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Structural insight into RNA synthesis by influenza D polymerase

Abstract

The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3ʹ-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.

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Fig. 1: Purification and biochemical characterization of FluDPol.
Fig. 2: Overall structure of FluDPol.
Fig. 3: Comparison of the apo and promoter-bound FluDPol structures.
Fig. 4: Interactions between the vRNA/cRNA promoter and FluDPol in different conformations.
Fig. 5: Transcription and replication activities of wild-type and mutant FluDPol in vivo and in vitro.
Fig. 6: Proposed working model of the different promoter conformations for RNA synthesis by FluPol.

Data availability

The density maps have been deposited to the Electron Microscopy Data Bank under the accession numbers EMD-9577 (apo FluDPol), EMD-9578 (vRNA promoter-bound class A1), EMD-9579 (class A2), EMD-9581 (class B1), EMD-9580 (class B2), EMD-9582 (class B3), EMD-9887 (cRNA promoter-bound class 1) and EMD-9888 (class 2). The coordinates of the corresponding atomic models have been deposited to the Protein Data Bank with the entries 6AB7 (apo FluDPol), 6ABB (promoter-bound class A1), 6ABD (class A2), 6ABF (class B1), 6ABE (class B2), 6ABG (class B3), 6JU2 (cRNA promoter-bound class 1) and 6JU3 (class 2).

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Acknowledgements

We thank all staff at the National Center for Protein Science Shanghai cryo-EM department and the Center of Biological Imaging, Institute of Biophysics for assistance with data collection. We are grateful to G. Wang (The Core Facilities at School of Life Sciences, Peking University), T. Yang and the staff in the EM department of the State Key Laboratory of Membrane Biology, Institute of Zoology, CAS for their technical support in the operation of the electron microscope. The ForteBio Octet experiment was supported by the Research Facility Center at Beijing Institutes of Life Science, CAS. This study was supported by the Strategic Priority Research Program of the CAS (grant no. XDB29010000), the National Science and Technology Major Project (grant no. 2018ZX10101004) and the External Cooperation Program of the CAS (grant no. 153211KYSB20160001). R.P. was supported by the Young Elite Scientist Sponsorship Program from the China Association for Science and Technology (grant no. 2018QNRC001). M.W. and J.Y. were also supported by the National Science and Technology Major Project (grant no. 2018ZX09711003). G.F.G. was partly supported as a leading principal investigator of the NSFC Innovative Research Group (grant no. 81621091). Y.S. was supported by the Excellent Young Scientist Program from the National Natural Science Foundation of China (grant no. 81622031), the Excellent Young Scientist Program of the CAS and the Youth Innovation Promotion Association CAS (grant no. 2015078).

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Contributions

Y.S., R.P. and G.F.G. designed the project. Q.P. and Y.L. purified the protein samples and conducted the biochemical studies. Q.P., R.P. and S.L. prepared the cryo-EM samples and collected the data. R.P. and X.Z. performed image processing and reconstruction. R.P. and J.Q. built the atomic models. Q.P., R.P., M.W. and Y.S. analysed the structure. Q.P., Y.L., Y.C., H.S. and M.H. conducted the radioactive-labelled polymerase activity assays. Q.P., M.W., W.Y. and T.D. performed the replicon-based polymerase activity assays. Y.S., R.P. and G.F.G. wrote the paper. M.W., M.H., H.S., T.D., P.W., J.Y. and B.Z. revised the manuscript and were involved in intensive discussions of the data. Y.S. supervised all of the research.

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Correspondence to George F. Gao or Yi Shi.

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Peng, Q., Liu, Y., Peng, R. et al. Structural insight into RNA synthesis by influenza D polymerase. Nat Microbiol 4, 1750–1759 (2019). https://doi.org/10.1038/s41564-019-0487-5

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