The role of the priming loop inInfluenza A virusRNA synthesis.
RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1. They adopt a closed, right-handed fold with conserved subdomains called palm, fingers, and thumb1,2. Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs, and magnesium ions to facilitate nucleotide condensation1. For the initiation of RNA synthesis, most RdRps use either a primer-dependent orde novomechanism3. TheInfluenza A virusRdRp in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internalde novoinitiation for replication4. To understand how theInfluenza A virusRdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation, and single-molecule FRET assays. Our data shows that this β-hairpin is essential for terminal initiation during replication, but auxiliary 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 synthesisin vitroand in cell culture. Overall, this work advances our understanding ofInfluenza A virusRNA synthesis and identifies the initiation platform of viral replication.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, Oxford, United Kingdom; Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, OX1 3PU, United Kingdom.
Aartjan J W
AJ
Te Velthuis
Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, OX1 3PU, United Kingdom.
Nicole C
NC
Robb
Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, OX1 3PU, United Kingdom.
Achillefs N
AN
Kapanidis
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE, Oxford, United Kingdom.
Ervin
E
Fodor
Influenza virus; RNA-dependent RNA polymerase; priming loop; replication; transcription