in a self-stalled elongation state were then subjected to a last non-uniform refinement. Post-processing was performed in Relion 4.0 using a manually determined B-factor. Reported global resolution is based on the FSC 0.143 cut-off criteria. Local resolution variations were estimated in Relion 4.0. Detailed image processing information is shown in Fig. S9.

For sample 3, all remaining particles were subjected to an 'Heterogeneous refinement' job, with three symmetrical dimers and three monomers as initial 3D models. Particles from well-resolved dimeric 3D classes were subjected to a final non-uniform refinement. Post-processing was performed in Relion 4.0 using an automatically determined B-factor. Reported global resolution is based on the FSC 0.143 cut-off criteria. Local resolution variations were estimated in Relion 4.0. Detailed image processing information is shown in Fig. S10.

## Model building and refinement

Using the FluPol Zhejiang-H7N9 elongation complex as starting point (PDB: 7QTL)⁴³, atomic models were constructed by iterative rounds of manual model building with COOT (version 0.9.8) and real-space refinement using Phenix (version 1.20.1)⁸³. Validation was performed using Phenix (version 1.20.1). Model resolution according to the cryo-EM map was estimated at the 0.5 FSC cutoff. Figures were generated using ChimeraX (version 1.6.1)⁸⁴.

## Statistics

Statistics were performed with the GraphPad Prism software (Version 9.5.1 (528)), as indicated in the figure legends.

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

The coordinates and EM maps generated in this study have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank: Influenza A/Zhejiang-H7N9 polymerase (4 M mutant) in replicase-like conformation in pre-initiation state with RNAP II pS5 CTD peptide mimic bound in site 1A/2A, PDB 8PMO and EMD-17755; Influenza A/Zhejiang-H7N9 polymerase (4 M mutant) in pre-initiation state with continuous RNAP II pS5 CTD peptide mimic bound in site 1A/2A PDB 8PNP and EMD-17782; Influenza A/Zhejiang-H7N9 polymerase (4 M mutant) in elongation state with continuous RNAP II pS5 CTD peptide mimic bound in site 1A/2A PDB 8PNQ and EMD-17783; Influenza A/Zhejiang-H7N9 polymerase (PA K289A+C489R) symmetric dimer bound to the promoter PDB 8POH and EMD-17792. Influenza A/H7N9 polymerase in pre-initiation state, intermediate conformation (I) with PB2-C(I), ENDO(T), and Pol II pS5 CTD peptide mimic bound in site 1A/2A PDB 8R3L and EMD-18872; Influenza A/H7N9 polymerase in self-stalled pre-termination state, with Pol II pS5 CTD peptide mimic bound in site 1A/2A PDB 8R3K and EMD-18871. The NGS raw reads generated in this study have been deposited in the European nucleotide archive under accession code ERP149587 [https://www.ebi.ac.uk/ena/browse/view/PRJEB64419]. The sequence files of plasmids used in this study have been deposited in the Zenodo repository [https://doi.org/10.5281/zenodo.10462746]. The raw data generated in this study are provided in the Source Data File. Source data are provided with this paper.

## References

1. Krammer, F. et al. Influenza. *Nat. Rev. Dis. Prim.* **4**, 3 (2018).

2. Harrington, W. N., Kackos, C. M. & Webby, R. J. The evolution and future of influenza pandemic preparedness. *Exp. Mol. Med.* **53**, 737-749 (2021).

3. Mifsud, E. J., Hayden, F. G. & Hurt, A. C. Antivirals targeting the polymerase complex of influenza viruses. *Antivir. Res.* **169**, 1045-1054 (2019).

4. Wandzik, J. M., Kouba, T. & Cusack, S. Structure and function of influenza polymerase. *Cold Spring Harb. Perspect. Med.* **11**, a038372 (2021).

5. Eisfeld, A. J., Neumann, G. & Kawaoka, Y. At the centre: influenza A virus ribonucleoproteins. *Nat. Rev. Microbiol.* **13**, 28–41 (2015).

6. Plotch, S. J., Bouloy, M. & Krug, R. M. Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro. *Proc. Natl Acad. Sci. USA* **76**, 1618–1622 (1979).

7. Deng, T., Vreede, F. T. & Brownlee, G. G. Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication. *J. Virol.* **80**, 2337–2348 (2006).

8. Dias, A. et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. *Nature* **458**, 914–918 (2009).

9. Guilligay, D. et al. The structural basis for cap binding by influenza virus polymerase subunit PB2. *Nat. Struct. Mol. Biol.* **15**, 500–506 (2008).

10. Kouba, T., Drncová, P. & Cusack, S. Structural snapshots of actively transcribing influenza polymerase. *Nat. Struct. Mol. Biol.* **26**, 460–470 (2019).

11. Pflug, A. et al. Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors. *Nucleic Acids Res.* **46**, 956–971 (2018).

12. Wandzik, J. M. et al. A structure-based model for the complete transcription cycle of influenza polymerase. *Cell* **181**, 877–893.e21 (2020).

13. Carrique, L. et al. Host ANP32A mediates the assembly of the influenza virus replicase. *Nature* **587**, 638–643 (2020).

14. Keown, J. R. et al. Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies. *Nat. Commun.* **13**, 251 (2022).

15. Wang, F. et al. The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome. *Nucleic Acids Res.* **50**, 5713–5725 (2022).

16. Camacho-Zarco, A. R. et al. Multivalent dynamic colocalization of avian influenza polymerase and nucleoprotein by intrinsically disordered ANP32A reveals the molecular basis of human adaptation. *J. Am. Chem. Soc.* **145**, 20985–21001 (2023).

17. Poon, L. L., Pritlove, D. C., Fodor, E. & Brownlee, G. G. Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. *J. Virol.* **73**, 3473–3476 (1999).

18. Zhu, Z., Fodor, E. & Keown, J. R. A structural understanding of influenza virus genome replication. *Trends Microbiol.* **31**, 308–319 (2023).

19. Oymans, J. & Te Velthuis, A. J. W. A mechanism for priming and realignment during influenza A virus replication. *J. Virol.* **92**, e01773–17 (2018).

20. Zhang, S., Wang, J., Wang, Q. & Toyoda, T. Internal initiation of influenza virus replication of viral RNA and complementary RNA in vitro. *J. Biol. Chem.* **285**, 41194–41201 (2010).

21. York, A., Hengrung, N., Vreede, F. T., Huiskonen, J. T. & Fodor, E. Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus. *Proc. Natl Acad. Sci. USA* **110**, E4238–E4245 (2013).

22. Krischuns, T. et al. Type B and type A influenza polymerases have evolved distinct binding interfaces to recruit the RNA polymerase II CTD. *PLoS Pathog.* **18**, e1010328 (2022).

23. Lukarska, M. et al. Structural basis of an essential interaction between influenza polymerase and Pol II CTD. *Nature* **541**, 117–121 (2017).

24. Rialdi, A. et al. The RNA exosome syncs IAV-RNAPII transcription to promote viral ribogenesis and infectivity. *Cell* **169**, 679–692.e14 (2017).
in a self-stalled elongation state were then subjected to a last non-uniform refinement. Post-processing was performed in Relion 4.0 using a manually determined B-factor. Reported global resolution is based on the FSC 0.143 cut-off criteria. Local resolution variations were estimated in Relion 4.0. Detailed image processing information is shown in Fig. S9.

For sample 3, all remaining particles were subjected to an 'Heterogeneous refinement' job, with three symmetrical dimers and three monomers as initial 3D models. Particles from well-resolved dimeric 3D classes were subjected to a final non-uniform refinement. Post-processing was performed in Relion 4.0 using an automatically determined B-factor. Reported global resolution is based on the FSC 0.143 cut-off criteria. Local resolution variations were estimated in Relion 4.0. Detailed image processing information is shown in Fig. S10.

## Model building and refinement

Using the FluPol Zhejiang-H7N9 elongation complex as starting point (PDB: 7QTL)⁴³, atomic models were constructed by iterative rounds of manual model building with COOT (version 0.9.8) and real-space refinement using Phenix (version 1.20.1)⁸³. Validation was performed using Phenix (version 1.20.1). Model resolution according to the cryo-EM map was estimated at the 0.5 FSC cutoff. Figures were generated using ChimeraX (version 1.6.1)⁸⁴.

## Statistics

Statistics were performed with the GraphPad Prism software (Version 9.5.1 (528)), as indicated in the figure legends.

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

The coordinates and EM maps generated in this study have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank: Influenza A/Zhejiang-H7N9 polymerase (4 M mutant) in replicase-like conformation in pre-initiation state with RNAP II pS5 CTD peptide mimic bound in site 1A/2A, PDB 8PMO and EMD-17755; Influenza A/Zhejiang-H7N9 polymerase (4 M mutant) in pre-initiation state with continuous RNAP II pS5 CTD peptide mimic bound in site 1A/2A PDB 8PNP and EMD-17782; Influenza A/Zhejiang-H7N9 polymerase (4 M mutant) in elongation state with continuous RNAP II pS5 CTD peptide mimic bound in site 1A/2A PDB 8PNQ and EMD-17783; Influenza A/Zhejiang-H7N9 polymerase (PA K289A+C489R) symmetric dimer bound to the promoter PDB 8POH and EMD-17792. Influenza A/H7N9 polymerase in pre-initiation state, intermediate conformation (I) with PB2-C(I), ENDO(T), and Pol II pS5 CTD peptide mimic bound in site 1A/2A PDB 8R3L and EMD-18872; Influenza A/H7N9 polymerase in self-stalled pre-termination state, with Pol II pS5 CTD peptide mimic bound in site 1A/2A PDB 8R3K and EMD-18871. The NGS raw reads generated in this study have been deposited in the European nucleotide archive under accession code ERP149587 [https://www.ebi.ac.uk/ena/browse/view/PRJEB64419]. The sequence files of plasmids used in this study have been deposited in the Zenodo repository [https://doi.org/10.5281/zenodo.10462746]. The raw data generated in this study are provided in the Source Data File. Source data are provided with this paper.

## References

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3. Mifsud, E. J., Hayden, F. G. & Hurt, A. C. Antivirals targeting the polymerase complex of influenza viruses. *Antivir. Res.* **169**, 1045-1054 (2019).

4. Wandzik, J. M., Kouba, T. & Cusack, S. Structure and function of influenza polymerase. *Cold Spring Harb. Perspect. Med.* **11**, a038372 (2021).

5. Eisfeld, A. J., Neumann, G. & Kawaoka, Y. At the centre: influenza A virus ribonucleoproteins. *Nat. Rev. Microbiol.* **13**, 28–41 (2015).

6. Plotch, S. J., Bouloy, M. & Krug, R. M. Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro. *Proc. Natl Acad. Sci. USA* **76**, 1618–1622 (1979).

7. Deng, T., Vreede, F. T. & Brownlee, G. G. Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication. *J. Virol.* **80**, 2337–2348 (2006).

8. Dias, A. et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. *Nature* **458**, 914–918 (2009).

9. Guilligay, D. et al. The structural basis for cap binding by influenza virus polymerase subunit PB2. *Nat. Struct. Mol. Biol.* **15**, 500–506 (2008).

10. Kouba, T., Drncová, P. & Cusack, S. Structural snapshots of actively transcribing influenza polymerase. *Nat. Struct. Mol. Biol.* **26**, 460–470 (2019).

11. Pflug, A. et al. Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors. *Nucleic Acids Res.* **46**, 956–971 (2018).

12. Wandzik, J. M. et al. A structure-based model for the complete transcription cycle of influenza polymerase. *Cell* **181**, 877–893.e21 (2020).

13. Carrique, L. et al. Host ANP32A mediates the assembly of the influenza virus replicase. *Nature* **587**, 638–643 (2020).

14. Keown, J. R. et al. Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies. *Nat. Commun.* **13**, 251 (2022).

15. Wang, F. et al. The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome. *Nucleic Acids Res.* **50**, 5713–5725 (2022).

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19. Oymans, J. & Te Velthuis, A. J. W. A mechanism for priming and realignment during influenza A virus replication. *J. Virol.* **92**, e01773–17 (2018).

20. Zhang, S., Wang, J., Wang, Q. & Toyoda, T. Internal initiation of influenza virus replication of viral RNA and complementary RNA in vitro. *J. Biol. Chem.* **285**, 41194–41201 (2010).

21. York, A., Hengrung, N., Vreede, F. T., Huiskonen, J. T. & Fodor, E. Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus. *Proc. Natl Acad. Sci. USA* **110**, E4238–E4245 (2013).

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39. Chen, K.-Y., Santos Afonso, E. D., Enouf, V., Isel, C. & Naffakh, N. Influenza virus polymerase subunits co-evolve to ensure proper levels of dimerization of the heterotrimer. *PLoS Pathog.* **15**, e1008034 (2019).

40. Chang, S. et al. Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution. *Mol. Cell* **57**, 925–935 (2015).

41. Fan, H. et al. Structures of influenza A virus RNA polymerase offer insight into viral genome replication. *Nature* **573**, 287–290 (2019).

42. Sheppard, C. M. et al. An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization. *Nat. Commun.* **14**, 6135 (2023).

43. Kouba, T. et al. Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106. *Cell Rep.* **42**, 111901 (2023).

44. Chen, Y. et al. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. *Lancet* **381**, 1916–1925 (2013).

45. Li, H. et al. An intermediate state allows influenza polymerase to switch smoothly between transcription and replication cycles. *Nat. Struct. Mol. Biol.* **30**, 1183–1192 (2023).

46. Thierry, E. et al. Influenza polymerase can adopt an alternative configuration involving a radical repacking of PB2 domains. *Mol. Cell* **61**, 125–137 (2016).

47. Vreede, F. T. & Brownlee, G. G. Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro. *J. Virol.* **81**, 2196–2204 (2007).

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## Acknowledgements

We thank Ron Fouchier (Erasmus MC Department of Viroscience, Netherlands), George Brownlee (Oxford University), Bernard Delmas (INRAE, France), Robert-Jan Lebbink (Utrecht University, Netherlands) and Sandie Munier (Institut Pasteur, France) for sharing plasmids and antibodies. We thank Martin Pelosse for support in using the Eukaryotic Expression Facility at EMBL Grenoble; Aldo R. Camacho-Zarco and Martin Blackledge for providing huANP32A truncated constructs proteins; Daphne Walter and Vladimir Benes from the EMBL GeneCore facility for the next generation sequencing data; Wojtek Galej, Sarah Schneider and Romain Linares for access to the Glacios at EMBL Grenoble; Guy Schoehn and Eleftherios Zarkadas for access to the Glacios at IBS Grenoble; Aymeric Peuch for support with using the joint EMBL-IBS computer clusters. We acknowledge the European Synchrotron

Radiation Facility and PSB for access to the Titan Krios CM01, and Michael Hons for assistance with data collection. We thank the Pasteur International Bioresources Network (PIBNet) for genome sequencing of recombinant viruses. This work was funded by the ANR grant FluTranscript (ANR-18-CE11-0028, S.C. and N.N.), the ANR LabEx IBEID (ANR-10-LABX-62-IBEID, N.N. and T.K.), and the German Research Foundation (DFG) grant SFB-TR84 (project B2, T.W.). This work used the platforms at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

## Author contributions

TK, SC and NN conceived the project. TK and BA performed most of the experiments. CI performed serial passaging of mutant viruses. SP provided research assistance. MB and TW generated and provided essential reagents. TK, BA, CI, SC and NN oversaw and interpreted data. TK, BA, SC and NN wrote the manuscript with input from all authors. All authors read and approved the final manuscript.

## Competing interests

The authors declare no competing interests.

## Additional information

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-45205-2.

**Correspondence** and requests for materials should be addressed to Tim Krischuns, Stephen Cusack or Nadia Naffakh.

**Peer review information** *Nature Communications* thanks Alex Chin, Haitian Fan and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

**Reprints and permissions information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024
69. Zheng, L., Baumann, U. & Reymond, J.-L. An efficient one-step site-directed and site-saturation mutagenesis protocol. *Nucleic Acids Res.* **32**, e115 (2004).

70. van Diemen, F. R. et al. CRISPR/Cas9-mediated genome editing of herpesviruses limits productive and latent infections. *PLoS Pathog.* **12**, e1005701 (2016).

71. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. *Methods* **25**, 402–408 (2001).

72. Krischuns, T. et al. Phosphorylation of TRIM28 enhances the expression of IFN-β and proinflammatory cytokines during HPAIV Infection of human lung epithelial cells. *Front. Immunol.* **9**, 2229 (2018).

73. Da Costa, B. et al. Temperature-sensitive mutants in the influenza A virus RNA polymerase: alterations in the PA linker reduce nuclear targeting of the PB1-PA dimer and result in viral attenuation. *J. Virol.* **89**, 6376–6390 (2015).

74. Matrosovich, M., Matrosovich, T., Garten, W. & Klenk, H.-D. New low-viscosity overlay medium for viral plaque assays. *Virol. J.* **3**, 63 (2006).

75. Watson, S. J. et al. Viral population analysis and minority-variant detection using short read next-generation sequencing. *Philos. Trans. R. Soc. Lond. B Biol. Sci.* **368**, 20120205 (2013).

76. Sjaastad, L. E. et al. Distinct antiviral signatures revealed by the magnitude and round of influenza virus replication in vivo. *Proc. Natl Acad. Sci. USA* **115**, 9610–9615 (2018).

77. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. *Nat. Methods* **9**, 676–682 (2012).

78. Camacho-Zarco, A. R. et al. Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A. *Nat. Commun.* **11**, 3656 (2020).

79. Huyton, T. & Wolberger, C. The crystal structure of the tumor suppressor protein pp32 (Anp32a): structural insights into Anp32 family of proteins. *Protein Sci.* **16**, 1308–1315 (2007).

80. Kechin, A., Boyarskikh, U., Kel, A. & Filipenko, M. cutPrimers: a new tool for accurate cutting of primers from reads of targeted next generation sequencing. *J. Comput Biol.* **24**, 1138–1143 (2017).

81. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. *Nat. Methods* **14**, 331–332 (2017).

82. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoS-PARC: algorithms for rapid unsupervised cryo-EM structure determination. *Nat. Methods* **14**, 290–296 (2017).

83. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. *Acta Crystallogr D. Struct. Biol.* **74**, 531–544 (2018).

84. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. *Protein Sci.* **27**, 14–25 (2018).

## Acknowledgements

We thank Ron Fouchier (Erasmus MC Department of Viroscience, Netherlands), George Brownlee (Oxford University), Bernard Delmas (INRAE, France), Robert-Jan Lebbink (Utrecht University, Netherlands) and Sandie Munier (Institut Pasteur, France) for sharing plasmids and antibodies. We thank Martin Pelosse for support in using the Eukaryotic Expression Facility at EMBL Grenoble; Aldo R. Camacho-Zarco and Martin Blackledge for providing huANP32A truncated constructs proteins; Daphne Walter and Vladimir Benes from the EMBL GeneCore facility for the next generation sequencing data; Wojtek Galej, Sarah Schneider and Romain Linares for access to the Glacios at EMBL Grenoble; Guy Schoehn and Eleftherios Zarkadas for access to the Glacios at IBS Grenoble; Aymeric Peuch for support with using the joint EMBL-IBS computer clusters. We acknowledge the European Synchrotron

Radiation Facility and PSB for access to the Titan Krios CM01, and Michael Hons for assistance with data collection. We thank the Pasteur International Bioresources Network (PIBNet) for genome sequencing of recombinant viruses. This work was funded by the ANR grant FluTranscript (ANR-18-CE11-0028, S.C. and N.N.), the ANR LabEx IBEID (ANR-10-LABX-62-IBEID, N.N. and T.K.), and the German Research Foundation (DFG) grant SFB-TR84 (project B2, T.W.). This work used the platforms at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

## Author contributions

TK, SC and NN conceived the project. TK and BA performed most of the experiments. CI performed serial passaging of mutant viruses. SP provided research assistance. MB and TW generated and provided essential reagents. TK, BA, CI, SC and NN oversaw and interpreted data. TK, BA, SC and NN wrote the manuscript with input from all authors. All authors read and approved the final manuscript.

## Competing interests

The authors declare no competing interests.

## Additional information

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-45205-2.

**Correspondence** and requests for materials should be addressed to Tim Krischuns, Stephen Cusack or Nadia Naffakh.

**Peer review information** *Nature Communications* thanks Alex Chin, Haitian Fan and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

**Reprints and permissions information** is available at http://www.nature.com/reprints

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2024