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 or de novo mechanism3. 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 replication4. 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.
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This work was supported by a Wellcome Trust grant 098721/Z/12/Z (to A.J.W.t.V.), a Netherlands Organization for Scientific Research (NWO) grant 825.11.029 (to A.J.W.t.V.), a Medical Research Council (MRC) grant MR/K000241/1 (to E.F.), a European Commission Seventh Framework Program grant FP7/2007-2013 HEALTH-F4-2008-201418 (to A.N.K.), a Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/J001694/1 (to A.N.K. and E.F.), and a Kemp postdoctoral fellowship from Lincoln College Oxford (to A.J.W.t.V.).
The authors declare no competing financial interests.
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te Velthuis, A., Robb, N., Kapanidis, A. et al. The role of the priming loop in influenza A virus RNA synthesis. Nat Microbiol 1, 16029 (2016). https://doi.org/10.1038/nmicrobiol.2016.29
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