilligay
authors.affiliation: Grenoble Outstation, European Molecular Biology Laboratory, Grenoble 38042, France.|Unit of Virus-Host Cell Interactions, EMBL-UGA-CNRS, Grenoble 38042, France.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
mesh_terms: D020029:Base Pairing; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D009981:Influenza B virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases