Masking of double-stranded RNA by the influenza A virus NS1 protein

• Project/Area Number: 19K07575
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 49060:Virology-related
• Research Institution: Kyoto University
• Principal Investigator: Nakano Masahiro 京都大学, ウイルス・再生医科学研究所, 助教 (90456997)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2020: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: インフルエンザウイルス / 自然免疫 / 二本鎖RNA / マスキング / 高速原子間力顕微鏡 / 免疫蛍光染色 / リボヌクレオタンパク質 / 自然免疫応答 / リボヌクレオタンパク質複合体 / 原子間力顕微鏡

Outline of Research at the Start

申請者はこれまでに、インフルエンザウイルスのリボヌクレオタンパク質がin vitroでループ状の二本鎖RNAを合成することを明らかにしてきた。本課題ではまず、NS1欠損ウイルス感染細胞内で二本鎖RNAがマスクされることなく検出されるかどうかを確認する。次に、組み換えNS1タンパク質を用いてNS1の二本鎖RNAへの結合・マスキングに重要なアミノ酸を同定し、そこで得られた情報をもとにNS1変異ウイルスを作成して二本鎖RNAが検出されるかを解析する。さらに、NS1の二本鎖RNAへの結合の過程を原子間力顕微鏡を用いてリアルタイムでイメージングすることによって、そのマスキング機構の解明を試みる。

Outline of Final Research Achievements

It is widely known that dsRNAs produced by viral infection induce innate immune response in host cells. In this study, we focused on the influenza virus protein NS1 and tried to elucidate the mechanism for masking of dsRNA by NS1. First, we prepared the recombinant NS1 protein and observed the masking of dsRNA which was produced by in vitro RNA synthesis. The results suggested the interaction between NS1 and other viral proteins (nucleoproteins or RNA polymerases) was important for the masking. Next, we produced the NS1-deletion virus and detected dsRNAs in its infected cells. It was suggested that NS1 masked dsRNA in the virus-infected cells. Furthermore, the dsRNA was thought to induce innate immune response since it was detected not only in the nucleus but also in the cytoplasm. Finally, we showed that amino acids in the C-terminal region of NS1 were important for the masking of dsRNA.

Academic Significance and Societal Importance of the Research Achievements

ウイルス感染により生じる二本鎖RNAは、宿主の自然免疫応答を引き起こすことが古くから知られている。しかし、これまでインフルエンザウイルスに関してはその感染細胞内で二本鎖RNAを形成しないと考えられていたことから、インフルエンザウイルスが自然免疫を誘導する仕組みや、どのように自然免疫を回避しているかは不明な点も多く残されていた。今回の研究成果では、インフルエンザウイルスタンパク質NS1が二本鎖RNAを感染細胞内でもマスクすることや、そのメカニズムについてもマスキングに必要なNS1の領域などの重要な学術的知見が得られた。これは新たなタイプの治療薬の開発へとつながりその社会的意義も大きい。

Research Products

• [Journal Article] Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation (2022)
  • Author(s): Sho Miyamoto, Masahiro Nakano, Takeshi Morikawa, Ai Hirabayashi, Ryoma Tamura, Yoko Fujita-Fujiharu, Nanami Hirose, Yukiko Muramoto, and Takeshi Noda
  • Journal Title: mBio Volume: 13 Issue: 1
  • DOI: 10.1128/mbio.03315-21
  • Peer Reviewed / Open Access
• [Journal Article] Amplification-free RNA detection with CRISPR?Cas13 (2021)
  • Author(s): Shinoda Hajime、Taguchi Yuya、Nakagawa Ryoya、Makino Asami、Okazaki Sae、Nakano Masahiro、Muramoto Yukiko、Takahashi Chiharu、Takahashi Ikuko、Ando Jun、Noda Takeshi、Nureki Osamu、Nishimasu Hiroshi、Watanabe Rikiya
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 476-476
  • DOI: 10.1038/s42003-021-02001-8
  • Peer Reviewed / Open Access
• [Journal Article] Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis (2021)
  • Author(s): Masahiro Nakano, Yukihiko Sugita, Noriyuki Kodera, Sho Miyamoto, Yukiko Muramoto, Matthias Wolf, and Takeshi Noda
  • Journal Title: Commun. Biol. Volume: 4 Issue: 1 Pages: 858-858
  • DOI: 10.1038/s42003-021-02388-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Acetylation of the influenza A virus polymerase subunit PA in the N‐terminal domain positively regulates its endonuclease activity (2021)
  • Author(s): Hatakeyama Dai、Shoji Masaki、Ogata Seiryo、Masuda Takeshi、Nakano Masahiro、Komatsu Tsugunori、Saitoh Ayaka、Makiyama Kyoko、Tsuneishi Hazuki、Miyatake Asuka、Takahira Mizuki、Nishikawa Erina、Ohkubo Ayana、Noda Takeshi、Kawaoka Yoshihiro、Ohtsuki Sumio、Kuzuhara Takashi
  • Journal Title: The FEBS Journal Volume: 289 Issue: 1 Pages: 231-245
  • DOI: 10.1111/febs.16123
  • Peer Reviewed / Open Access
• [Journal Article] CP100356 Hydrochloride, a P-Glycoprotein Inhibitor, Inhibits Lassa Virus Entry: Implication of a Candidate Pan-Mammarenavirus Entry Inhibitor (2021)
  • Author(s): Takenaga T, Zhang Z, Muramoto Y, Fehling SK, Hirabayashi A, Takamatsu Y, Kajikawa J, Miyamoto S, Nakano M, Urata S, Groseth A, Strecker T, Noda T.
  • Journal Title: Viruses Volume: 13 Issue: 9 Pages: 1763-1763
  • DOI: 10.3390/v13091763
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis (2021)
  • Author(s): Sharma Mahima、Abayakoon Palika、Epa Ruwan、Jin Yi、Lingford James P.、Shimada Tomohiro、Nakano Masahiro、Mui Janice W.-Y.、Ishihama Akira、Goddard-Borger Ethan D.、Davies Gideon J.、Williams Spencer J.
  • Journal Title: ACS Central Science Volume: 7 Issue: 3 Pages: 476-487
  • DOI: 10.1021/acscentsci.0c01285
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Influenza A virus NS1 optimises virus infectivity by enhancing genome packaging in a dsRNA-binding dependent manner (2020)
  • Author(s): Sha Tim Wai、Weber Michaela、Kasumba Dacquin M.、Noda Takeshi、Nakano Masahiro、Kato Hiroki、Fujita Takashi
  • Journal Title: Virology Journal Volume: 17 Issue: 1 Pages: 107-107
  • DOI: 10.1186/s12985-020-01357-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] vRNA-vRNA interactions in influenza A virus HA vRNA packaging. (2020)
  • Author(s): Miyamoto, S., Muramoto, Y., Shindo, K., Fujita, Y., Morikawa, T., Tamura, R., Gilmore, J.L., Nakano, M., Noda, T.
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/2020.01.15.907295
  • Open Access
• [Journal Article] Generation of a purely clonal defective interfering influenza virus. (2019)
  • Author(s): Yamagata Y, Muramoto Y, Miyamoto S, Shindo K, Nakano M, Noda T.
  • Journal Title: Microbiol Immunol. Volume: 63 Issue: 5 Pages: 164-171
  • DOI: 10.1111/1348-0421.12681
  • NAID: 40021927501
  • Peer Reviewed / Open Access
• [Journal Article] Microtubule-dependent transport of arenavirus matrix protein demonstrated using live-cell imaging microscopy. (2019)
  • Author(s): Takamatsu Y, Kajikawa J, Muramoto Y, Nakano M, Noda T.
  • Journal Title: Microscopy (Oxf) Volume: 68 Pages: 450-456
  • DOI: 10.1093/jmicro/dfz034
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Roles of non-structural proteins against production of double-stranded RNA in influenza virus-infected cells (2021)
  • Author(s): Masahiro Nakano, S Junichi Kajikawa, Sho Miyamoto, Chiho Ohnishi, Yukiko Muramoto, and Takeshi Noda
  • Organizer: 第68回 日本ウイルス学会学術集会
• [Presentation] NS1欠損インフルエンザウイルス感染により誘導される二本鎖RNAの合成とその細胞内局在 (2020)
  • Author(s): 中野 雅博，宮本 翔，梶川 純一，村本 裕紀子，野田 岳志
  • Organizer: 9th Negative Strand Virus-Japan Symposium
• [Presentation] Identification of amino acid residues of influenza virus NS1 important for masking of double-stranded viral RNAs (2019)
  • Author(s): Masahiro Nakano, Sho Miyamoto, Junichi Kajikawa, Yukiko Muramoto, and Takeshi Noda
  • Organizer: 第67回 日本ウイルス学会学術集会
• [Book] パンデミック時代の感染症研究 (2021)
  • Author(s): 嘉糠 洋陸
  • Total Pages: 211
  • Publisher: 羊土社
  • ISBN: 9784758103923