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Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis

Nakano, Masahiro Sugita, Yukihiko Kodera, Noriyuki Miyamoto, Sho Muramoto, Yukiko Wolf, Matthias Noda, Takeshi 京都大学 DOI:10.1038/s42003-021-02388-4

2021

概要

The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.

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Acknowledgements

We thank Yousuke Furuta for providing us with T-705RTP; Yoshihiro Kawaoka for

providing us with plasmids; Ichiro Taniguchi, Keiko Shindo, Akiko Makino, and Keizo

Tomonaga for technical assistance; Akira Ishihama for helpful discussion; Toshio Ando

and Takayuki Uchihashi for technical support and valuable discussions at Bio-AFM

Summer School 2014 held at Kanazawa University. We also thank Editage (www.editage.

com) for English language editing. We thank Steven D. Aird for technical editing (www.

sda-technical-editor.org). This work was supported by a JSPS Grant-in-Aid for Scientific

Research (C) (16K08808, 19K07575) (to M.N.), a JSPS Grant-in-Aid for Early-Career

Scientists (19K16667), a Research Grant from the Kazato Research Encouragement Prize

(to Y.S.), an AMED Platform project for Supporting Drug Discovery and Life Science

Research (BINDS) (JP18am0101076) (to M.W.), a Japan Science and Technology Agency

PRESTO grant (JPMJPR13L9), a JSPS Grant-in-Aid for Scientific Research (B)

(17H04082, 20H03494), a JSPS Grant-in-Aid for Challenging Research (Exploratory)

(19K22529), JSPS Core-to-Core Program A, a MEXT Grant-in-Aid for Scientific

Research on Innovative Area (19H04831), an AMED Research Program on Emerging

and Re-emerging Infectious Disease grants (19fk0108113, 20fk0108270h0001), a Grant

from the Daiichi Sankyo Foundation of Life Science, and the Uehara Memorial Foundation (to T.N.), and grants from the Joint Research Project of the Institute of Medical

Science, the University of Tokyo, the Joint Usage/Research Center program of Institute

for Frontier Life and Medical Sciences Kyoto University, and the Takeda Science

Foundation (to Y.S. and T.N.). M.W. was supported by direct funding from Okinawa

Institute of Science and Technology Graduate University.

Author contributions

M.N. and T.N. designed the study. M.N., Y.S., N.K., S.M. and Y.M. performed the

experiments. M.N., Y.S., N.K., S.M. and T.N. analysed data. M.N., Y.S., M.W. and T.N.

wrote the manuscript. All authors reviewed and approved the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s42003-021-02388-4.

Correspondence and requests for materials should be addressed to T.N.

Peer review information Communications Biology thanks the anonymous reviewers for

their contribution to the peer review of this work. Primary Handling Editors: Anam

Akhtar. Peer reviewer reports are available.

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