Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic viral RNA (vRNA) into messenger RNA. The polymerase is initially bound to a promoter comprising the partially base-paired 3′ and 5′ extremities of the RNA. A short, capped primer, ′cap-snatched′ from a nascent host polymerase II transcript, is directed towards the polymerase active site to initiate RNA synthesis. Here we present structural snapshots, as determined by X-ray crystallography and cryo-electron microscopy, of actively initiating influenza polymerase as it transitions towards processive elongation. Unexpected conformational changes unblock the active site cavity to allow establishment of a nine-base-pair template–product RNA duplex before the strands separate into distinct exit channels. Concomitantly, as the template translocates, the promoter base pairs are broken and the template entry region is remodeled. These structures reveal details of the influenza polymerase active site that will help optimize nucleoside analogs or other compounds that directly inhibit viral RNA synthesis.
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Coordinates and structure factors or maps have been deposited in the Protein Data Bank or the Electron Microscopy Data Bank.
FluB polymerase-initiation complex 15-mer primer (X-ray) PDB ID 6QCX
FluB polymerase-initiation complex 14-mer primer (X-ray) PDB ID 6QCW
FluB polymerase-initiation complex 14-mer primer + CTP (X-ray) PDB ID 6QCV
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We thank EMBL (especially M. Bowler) and ESRF staff for access to X-ray beamlines; the ESRF (especially E. Kandiah), IBS and EMBL staff for access to the ESRF Krios beamline CM01; the EMBL eukaryotic expression and high-throughput crystallization facilities and the biophysical platform within the EMBL–ESRF–ILL–IBS Partnership for Structural Biology. We thank J. Wandzik (EMBL) for help with transcription experiments and electron microscopy data collection. This work was partly supported by ERC Advanced Grant V-RNA (No. 322586) to S.C. and an ANR grant (No. ANR-18-CE11-0028) to S.C. T.K. holds a fellowship from the EMBL Interdisciplinary Postdocs (EI3POD) initiative, co-funded by Marie Skłodowska-Curie grant agreement No. 664726.
Integrated supplementary information
a, X-ray electron density for the primer-template duplex RNA in the crystal structure of the mixed initiation state with 15-mer capped primer. Fo-Fc omit map contoured at 3 σ. b, c and d, CryoEM density for the 5′ vRNA hook and primer-product-template duplex. Fit of the 5′ vRNA hook A1-G12 (b) and product-template duplex (c) into the cryoEM density of the elongation state filtered at 3.2 Å resolution. d, Verification of base pair identity of the product-template duplex using the cryoEM density. e, CTP electron density. Fo − Fc omit map electron density, contoured at 3σ, for the soaked CTP in the crystal structure of the mixed initiation state with the 14-mer capped primer.
Supplementary Figure 2 Both 18-mer and 18 + 3-mer 3′ vRNA template are functional for transcription initiation with 14-mer or 15-mer capped primers.
a, Cap dependent transcription assay (n=3) showing that 18 or 18+3 3′ vRNA templates are both active and produce the same transcription products. Read-through products and endonuclease cleavage products are indicated. Radiolabeled capped RNA markers, of size indicated, are at the left. An incubation time of four hours was chosen for the cryo-EM experiment as beyond that time there was no increase of product. b, Quantitative activity comparison of the 18 or 18+3 3′ vRNA templates after four hours in presence of ATP and GTP and 15-mer capped primer. The activity of 18+3 3′ vRNA is 72.8% ± 0.03 (n=6, mean and s.d.) of the activity of native 18 3′ vRNA. The read-through activity, defined by ratio of the read-through product to the desired capped transcription product, are 15% different for the two vRNA templates (n=6, mean and s.d., two-tail t-test). c, Cap dependent transcription assay showing products of 18 or 18+3 3′ vRNA templates with 14- and 15-mer cap primers. The capped 14-mer requires CTP in the reaction mixture to function as a primer, in accord with CTP serving as initiating nucleotide for this primer (although with the 18+3 template some mis-initiation seems to occurs, maybe because the primer and template anneal more readily).
a, Representative micrograph of transcribing FluB polymerase complexes in free standing ice after MotionCor2 correction at defocus of ~ 2.5 µm. b, 2D-class averages of the transcribing FluB polymerase complexes. c and d, Angular distribution for particle projections of the pre-initiation and elongation state, respectively, visualized on a globe like plane. CryoEM particle orientation efficiency scores of 0.57 and 0.6, respectively, suggest a sufficient Fourier space coverage. Distribution of local resolution of the pre-initiation e, and elongation f, state cryoEM maps and two perpendicular slices. Maps are coloured according to the local resolution calculated within RELION software package. Resolution is as indicated in the color bar. g, Fourier shell correlation (FSC) curves for the pre-initiation and elongation states. The plot of the FSC between two independently refined half-maps shows the overall resolution of the two maps as indicated by the gold standard FSC 0.143 cut-off criteria. h, FSC between the FluB polymerase complexe atomic models and the cryoEM density maps. The FSC 0.5 cut-off criteria is indicated.
Summary of the cryoEM 3D classification and refinement scheme for FluB polymerase complexes. Initially, ~ 615k particles were classified into eight 3D classes with angular assignment (upper lane, number of particles in each class is indicated in thousands). Incomplete, low resolution, and damaged particle classes were excluded from further data analysis. The four most prominent 3D classes of the complete FluB polymerase were merged into a global consensus model (second line) and further classified into six classes (third line). Atomic resolution cryoEM maps for pre-initiation and elongation states were refined and post-processed with their respective masks in RELION (bottom line right) and amplitude scaled in LocScale (bottom line left).
Supplementary Figure 5 Global changes in polymerase structure upon transition to the elongation state.
A comparison of the PB1 structure in the pre-initiation (grey) and elongation state (colors) as analyzed with DynDom (http://dyndom.cmp.uea.ac.uk/dyndom/). The thumb domain (~ 509–670) together with the PB2 N1 and N2 domains (PB2/54-153, ruby) rotate outwards by ~ 4.5° compared to the superposed palm domain (cyan). The movement is mediated by a hinge between the palm and thumb domains involving PB1 residues 508–509 and 533–538 (green). There is also a slight shift of the PA-C domain (not shown). We also quantified the consequence of this conformational change on the width of the active site cavity taken as the distance between the Cα atoms of PB1/Glu256 of helix α9 and PB1/Ile524 of helix α17. The width increases from ~ 30.7 Å in the pre-initiation state to ~ 31.4 Å in the initiation state and ~ 32.1 Å in the elongation state. The cavity is almost wide enough to fit A-form duplex RNA even in the pre-initiation state but must splay by ~ 1.4 Å to accommodate the extended product-template RNA duplex.
Supplementary Figure 6 The priming loop remodels during the pre-initiation to elongation transition.
a, Detailed view of the primer (blue)-template (yellow) duplex and partially extruded priming loop (grey) in the mixed-initiation state. Close packed bases of -5U, A8 and A10 butt against residues 652–655 (grey sticks) of the repositioned priming loop and a salt-bridge between conserved PB1 Asp655 and PB2 Arg218 (red sticks). b, Superposition of priming loop conformations as seen in the two pre-initiation state (grey, cryoEM and cyan, PDB entry 5MSG), mixed-initiation state (green) and elongation state (orange). The left view is orientated as in Fig. 2 and the right view is vertically rotated by 50°. c, Fit of the priming loop residues PB1/631-661 into the cryoEM density of the pre-initiation state filtered at 3.16 Å resolution. d, Fit of the priming loop residues PB1/631-661 into Fo-Fc omit electron density map of the 15-mer capped primer mixed-initiation state crystal structure contoured at 3 σ. e, Fit of the priming loop residues PB1/631-661 into the cryoEM density of the elongation state filtered at 3.20 Å resolution.
Interactions of the distal part of the capped RNA primer (m7GpppGAAUGC…) with specific residues (yellow sticks) of the cap-binding (orange), PB2-midlink (violet) and PB2-N (ruby) domains as observed in the mixed initiation state structures. Nucleotides 2-AAU-4 are stacked on each other and sandwiched between PB2/Tyr434 of the cap-binding domain and a salt-bridge between PB2/Arg217 and Glu155 of the PB2 N2 domain. G5 is in a separate pocket stacking between of PB2/Arg146 and Arg425 and makes base contacts with PB1/Glu227.
a, Superposition of the methionine-rich motif B loop conformations as observed in the pre-translocate state (light grey), post-translocated state (dark grey) and intermediate positions as observed in two different pre-initiation state structures (this study and PDB entry 5MSG). PB1/Met410 gradually flips (gradient of greys) from the position where it contacts (orange dashed lines) the product base at the +1 position (marked by blue 15C) towards a disengaged position when there is no base. Notably, the pathway would clash (red dashed line) with the +1 template position (marked by yellow +1G) position in both pre and post translocated states. b and c, Details of the methionine-rich motif B loop conformations when the incoming NTP site is vacant or occupied, respectively, and their superposition d, showing that PB1/Phe344 and Phe413 both reorient to accommodate Met410 in a distinct hydrophobic pocket when the incoming NTP site is vacant. e and f, Fit of the methionine-rich motif B loop residues into the Fo − Fc omit map electron density of the vacant (capped 14-mer X-ray structure) and occupied (capped 15-mer X-ray structure) states of the active site at 3σ.