Structure of influenza A polymerase bound to the viral RNA promoter

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Abstract

The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3′ and 5′ extremities of the viral RNA. Here we present the crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. PB1 contains a canonical RNA polymerase fold that is stabilized by large interfaces with PA and PB2. The PA endonuclease and the PB2 cap-binding domain, involved in transcription by cap-snatching, form protrusions facing each other across a solvent channel. The 5′ extremity of the promoter folds into a compact hook that is bound in a pocket formed by PB1 and PA close to the polymerase active site. This structure lays the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase, and opens new opportunities for anti-influenza drug design.

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Figure 1: Overall structure of the bat influenza A polymerase complex with the vRNA promoter.
Figure 2: PB1 structure and comparison with other RNA virus polymerases.
Figure 3: PA and PB2 structure and the PA-linker–PB1 interface.
Figure 4: PB1 functional regions.
Figure 5: Structure of the vRNA promoter and how it binds to the polymerase.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Structure factors and coordinates for Bat FluA have been deposited in the Protein Data Bank (PDB) under accession 4WSB.

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Acknowledgements

We thank members of the ESRF-EMBL Joint Structural Biology Group for access to European Synchrotron Radiation Facility (ESRF) beamlines, staff of the European Molecular Biology Laboratory (EMBL) eukaryotic expression and high-throughput crystallization facilities within the Partnership for Structural Biology (PSB), D. Hart for help with construct design, and H. Malet for electron microscopy. This work was supported by ERC Advanced Grant V-RNA (322586) to S.C.

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Contributions

A.P. did protein expression, purification and crystallization, with help from S.R. and D.G. who also did activity assays. A.P. did X-ray data collection and, together with S.C., did crystallographic analysis. S.C. supervised the project and wrote the paper with input from the other authors.

Corresponding author

Correspondence to Stephen Cusack.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Production of influenza A polymerase heterotrimer.

a, The heterotrimeric bat polymerase was recombinantly expressed in insect cells as a self-cleaving polyprotein. N-terminally it encodes the tobacco etch virus (TEV) protease that cleaves C-terminal to the amino-acid sequence ENLYFQ (in italics), and releases N-terminally His-tagged PA, PB1, C-terminally strep-tagged PB2 and cyan fluorescent protein (CFP) for facilitated monitoring of expression. Arrows indicate the N-to-C-terminal direction and the termini of each mature protein. The histidine and streptavidin tags are underlined. b, After ammonium sulphate precipitation, immobilized metal ion affinity chromatography, engineered streptavidin (strep-tactin) affinity and heparin chromatography, the final purification step consisted of size-exclusion chromatography. The elution profile (monitored by the absorbance at 280 nm) with a single and nearly symmetric peak suggests a homogeneous and monomeric polymerase complex. mAU, milli-absorption unit. c, Fractions of the final size-exclusion chromatography were subjected to 10% SDS–PAGE followed by Coomassie blue staining. Lane 1 contains the molecular mass markers and lanes 2–7 the eluate with PA (85.4 kilodaltons (kDa)), PB1 (87.8 kDa) and PB2 (91.0 kDa). d, Recombinant bat FluA polymerase was visualized by electron microscopy following negative staining with sodium silico-tungstate of a 0.02 mg ml−1 protein sample. The image demonstrates that the sample is homogeneous and monodisperse with a V- or doughnut-like shape and central cavity.

Extended Data Figure 2 Endonuclease, RNA transcription and RNA replication activities of recombinant FluA polymerase.

a, Mini-panhandle vRNA: 5′-pppAGUAGUAACAAGAGGGUAUUGUAUACCUCUGCUUCUGCU-3′. b, Separate 5′ and 3′ ends: 5′: 5′-pAGUAGUAACAAGAGGGUA-3′; 3′: 5′-UAUACCUCUGCUUCUGCU-3′. c, Endonuclease, cap-dependent transcription and ApG-primed replication assays. Cleavage of the cap donor is visible in lanes 2–6. Capped transcripts are visible in lanes 10 (from vRNA panhandle template) and 13 (from separated 5′ and 3′ vRNA ends) as well as cRNA produced in lanes 17 and 20. Markers, with size shown on the left, are RNA ladders labelled with 32P-pCp nucleotide. d, e, Time course of unprimed (d) and ApG-primed (e) vRNA replication by bat influenza A polymerase. The products of replication (cRNA) are indicated with an arrow. Ladders (lanes L) are 32P-pCp nucleotide-labelled RNA oligomers. ApG-primed replication is more efficient than unprimed replication.

Extended Data Figure 3 Surface views of the FluA heterotrimer with bound vRNA promoter.

ad, Four surface views at roughly 0° (a), 180° (b), 110° (c) and 290° (d) rotations with PA, PB1 and PB2 uniformly green, cyan and red, respectively. Major subdomains are labelled. The vRNA 5′ and 3′ ends are pink and yellow, respectively.

Extended Data Figure 4 PA and PB2 structure and new inter-subunit interactions.

a, Interactions of the PA-linker (green tube) with the outer surface of the fingers (pale cyan) and palm (pale salmon) domains of PB1. Contacts are mediated by both highly conserved hydrophobic residues (for example, PA residues Phe 205, Phe 211, Leu 214, Pro 220, Tyr 226, Phe 229, Tyr 232, Val 233, Ile 242, Leu 246, Met 249 and Val 253) and polar interactions (for example, PA Glu 203, Lys 230, Glu 243 and Lys 245 to PB1 Arg 162, Glu 331, His 465 and Asp 86, respectively). b, Transparent surface diagram showing the anchoring of the PA endonuclease domain (forest green) onto the PB1-Cter–PB2-Nter interface region (cyan/red) and its position relative to the PB2 cap-binding domain (orange). The nuclease helix α4 packs parallel to the penultimate PB1 helix α21 involving both hydrophobic (for example, PA Ile 86, Ile 90 and Ile 94 with PB1 Ser 720, Ile 724 and Ile 728, respectively) and polar interactions (for example, PA Glu 77 with PB1 Arg 727). Other contacts include the PB2 170-loop interacting with the same PA helix α4 in the vicinity of Trp 88. Also the endonuclease insertion (PA 70-loop, residues 67–74) packs on the first part of the last PB1 helix α22. The total buried surface area between the endonuclease and PB1/PB2 is 2,265 Å2.

Extended Data Figure 5 NTP and template tunnels in PB1.

a, View straight along the putative NTP entrance tunnel towards the putative priming loop (magenta) in the internal cavity. The NTP channel is lined with basic residues from the fingertips (Lys 235, Lys 237 and Arg 239, blue), fingers (Arg 45, cyan) and palm (Lys 308, Lys 480 and Lys 481, red) that are absolutely conserved in all influenza strains. The fingertips are in close proximity to PA helices α20 and α21 and to the loop of the 5′ hook. b, Surface view as in a showing that the putative priming loop in the interior cavity is visible through the NTP tunnel. c, View straight along the template entrance tunnel towards the priming loop (magenta) in the internal cavity. The tunnel is lined by residues conserved in all influenza strains and from all three subunits, Arg 507 and Asp 509 from PA (green), Tyr 30, Arg 126, Met 227, Lys 229 and Asp 230 from PB1 (cyan), and Arg 38, Lys 41 and Asn 42 from PB2 (red). d, Surface view as in c showing that the internal priming loop is visible through the template tunnel.

Extended Data Figure 6 Recognition of the vRNA 3′ end.

Protein interactions of the distal 3′ end showing the role of PB2-Nter (red). PB2 residues Arg 46 and Trp 49 and PA residue Lys 567 stabilize the sharp turn between 3′ nucleotides C8 and G9. PB2 Arg 38 and PB1-Cter residues Asn 671, Arg 672 and Asn 676 also bind the 3′ end. In the accompanying paper18, Fig. 2a shows the interactions with the complete 3′ end as observed in the FluB vRNA complex.

Extended Data Figure 7 vRNA arrangement in the bat polymerase crystals.

Simplified diagram showing vRNA sequence and secondary structure in the bat FluA crystals including vRNA-mediated crystal contact (inverted sequences) that forms an extended duplex. Crystals were grown with 3′-end nucleotides 1–18 or 3–18, but only those from 6–18 were visible (hence 1–5 are in italics).

Extended Data Table 1 Data collection and refinement statistics for bat FluA polymerase
Extended Data Table 2 Direct polar polymerase–vRNA contacts for the bat FluA structure

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, Supplementary Discussions and Supplementary References. (PDF 1975 kb)

360° rotation of bat FluA structure in ribbon representation about the vertical axis

View and colouring as in Fig. 1 (AVI 16280 kb)

360° rotation of bat FluA structure in surface representation about the vertical axis

View and colouring as in Fig. 1. (AVI 24293 kb)

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Pflug, A., Guilligay, D., Reich, S. et al. Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355–360 (2014). https://doi.org/10.1038/nature14008

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