title: The role of the priming loop in influenza A virus RNA synthesis.
abstract: RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes(1). They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb(1,2). Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation(1). For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism(3). The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication(4). To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.
authors: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.|Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors: Aartjan J W
authors: http://orcid.org/0000-0002-5129-3953
authors: AJ
authors: Te Velthuis
authors: Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors: Nicole C
authors: NC
authors: Robb
authors: Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.
authors: Achillefs N
authors: AN
authors: Kapanidis
authors: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors: Ervin
authors: E
authors: Fodor
mesh_terms: D004252:DNA Mutational Analysis; D009980:Influenza A virus / Q000201:enzymology* / Q000378:metabolism*; D011487:Protein Conformation; D000072417:Protein Domains; D012367:RNA, Viral / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1