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Host ANP32A mediates the assembly of the influenza virus replicase

Nature volume 587, pages 638–643 (2020) Cite this article

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Abstract

Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge1. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes2,3. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity4. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.

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Fig. 1: Structures of dimers of FluPolC heterotrimers with or without bound ANP32A.
The alternative text for this image may have been generated using AI.
Fig. 2: FluPolC–FluPolC and ANP32A–FluPolC interaction interfaces.
The alternative text for this image may have been generated using AI.
Fig. 3: Interaction of ANP32LCAR with FluPolC and the effect of ANP32A on FluPolA activity.
The alternative text for this image may have been generated using AI.
Fig. 4: Functional implications of the FluPolC–ANP32A complex.
The alternative text for this image may have been generated using AI.

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Data availability

All data are available from the corresponding authors and/or included in the manuscript or Supplementary Information. Cryo-EM density maps with the corresponding atomic coordinates have been deposited in the Electron Microscopy Data Bank with accession codes EMD-10665 (FluPolC–huANP32A subclass 1), EMD-10667 (FluPolC–huANP32A subclass 2), EMD-10666 (FluPolC–chANP32A subclass 1), EMD-10659 (FluPolC–chANP32A subclass 2), EMD-10662 (FluPolC–chANP32A subclass 3) and EMD-10664 (FluPolC–chANP32A subclass 4), and the Protein Data Bank with accession codes 6XZQ (FluPolC–huANP32A subclass 1), 6Y0C (FluPolC-huANP32A subclass 2), 6XZR (FluPolC–chANP32A subclass 1), 6XZD (FluPolC–chANP32A subclass 2), 6XZG (FluPolC–chANP32A subclass 3) and 6XZP (FluPolC–chANP32A subclass 4).

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Acknowledgements

We thank N. Naffakh, P.-C. Shaw, G. G. Brownlee and F. Vreede for plasmids; I. Berger for the MultiBac system; D. Karia, A. Howe and D. Clare for assistance with cryo-EM; and G. G. Brownlee and members of the Fodor and Grimes laboratories for helpful comments and discussions. This work was supported by Medical Research Council (MRC) programme grant MR/R009945/1 (to E.F.), Wellcome Investigator Awards 200835/Z/16/Z (to J.M.G.) and 205100/Z/16/Z (to W.S.B.), MRC Studentship (to A.P.W.) and Imperial College President’s PhD Scholarship (to E.S.). We thank Diamond Light source for access and support of the cryo-EM facilities at the UK national Electron Bio-Imaging Centre (eBIC) (proposal EM20223), funded by the Wellcome, MRC and BBSRC. Further electron microscopy provision was provided through the OPIC electron microscopy facility, which was founded by a Wellcome JIF award (060208/Z/00/Z) and is supported by a Wellcome equipment grant (093305/Z/10/Z). Computation was performed at the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Part of this work was supported by Wellcome administrative support grant (203141/Z/16/Z).

Author information

Author notes

  1. Ecco Staller

    Present address: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

  2. These authors contributed equally: Loïc Carrique, Haitian Fan, Alexander P. Walker, Jeremy R. Keown

  3. These authors jointly supervised this work: Jonathan M. Grimes, Ervin Fodor

Authors and Affiliations

  1. Division of Structural Biology, University of Oxford, Oxford, UK

    Loïc Carrique, Jeremy R. Keown & Jonathan M. Grimes

  2. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Haitian Fan, Alexander P. Walker, Jane Sharps & Ervin Fodor

  3. Section of Molecular Virology, Imperial College London, London, UK

    Ecco Staller & Wendy S. Barclay

  4. Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, UK

    Jonathan M. Grimes

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Contributions

L.C., H.F., A.P.W., J.R.K., E.F. and J.M.G. conceived and designed the study. H.F., L.C. and J.R.K. carried out cloning of recombinant baculoviruses and protein purification, collected and processed electron microscopy data and built and refined models. A.P.W. and J.S. performed functional assays and analysed data. E.S. and W.S.B. provided plasmids and cell lines. J.M.G. and E.F. supervised the structural and functional studies, respectively. L.C., H.F., A.P.W., J.R.K., E.F. and J.M.G. wrote the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Ervin Fodor or Jonathan M. Grimes.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Julien Lescar, Yi Shi, Xiu-Feng Wan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 FluPolC activity depends on ANP32A and alignment of ANP32 proteins.

a, b, Luciferase reporter gene activities reflecting FluPolC activity in control (a) and dKO (b) eHAP cells in the presence or absence of overexpressed huANP32A, huANP32B or chANP32A. Data are presented as mean values ± s.e.m. n = 3 biologically independent samples from n = 3 independent experiments. Ordinary one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. P < 0.05 is considered significant to reject the null hypothesis. c, Sequence alignment of huANP32A, huANP32B, chANP32A, chANP32B. Residues involved in hydrogen bonding interactions with FluPolC are indicated in orange. The chANP32A avian-specific 33 amino acid insertion is highlighted in cyan. The SUMO interaction motif (SIM) sequence is indicated by black triangles. The figure was prepared with Espript 3.060.

Extended Data Fig. 2 Data collection, processing and analysis scheme.

a, b, Flowchart for the processing and the classification of the FluPolC-huANP32A complex (a) and FluPolC-chANP32A complex (b).

Extended Data Fig. 3 Single-particle cryo-EM analysis of FluPolC-huANP32A and FluPolC-chANP32A complexes.

a, e, Representative micrograph of FluPolC-huANP32A (a) and FluPolC-chANP32A (e) embedded in vitreous ice. Scale bar, 200 Å. b, f, Representative 2D class averages of FluPolC-huANP32A (b) and FluPolC-chANP32A (f). c, d, Data analysis for FluPolC-huANP32A Subclass1 (c) and Subclass2 (d). 3D reconstruction locally filtered and coloured according to RELION local resolution (left panel). FSC curve indicating overall map resolution and model-to-map FSC (middle panel). Curves are shown for phase randomization, unmasked, masked and phase-randomization-corrected masked maps. Angular distribution of particle projections with the cryo-EM map shown in grey (right panel). g–j, Data analysis for FluPolC-chANP32A Subclass1 (g), Subclass2 (h), Subclass3 (i) and Subclass4 (j). 3D reconstruction locally filtered and coloured according to RELION local resolution (top panel). FSC curve indicating overall map resolution and the model-to-map FSC (middle panel). Curves are shown for phase randomization, unmasked, masked and phase-randomization-corrected masked maps. Angular distribution of particle projections with the cryo-EM map shown in grey (bottom panel).

Extended Data Fig. 4 Comparison of FluPolR and FluPolE structures with the transcriptase and apo conformations of FluPol.

a–d, Comparison of structures of human influenza A/NT/60/68 (H3N2) bound to vRNA and capped RNA in the transcriptase conformation (PDB: 6RR7) (a) and human influenza C/Johannesburg/1/66 in the apo conformation (PDB: 5D98) (b) with structures of FluPolR (c) and FluPolE (d) in the FluPolC-chANP32A complex. e–h, Comparison of the PB2 domain arrangements in the complexes shown in a–d.

Extended Data Fig. 5 Close-up view of the interaction of 5′ and 3′ vRNA termini with FluPolR.

a, c, Close-up view of the 3′ vRNA pointing towards the active site in the FluPolC-huANP32A (a) and FluPolC-chANP32A (c) structures. b, d, Close-up view of the 3′ vRNA binding in a groove located between P3CTD and the PB1thumb and PB2N1 subdomains in the FluPolC-huANP32A (b) and FluPolC-chANP32A (d) structures.

Extended Data Fig. 6 Effect of FluPolR-FluPolE dimer interface mutations on FluPolA activity.

a, b, Effect of mutations at the FluPolR-FluPolE dimer interface on FluPolA activity in viral minigenome assays (a) and cRNA encapsidation by FluPolA (b). Data are presented as mean values ± s.e.m. n = 3 biologically independent samples from n = 3 independent experiments. Ordinary one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. P < 0.05 is considered significant to reject the null hypothesis. Western blot analyses were repeated from n = 3 independent experiments with similar results. For gel source data, see Supplementary Fig. 2.

Extended Data Fig. 7 Effect of FluPolA mutations at the FluPolA-ANP32A interface on FluPolA activity and interaction with huANP32A.

a, b, Effect of FluPolA mutations at the FluPolA-ANP32A interface on FluPolA activity in viral minigenome assays on (a) and FluPolA-ANP32A interaction (b). Data are presented as mean values ± s.e.m. n = 3 biologically independent samples from n = 3 independent experiments. Ordinary one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. P < 0.05 is considered significant to reject the null hypothesis. Western blot analyses were repeated from n = 3 independent experiments with similar results. For gel source data, see Supplementary Fig. 2.

Extended Data Fig. 8 Structural comparison of PB2627 domains of FluPolA and FluPolC.

Structures of the PB2627 domains from crystal structures of FluPol from influenza C/Johannesburg/1/1966 (a, PDB ID: 5D98) and A/NT/60/1968 (H3N2) (b, PDB ID: 6QNW) viruses are aligned and shown in cartoon mode. Residues discussed in this study are highlighted in stick mode and coloured in orange.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Figure (download PDF )

Supplementary Fig. 1 Sequence alignment of FluPolA and FluPolC. a - c, Sequence alignment of FluPol subunits (a, PA/P3; b, PB1; c, PB2) from influenza A/WSN/33 (H1N1) and C/Johannesburg/1/1966 viruses. Residues involved in forming the asymmetric FluPol dimer interface are highlighted in cyan, and residues involved in ANP32A binding are highlighted in orange. The figure was prepared with Espript 3.060.

Reporting Summary (download PDF )

Supplementary Figure (download PDF )

Supplementary Fig. 2 Source data (gels).

Peer Review File (download PDF )

Video 1 (download MP4 )

Overview of the FluPolC-chANP32A structure. The movie shows how vRNA-bound FluPolC (FluPolR) assembles with an apo FluPolC (FluPolE) to form an asymmetric dimer stabilized by ANP32A.

Video 2 (download MP4 )

Cryo-EM map of the FluPolC-chANP32A complex. The movie shows the repositioning of the reconstructed densities, corresponding to the PB2627 domains of both polymerases and chANP32A, along the third eigenvector.

Video 3 (download MP4 )

Cryo-EM map of the FluPolC-chANP32A complex. The movie shows the cryo-EM density of the FluPolC-chANP32A complex, highlighting the proximity of the product exit channel of FluPolR to the 5’ RNA binding site of FluPolE.

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Carrique, L., Fan, H., Walker, A.P. et al. Host ANP32A mediates the assembly of the influenza virus replicase. Nature 587, 638–643 (2020). https://doi.org/10.1038/s41586-020-2927-z

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