The influenza virus genome consists of eight viral ribonucleoproteins (vRNPs), each consisting of a copy of the polymerase, one of the genomic RNA segments and multiple copies of the nucleoprotein arranged in a double helical conformation. vRNPs are macromolecular machines responsible for messenger RNA synthesis and genome replication, that is, the formation of progeny vRNPs. Here, we describe the structural basis of the transcription process. The mechanism, which we call the ‘processive helical track’, is based on the extreme flexibility of the helical part of the vRNP that permits a sliding movement between both antiparallel nucleoprotein-RNA strands, thereby allowing the polymerase to move over the genome while bound to both RNA ends. Accordingly, we demonstrate that blocking this movement leads to inhibition of vRNP transcriptional activity. This mechanism also reveals a critical role of the nucleoprotein in maintaining the double helical structure throughout the copying process to make the RNA template accessible to the polymerase.
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The EM maps have been deposited with the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb) under accession numbers EMD-0175, EMD-4412, EMD-4423, EMD-4426 and EMD-4430. The atomic coordinates of the docking have been deposited with the Protein Data Bank (https://www.rcsb.org/) under accession numbers 6H9G, 6I54, 6I7B, 6I7M and 6I85.
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We thank S.H.W. Scheres, C. Savva and the Laboratory of Molecular Biology (Cambridge) for access to the Titan Krios microscope and technical assistance. We thank E. Sahagún for the generation of the Supplementary video (https://scixel.es/). The professional editing service NB Revisions (https://www.nbrevisions.com/) was used for technical editing of the manuscript before submission. We acknowledge the cryo-EM facility of the CNB–CIB (CSIC) for its technical advice and support throughout this work. This work was supported by the Spanish Ministry of Science, Innovation and Universities (Ministerio de Ciencia, Innovación y Universidades) grant nos. BFU2017-90018-R and BFU2011-25090/BMC (J.M.-B.) and Integrative Biology of Emerging Infectious Diseases LabEx grant no. 10-LABX-0062 (N.N.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Cryo-EM image of isolated vRNPs showing the extreme flexibility of the particles. b, Gallery of 2D averages with some examples of the different structures of the helical part of influenza vRNPs. Scale bars represent 100Å.
Extended Data Fig. 2 Variation of the relative position of two nucleoprotein monomers extracted from opposite strands of the helixes shown in Fig. 1 a-d of the main text.
For clarity, the equatorial region between the head (yellow) and body (green) domains has been marked with a red line, with the arrows indicating the direction of displacement. In all cases the position of the helical axis of the vRNP is represented with a vertical black line and the position of the dihedral axis is perpendicular to the plane of the figure and depicted by the () symbol.
Extended Data Fig. 3 Docking reliability of the nucleoprotein atomic structure (pdb 2IQH) into the cryo-EM density maps.
From top to bottom, six views representing two rotated positions (top and bottom raw for each case) of the cryo-EM density maps shown at three different thresholds (2.2, 2.8 and 3.2 σ) corresponding to the volumes shown in Fig. 1a–c, respectively. It is important to notice that cryo-EM maps contain the genomic ARN not present in the atomic structure. In all cases, the quality of the fit of the nucleoprotein atomic structure into the map is evident.
Extended Data Fig. 4 Negative staining electron microscopy characterization of nucleozin-treated vRNPs.
a, After nucleozin treatment, vRNPs appear as straight structures with a characteristic dark centerline along the helix. b, Longer vRNPs appear as broken helixes showing sharp corners. These structures probably formed from the initial binding of nucleozin at different points of one long helix followed by a cooperative extension of the conformational change induced by the drug; the growth of the straight segments generates these striking bends in the joint zones (see Extended Data Fig. 5 for more information). c, Gallery of averaged kinks selected from the nucleozin-treated vRNPs. The treated particles always show a characteristic central line and an increase in the diameter of the helix is clearly visible (compare with Extended Data Fig. 1b), indicating that a conformational change has occurred. Representative individual images of each class are shown on the right. Scale bar represents 200 Å in a and b and 150 Å in c.
a, Another structure obtained by cryo-EM after incubation of native vRNPs in the presence of nucleozin; the nucleozin binding site is marked with magenta circles. The structural changes are manifested in a decrease of z-rise, from 28–35 Å in untreated vRNPs to approximately 20 Å in the treated ones. Additionally, the phi angle drops from about 55–60 degrees to around 20 degrees. In consequence, the diameter of the helix increases (compare Fig. 3c and Extended Data Fig. 5a with Fig. 1a–d) and the vRNPs become shorter. Scale bar represents 50 Å. b, Scheme of the collapse of the major groove upon nucleozin treatment. On the left side, a draft of the wild type vRNP structure is shown, the upper (red) and lower (blue) strands and two pairs of nucleoproteins have been outlined, indicating the position of the nucleozin binding sites (magenta circles). In most cases, nucleozin probably binds to one nucleoprotein and the flexibility of the helix allows contact with the neighboring nucleoprotein of the contiguous turn, which produces the cross-linkage of both nucleoproteins, collapsing the major groove of the structure and opening the minor one. The right side of this panel shows two schemes for the helix after nucleozin binding corresponding to the structures shown in Fig. 3c and Extended Data Fig. 5a, respectively. c, Despite the cross-linking produced by nucleozin, vRNPs retain their flexibility due to the possibility of small movements among nucleoprotein dimers along the z-axis (left) or residual ability of the monomers to rotate (right). In this scheme, the nucleozin binding sites have been removed for clarity.
Extended Data Fig. 6 Decreased in vitro transcriptional activity of vRNPs pre-incubated with nucleozin.
a, Gel and quantification of mRNA synthesized in 1 h of transcription after 10 min of incubation with different concentrations of nucleozin. b, Gel and quantification of the shorter mRNAs synthesized in 1 h of transcription after 10 min of incubation with different concentrations of nucleozin. c, Effect of duration of nucleozin pre-incubation on mRNA synthesis by vRNPs. For treatments with small amounts of nucleozin (left panel, 1μM), the pre-treatment time had little influence on activity, but again affected longer mRNAs to a greater extent. One result of three independent experiments is shown.
a, Image gallery of negatively stained vRNPs during the transcription process. In all cases a nucleoprotein loop is clearly visible at each end of the vRNP and the polymerase is located at some point along the helical part (blue arrows). The first two images show vRNPs where the His-tagged PB2 polymerase has been labeled with 5-nm Ni-NTA-Nanogold nanoprobe (black arrows). In some cases, an mRNA thread emerging from the polymerase was visible (green arrows). b, Gallery of non-activated control vRNPs showing the polymerase Ni-NTA-Nanogold labeled at one end. c, Average images obtained after alignment and classification of the region where the polymerase was located on the vRNPs during transcription. Each average was obtained from around 200 images; representative single images of the classes are shown on the right. Scale bars represent 150 Å.
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Coloma, R., Arranz, R., de la Rosa-Trevín, J.M. et al. Structural insights into influenza A virus ribonucleoproteins reveal a processive helical track as transcription mechanism. Nat Microbiol 5, 727–734 (2020). https://doi.org/10.1038/s41564-020-0675-3
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