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An intermediate state allows influenza polymerase to switch smoothly between transcription and replication cycles

Nature Structural & Molecular Biology volume 30, pages 1183–1192 (2023)Cite this article

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Abstract

Influenza polymerase (FluPol) transcribes viral mRNA at the beginning of the viral life cycle and initiates genome replication after viral protein synthesis. However, it remains poorly understood how FluPol switches between its transcription and replication states, especially given that the structural bases of these two functions are fundamentally different. Here we propose a mechanism by which FluPol achieves functional switching between these two states through a previously unstudied conformation, termed an ‘intermediate state’. Using cryo-electron microscopy, we obtained a structure of the intermediate state of H5N1 FluPol at 3.7 Å, which is characterized by a blocked cap-binding domain and a contracted core region. Structural analysis results suggest that the intermediate state may allow FluPol to transition smoothly into either the transcription or replication state. Furthermore, we show that the formation of the intermediate state is required for both the transcription and replication activities of FluPol, leading us to conclude that the transcription and replication cycles of FluPol are regulated via this intermediate state.

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Fig. 1: Transition from FluPolH5N1 to the T-state.
Fig. 2: Transition from the FluPolH5N1 to the R-state.
Fig. 3: The intermediate state is critical for polymerase activity.
Fig. 4: The contracted core of FluPolH5N1.
Fig. 5: The key role of the η7 loop in replication initiation of FluPol.
Fig. 6: Model of FluPol-mediated transitions in the viral life cycle.

Data availability

The cryo-EM maps of FluPolH5N1 complexes have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-34496 (core region) and EMD-34497 (full length). The coordinates for the atomic model of the FluPolH5N1 complex have been deposited in the Protein Data Bank under accession number 8H69. Source data are provided with this paper.

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Acknowledgements

We thank staff in the Center of Cryo-Electron Microscopy, Zhejiang University, for their assistance during data collection. We thank G. Ji, X. Huang, B. Zhu, L. Zhang and D. Fan in the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science, for their assistance during data collection and T. Niu for computational assistance. We thank Y. Ma and P. Xia for their help during sample screening at the State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences. We thank the use of Cryo-EM instruments in the Cryo-EM Facility Center of Southern University of Science and Technology. We thank H. Zhang (Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences) for assisting us with the detection of radioactivity. We thank the staff from BL19U2 beamline of National Facility for Protein Science in Shanghai at Shanghai Synchrotron Radiation Facility for assistance during data collection. This work was supported by the National Natural Science Foundation of China (31530015, 82071346, 32000860 and 32271321), ‘Pearl River Talent Plan’ Innovation and Entrepreneurship Team Project of Guangdong Province (2019ZT08Y464), Natural Science Foundation of Guangdong Province, China (2020B1515020035), the Key Fundamental Research Projects of Shenzhen Science and Technology Plan (JCYJ20200109142418595 and JCYJ20200109142412265), Fund of Shenzhen Key Laboratory (ZDSYS20220606100803007) and Shenzhen Science and technology planning project (RCBS20200714114922284).

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Author notes

  1. These authors contributed equally: Huanhuan Li, Yixi Wu, Minke Li, Lu Guo.

Authors and Affiliations

  1. Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, School of Medicine, Shenzhen Campus of Sun Yat-Sen University, Sun Yat-Sen University, Shenzhen, China

    Huanhuan Li, Yixi Wu, Minke Li & Yingfang Liu

  2. Department of General Surgery, Guangdong Institute of Gastroenterology, Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat‐sen University, Guangzhou, China

    Huanhuan Li, Lixin Zhu, Ping Lan & Yingfang Liu

  3. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Huanhuan Li, Lu Guo, Quan Wang & Yingfang Liu

  4. Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, School of Pharmaceutical Sciences (Shenzhen), Shenzhen Campus of Sun Yat-Sen University, Sun Yat-Sen University, Shenzhen, China

    Yaqi Gao, Jihua Zhang, Zhaohua Lai & Huanhuan Liang

  5. Departments of Biophysics and Pathology of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Xing Zhang

  6. Center of Cryo Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, China

    Xing Zhang

  7. Laboratory of Structural Biology, Tsinghua University, Beijing, China

    Zihe Rao

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  1. Huanhuan Li
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Contributions

H. Li, Y.W., M.L., L.G., Y.G., Q.W., J.Z., Z.L., X.Z., L.Z., P.L. and Z.R. performed the experiments. H. Li, Y.W., M.L., Y.L. and H. Liang wrote the paper. Y.L. and H. Liang designed the experiments. All authors reviewed the results and approved the final version of the paper.

Corresponding authors

Correspondence to Yingfang Liu or Huanhuan Liang.

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Nature Structural & Molecular Biology thanks Aartjan te Velthuis, Dong Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Beth Moorefield and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Cryo-EM analyses of FluPolH5N1 bound to vRNA promoter.

a. A representative cryo-EM micrograph of FluPolH5N1 bound to vRNA promoter. b, c. Representative 2D classifications of FluPolH5N1 complex particles. White arrows indicate the vRNA promoter and yellow arrows indicate the flexible domains above the core region. d. Euler angle distributions of FluPolH5N1 complex in the final 3D reconstructions. e. Local resolution evaluations of cryo-EM maps of FluPolH5N1 complex by ResMap. f, g. Gold standard Fourier shell correlation curves for resolution evaluation of core region (f) and full-length (g) at 0.143 FSC. The FSC curves of the final refined models versus cryo-EM maps at 0.5 FSC are also shown. h. Representative regions of the cryo-EM structure of FluPolH5N1 are shown as cartoon representation colored as same as those in Fig. 1a. The density maps of motif pre-A (residues 225-243) and motif C (residues 436-453) were shown. i. The density maps of 3′-vRNA and 5′-vRNA. J, k. Plots of the global half-map FSC (solid red line) and map-to-model FSC (dotted green line) of full-length (j) and core region (k) FluPolH5N1 and together with the spread of directional resolution values defined by ±1σ from the mean.

Extended Data Fig. 2 Data processing strategies for 3D reconstruction of Flu PolH5N1 complex bound to vRNA promoter.

The black boxes indicate the selected 3D classes during data processing. The red boxes indicate the final maps.

Extended Data Fig. 3 The 3′-vRNA promoter end resting outside in FluPolH5N1.

The structure of the vRNA promoter in FluPolH5N1 complex is shown as cartoon (left) and stick (right) representations. Interacting residues in FluPolH5N1 stabilized the 3′-vRNA promoter end are shown as stick representation. For clarity, only polar interactions are shown as black dashed lines. Colors are shown as same as those in Fig. 1a.

Extended Data Fig. 4 The density maps and expressions of mutants of FluPolH5N1.

a. The density maps of the hinge region, η7 loop in PB1 (left) and residues stabilizing the inactive conformation of PB2-C FluPolH5N1 (right). Colors are shown as same as those in Fig. 1a. b, c. SDS-PAGE analysis of purified mutant proteins expressed using the Bac-to-Bac expression system. Data shown are representative of two independent experiments with similar results. d, e. Western blot analysis of the expression of mutant proteins stabilizing the inactive conformation of PB2-C (d) and the PB2 G248A mutant (e) of FluPolH5N1. Data shown are representative of two independent experiments with similar results.

Source data

Extended Data Fig. 5 SAXS analysis of the experimental profiles and the calculated profiles.

a-c. The experimental SAXS scattering curve of wild-type FluPol H5N1 (gray) fits with the calculated curves of the intermediate FluPolH5N1 (a, black), transcriptase (b, blue, 5MSG) and replicase (c, green, 6XZG), respectively. d, e. The experimental profiles of E517A (d, red) and K586A/R589A (e, orange) fit the calculated profiles from transcriptase (blue, 5MSG). f, g. The experimental profiles of E517A (f, red) and K586A/R589A (g, orange) fit the calculated profiles from replicase (green, 6XZG). The χ2 value is shown as indicated. Data were analyzed by BioXTAS RAW and ATSAS packages.

Extended Data Fig. 6 Comparison of the mRNA cap analogs binding capacity.

a-c. m7GTP binding of WT PB2cap (a), H432A PB2cap (b) and R436A PB2cap (c) were analyzed by ITC. The raw data are shown at the top and the integrated data shown with continuous lines are in the bottom. d, e. Table (d) and histogram (e) summary of binding parameter KD. Data are shown as mean ± s.e.m form n = 3 biologically independent samples. ns, no significance. The P values was analyzed by one-way ANOVA.

Source data

Extended Data Fig. 7 Bio-layer interferometry (BLI) binding profiles of WT FluPol and mutants to 3′ vRNA.

a-c. The binding affinities between the WT FluPol (a), mutants M507A (b), E508A (c) and 3′ vRNA at indicated concentrations were determined by BLI experiment, respectively. d. The values of KD, KD error and Full R2 were shown in the table. The data shown are representative of two independent experiments with similar results.

Source data

Extended Data Fig. 8 Structures of core regions in product disassociation and recycling states during the end of transcription closely resemble the intermediate core of FluPolH5N1.

The secondary structures of catalytic cavities of FluPolH5N1 (in green), FluPol in transcription-product disassociation state (a, PDB: 6T0U, in pink), FluPol in transcription-recycling state (b, PDB: 6T2C, in wheat). Close-up views show the similar conformation of the η7 loop protruding towards the cavity in these core regions. The priming loop in FluPolH5N1 is shown while the priming loops in product disassociation and recycling states are extruded and invisible.

Extended Data Fig. 9 The η7 helix in the viral polymerase cavity is conserved among different viruses.

The RdRp regions in RABV (rabies virus, PDB: 6UEB), VSV (vesicular stomatitis virus, PDB: 6U1X), HRSV (human respiratory syncytial virus, PDB: 6PZK), HMPV (human metapneumovirus, PDB: 6U5O), HPIV (human parainfluenza virus, PDB: 6V85), LACV (La Crosse orthobunyavirus, PDB: 5AMQ), LASV (Lassa mammarenavirus, PDB: 6KLC), MACV (Machupo mammarenavirus, PDB: 6KLD) and SFTSV (thrombocytopaenia syndrome virus, PDB: 6Y6K) polymerases are shown as same orientation in cartoon representation (green). The positions of fingers, palm and thumb subdomains are labeled. The homologous structures of η7 helix (in blue) are found in all of these polymerases. Despite of sequence variability, bulky residues like phenylalanine, tryptophan and arginine, are observed at the homologous position of Met507 and Glu508 in influenza polymerase.

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 1. RNA sequences used for biochemical assays and cryo-EM. Supplementary Table 2. Primer sequences used for site-directed PCR mutagenesis to construct plasmids expressing mutant FluPol. Supplementary Table 3. P values for the statistical analyses in figures.

Supplementary Video 1

Transition from the intermediate conformation to replicase. FluPol is shown as a cartoon representation colored the same as in Fig. 1a. The 424-loop in the cap-binding domain and the PB2 helix α11 and hinge are colored in blue and black, respectively, for clarity. The transition from the intermediate FluPol to replicase is stabilized by newly synthesized FluPol (called encapsidating FluPol after replicase assembly) through interactions with the hinge region. After the rotation of the whole PB2-C, ANP32A induces the 627 domain to further rotate to an exposed position with host-specific residue 627 being highly accessible, thereby finishing the assembly of replicase. The trajectory of intermediate conformations was calculated by UCSF Chimera.

Supplementary Video 2

Transition from the intermediate conformation to transcriptase. FluPol is shown as a cartoon representation colored the same as in Fig. 1a. The 424-loop in the cap-binding domain and the PB2 helix α11 and hinge are colored in blue and black, respectively, for clarity. The transition from the intermediate FluPol to transcriptase is stabilized by the Pol II CTD peptide binding on the polymerase surface. Pol II CTD binding triggers conformational changes of the 627 domain, thereby releasing the cap-binding domain and activating the polymerase for ‘cap-snatching’ toward the transcription preinitiation state. The trajectory of intermediate conformations was calculated by UCSF Chimera.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Uncropped gel.

Source Data Fig. 3

Statistical source data.

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Statistical source data.

Source Data Fig. 5

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Source Data Extended Data Fig. 4

Uncropped gel and unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

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Li, H., Wu, Y., Li, M. et al. An intermediate state allows influenza polymerase to switch smoothly between transcription and replication cycles. Nat Struct Mol Biol 30, 1183–1192 (2023). https://doi.org/10.1038/s41594-023-01043-2

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