Peer reviewer reviews are available. Data availability All data are available from the corresponding authors and/or included in the paper or Supplementary Information. 7NHX, EMD-12342), class 2a (PDB 7NHA, EMD-12322), class 2b (PDB 7NHC, EMD-12323), class 3 (PDB 7NI0, EMD-12348). Cryo-EM density maps with the corresponding atomic coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank with the following accession codes for the complexes between the 1918 polymerase heterotrimer and nanobodies Nb8189 (PDB 7NIK, EMD-12361), Nb8190 (PDB 7NIL, EMD-12362), Nb8191 (PDB 7NIR, EMD-12363), Nb8192 (PDB 7NIs usually, EMD-12364), Nb8196 (PDB 7NJ3, EMD-12371), Nb8198 (PDB 7NJ4, EMD-12372), Nb8199 (PDB 7NJ5, EMD-12373), Nb8200 (PDB 7NJ7, EMD-12375), Nb8201 (PDB 7NK1, EMD-12428), Nb8202 (PDB 7NK2, EMD-12429), Nb8203 (PDB 7NK4, EMD-12430), Nb8204 (PDB 7NK6, EMD-12431), Nb8205 (PDB 7NK8, EMD-12433), Nb8206 (PDB 7NKA, EMD-12435), Nb8207 (PDB 7NKC, EMD-12437), Nb8209 (PDB 7NKI, EMD-12440), Nb8210 (PDB 7NKR, EMD-12447).?Source data are provided with this paper. Abstract Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase Hydroxyurea which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral Rabbit Polyclonal to SYT13 and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites around the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at Hydroxyurea these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is usually highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development. values are as follows: for Nb8190 thanks Maria Rosenthal and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Data availability All data are available from the corresponding authors and/or included in the paper or Supplementary Information. Crystallographic coordinates and maps generated in this study have been deposited in the PDB with accession codes 7NFQ, 7NFR, and 7NFT for the Nb8193, Nb8194, Nb8208 complexes, respectively. Cryo-EM density Hydroxyurea maps with the corresponding atomic coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank with the following accession codes for Hydroxyurea the 1918 polymerase heterotrimer class 1 (PDB 7NHX, EMD-12342), class 2a Hydroxyurea (PDB 7NHA, EMD-12322), class 2b (PDB 7NHC, EMD-12323), class 3 (PDB 7NI0, EMD-12348). Cryo-EM density maps with the corresponding atomic coordinates have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank with the following accession codes for the complexes between the 1918 polymerase heterotrimer and nanobodies Nb8189 (PDB 7NIK, EMD-12361), Nb8190 (PDB 7NIL, EMD-12362), Nb8191 (PDB 7NIR, EMD-12363), Nb8192 (PDB 7NIs usually, EMD-12364), Nb8196 (PDB 7NJ3, EMD-12371), Nb8198 (PDB 7NJ4, EMD-12372), Nb8199 (PDB 7NJ5, EMD-12373), Nb8200 (PDB 7NJ7, EMD-12375), Nb8201 (PDB 7NK1, EMD-12428), Nb8202 (PDB 7NK2, EMD-12429), Nb8203 (PDB 7NK4, EMD-12430), Nb8204 (PDB 7NK6, EMD-12431), Nb8205 (PDB 7NK8, EMD-12433), Nb8206 (PDB 7NKA, EMD-12435), Nb8207 (PDB 7NKC, EMD-12437), Nb8209 (PDB 7NKI, EMD-12440), Nb8210 (PDB 7NKR, EMD-12447).?Source data are provided with this paper. Competing interests The authors declare no competing interests. Footnotes Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Jeremy R. Keown, Zihan Zhu, Lo?c Carrique, Haitian Fan. These authors jointly supervised this work: Ervin Fodor, Jonathan M. Grimes. Contributor Information Ervin Fodor, Email: ku.ca.xo.htap@rodof.nivre. Jonathan M. Grimes, Email: ku.ca.xo.iburts@nahtanoj. Supplementary information The online version contains supplementary material available at 10.1038/s41467-021-27950-w..