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Structural insight into cap-snatching and RNA synthesis by influenza polymerase

Nature volume 516, pages 361–366 (2014)Cite this article

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Abstract

Influenza virus polymerase uses a capped primer, derived by ‘cap-snatching’ from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

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Figure 1: Structure of influenza B polymerase.
Figure 2: Promoter 3′-end binding and PB1 β-ribbon flexibility.
Figure 3: Model for replication initiation and elongation by influenza polymerase.
Figure 4: Cap-snatching and cap-dependent priming of transcription.
Figure 5: Model for cap-dependent transcription.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Structure factors and co-ordinates have been deposited in the Protein Data Bank (PDB) under the accessions 4WSA (FluB form 1) and 4WRT (FluB form 2).

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Acknowledgements

We thank the staff of the European Molecular Biology Laboratory (EMBL) eukaryotic expression and high-throughput crystallization facilities within the Partnership for Structural Biology (PSB) and members of the ESRF-EMBL Joint Structural Biology Group for help on European Synchrotron Radiation Facility (ESRF) beamlines. The work was supported by ERC Advanced Grant V-RNA (322586) and EU Grant FLU-PHARM (259751) to S.C. and partially by a Roche Postdoc Fellowship to S.R.

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Author notes

  1. Stefan Reich, Delphine Guilligay and Alexander Pflug: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France,

    Stefan Reich, Delphine Guilligay, Alexander Pflug, Hélène Malet, Imre Berger, Darren Hart, Thomas Lunardi, Max Nanao & Stephen Cusack

  2. University Grenoble Alpes-Centre National de la Recherche Scientifique-EMBL Unit of Virus Host-Cell Interactions, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France,

    Stefan Reich, Delphine Guilligay, Alexander Pflug, Hélène Malet, Imre Berger, Thibaut Crépin, Darren Hart, Thomas Lunardi, Max Nanao, Rob W. H. Ruigrok & Stephen Cusack

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Contributions

S.R., D.G. and T.L. did protein expression, purification, crystallization and activity assays. A.P. did crystallographic analysis. H.M. did electron microscopy and fitting to the mini-RNP electron microscopy map. M.N. calculated the first interpretable FluB polymerase electron density map. Using the polyprotein vector designed and provided by I.B., and with the help of D.H., S.C. designed the FluB polymerase construct. T.C., D.H., R.R. and S.C. have long-collaborated on studies of influenza polymerase. S.C. supervised the project, collected data, did crystallographic analysis and wrote the paper with input from S.R., D.G., A.P., H.M. and M.N.

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Correspondence to Stephen Cusack.

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Extended data figures and tables

Extended Data Figure 1 Production and characterization of influenza B polymerase heterotrimer.

a, Schematic of the self-cleaving polyprotein construct used to express recombinant influenza B heterotrimeric polymerase in insect cells. 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 expression monitoring. Arrows indicate the N-to-C-terminal direction and the termini of each mature protein. The histidine and streptavidin tags are underlined. b, Using the PB2 C-terminal strep-tag, most contaminating proteins could be separated from the polymerase as judged by 10% SDS–PAGE followed by Coomassie blue staining. Lanes ‘M’ contain the protein markers (molecular masses indicated); ‘in’, ‘ft’ and ‘w’ denote the input, flow-through and wash of the engineered streptavidin (strep-tactin) column, respectively, and ‘elution’ indicates the re-mobilization of bound heterotrimeric polymerase by a sharp gradient of d-desthiobiotin. The three subunits, PA (85.7 kDa), PB1 (86.1 kDa) and PB2 (90.8 kDa), run together on the gel. c, After ammonium sulphate precipitation, IMAC, strep-tactin affinity and heparin chromatography, the final purification step consists 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. d, Recombinant influenza B polymerase was analysed by electron microscopy following negative staining with sodium silico-tungstate of 0.02 mg ml−1 protein sample. The image demonstrates that the sample is homogeneous and monodisperse with a V- or doughnut-like shape with a central cavity.

Extended Data Figure 2 Endonuclease, transcription and replication activities of FluB polymerase.

a, Schematic of mini-panhandle vRNA: 5′-pppAGUAGUAACAAGAGGGUAUUGUAUACCUCUGCUUCUGCU-3′. b, Schematic of 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 and enhanced in the presence of the 5′ end, but not the 3′ end. 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 influenza B 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 Examples of electron density map for FluB polymerase.

a–c, Initial platinum SIRAS-phased and phase-extended experimental map at 3.6 Å resolution contoured at 1.1σ (brown) with superposed final model for the FluB1 crystal form. Also shown is the final model-phased selenium anomalous difference map at 4.1 Å resolution contoured at 3.2σ (purple) highlighting methionine positions. a, PB1 β-ribbon. b, vRNA 5′ hook. c, PA–PB1–PB2 helical interface. d, e, final 2Fo − Fc omit map at 2.7 Å resolution for the FluB2 crystal form contoured at 1.1σ. d, vRNA 5′ hook nucleotides 1–11. e, vRNA 3′ end nucleotides 1–9. Figures drawn with Bobscript52.

Extended Data Figure 4 Comparison of FluB and bat FluA polymerase structures.

a, Surface diagram of FluB1 structure coloured as in c except that PA-C, PB1and PB2-N are uniformly green, cyan and red, respectively. The bottom black arrow indicates the extra 12 C-terminal residues of FluB PA that extend the PA C-terminal helix compared to FluA, so that it directly contacts the PB2-NLS domain that is consequently orientated slightly differently from in FluA polymerase. b, As in a but for bat FluA structure. Arrows highlight the 70° difference in orientation of the cap-binding domain. The structural similarity between FluA and FluB polymerases (LSQMAN, cut-off 3.5 Å) is as follows. PA: 630 Cα atoms aligned, of which 38.6% are identical with root mean squared deviation (r.m.s.d.) 1.34 Å; PB1: 703 Cα atoms aligned, of which 61.3% are identical with r.m.s.d. 1.06 Å; PB2: 428 Cα atoms aligned, of which 40.6% are identical with r.m.s.d. 1.46 Å (excluding the cap-binding domain), and, taking into account the cap-binding domain rotation, 622 Cα atoms aligned, of which 39.0% are identical with r.m.s.d. 1.54 Å). c, Subunit domain structure of influenza B polymerase with names and extended colour scheme, showing the positions of the PB1 polymerase motifs. Note that for PB1, the FluB numbering compared to FluA is the same from 1–399 and is thereafter +1. For PB2, FluB is +2 from 1–469 and +1 from 470–628. For PA it is more complicated owing to several short insertions and deletions. See Supplementary Fig. 1.

Extended Data Figure 5 RNA–RNA crystal contact in FluB1 crystal form.

a, Cartoon of 5′ and 3′ vRNA ends (left, pink and yellow, respectively) interacting with crystallographic two-fold symmetry-related vRNA (right, pale pink and wheat, respectively). The PB1 β-ribbon (cyan) of the left-hand polymerase molecule interacts with the symmetry-related vRNA. b, Simplified diagram showing vRNA sequence and secondary structure in the FluB1 crystal form including vRNA-mediated crystal contact.

Extended Data Figure 6 Polymerase fitting into the mini-RNP electron microscopy map.

a, b, Top (a) and side (b) view of influenza A mini-RNP pseudo-atomic model with rescaled electron density39. PA, PB1 and PB2 (1–32 only) are shown as ribbons and coloured in green, cyan and rose, respectively. Unfilled electron density, likely to contain the rest of PB2, is shown in transparent rose. Nucleoproteins are shown in yellow ribbons, with the nucleoprotein–nucleoprotein interacting loop (residues 402–428) in orange. The vRNA 5′ and 3′ ends are shown in dark blue and red, respectively. c, Front view of influenza A mini-RNP pseudo-atomic model. The positions of antibody and tag labelling corresponding to domains of PA, PB1 and PB2 are shown as dark green, dark blue and dark rose spheres, respectively, as localized previously53. d, Close-up view of b. The PB1 β-ribbon (residues 177–214, purple) is located close to one of the proximal nucleoproteins and the vRNA. e, Putative interactions between the proximal nucleoprotein and polymerase. Nucleoprotein elements proposed for polymerase interaction are indicated in yellow, brown and orange. Polymerase interacting elements are shown in green, cyan, rose and magenta.

Extended Data Figure 7 Residual electron density in the FluB1 crystal form mimicking capped primer binding to the PB2 cap-binding domain.

Residual m2Fo − Fc (blue mesh at 0.9σ) and mFo − Fc (orange mesh at 2.5σ) electron density showing RNA-like density bound in the cap-binding site in the FluB1 crystal form. The low resolution and partial occupancy do not allow identification of the RNA and the discontinuous model shown is for illustrative purposes only. Owing to the rigorous purification procedure it is unlikely to be insect cell-capped RNA that is trapped on the polymerase. More likely it derives from the input vRNA used in crystallization, possibly partially digested by the endonuclease that generates 3′ ends. That this RNA could even be uncapped is explicable by the fact that the FluB cap-binding domain, unlike that of FluA, promiscuously binds both methylated and unmethylated guanosine54. Indeed, the density seems to be better fit with a free 3′ end sandwiched between Phe 406 and Trp 359 in the cap-binding site rather than a capped 5′ end. As the primer emerges from the cap-binding site it is initially channelled on one side by the base of the 424-loop, and on the other by residues 518–522 of the cap-627 linker. Further down, the extended 424-loop continues to guide the RNA, as well as, on the other side, the projecting N-terminal end of PB2 helix α9 (155-EMPPDE in FluB), with the double proline forcing the RNA into a ∼90° bend. Arg 425 and Arg 438 are well placed to interact with phosphates and one base seems to stack on the Glu 155–Arg 217 salt bridge. Conserved basic residues on PB2 N2 domain strands β7, 144-Arg-Lys-Arg (FluA 142-Arg-Lys-Arg), and β8, 216-Arg-Arg-Arg-Phe (FluA 214-Arg-Thr-Arg-Phe), are also likely to be involved. Straight-line distances from the cap-binding site to the bend and from the bend to the PB1 active site are indicated. See also Fig. 5.

Extended Data Figure 8 Schematic diagram of steps in cap-dependent transcription by influenza virus polymerase.

a, Cap-snatching from host pre-mRNA (red). The m7G cap is bound by the cap-binding domain (orange, orientated as in the FluA structure) and the pre-mRNA cleaved 10–14 nucleotides downstream by the endonuclease (green). The single-stranded vRNA genome is bound by its 5′ (hook, pink) and 3′ (template, yellow) ends to the polymerase (blue, depicted as a cutaway section). b, Transcription initiation. The cap-binding domain rotates to the position observed in the FluB1 structure directing the capped primer into the PB1 active site, where it potentially makes limited base pairs with the extremity of the template. Template-directed NTP addition (white) extends the host sequences (red) with virally encoded sequences (cyan). Note that in b–d additional conformational changes in the polymerase are expected, but not depicted since they are currently unknown. c, Transcription elongation. Transcription elongation proceeds, eventually leading to the release of the cap from the cap-binding domain (d) and the binding of host mRNP factors. d, Polyadenylation by stuttering. After most of the vRNA template has been translocated through the polymerase, only a tight turn connects it to the bound 5′-hook. The nucleotide sequence of this region is given at the bottom. This places the 5′ proximal oligo-U stretch in the PB1 active site allowing poly(A) tail synthesis by a stuttering mechanism in which the template is no longer translocated but the product strand is able to slip.

Extended Data Table 1 Data collection and refinement statistics for FluB polymerase structures
Full size table
Extended Data Table 2 Direct polar polymerase–vRNA contacts for the FluB2 structure
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, Supplementary Text and Supplementary References. (PDF 2312 kb)

Simulation of the putative rotation of the cap-binding domain (orange) by morphing between the domain positions seen in the FluA and FluB crystal structures.

The yellow spheres in the cap-binding domain correspond to the bound cap-analogue m7GTP. (MP4 557 kb)

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Reich, S., Guilligay, D., Pflug, A. et al. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361–366 (2014). https://doi.org/10.1038/nature14009

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