プラス鎖ＲＮＡウイルスの複製におけるウイルスと宿主の攻防

2013 Fiscal Year Annual Research Report

• Project Area: Molecular basis of host cell competency in virus infection
• Project/Area Number: 24115003
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: 脇田 隆字 国立感染症研究所, ウイルス第二部, 部長 (40280789)
• Co-Investigator(Kenkyū-buntansha): 竹安 邦夫 京都大学, 生命科学研究科, 教授 (40135695)
• Project Period (FY): 2012-06-28 – 2017-03-31
• Keywords: プラス鎖RNAウイルス / HCV / 宿主 / 原子間力顕微鏡

Research Abstract

HCVなどのプラス鎖RNAウイルスはウイルスゲノムが短く、コードされるウイルス遺伝子数は限られており、ウイルス複製増殖や病原性発現には、ウイルスタンパク質が多機能を有するとともに、多くの宿主因子を動員する必要がある。従って、HCVは、感染体と宿主の攻防を研究するために適したモデルである。網羅的な手法で見出した宿主因子について、ウイルス複製、粒子形成、成熟化における役割について解析していく。NS5A発現細胞からNS5Aに結合する膜蛋白を精製し, プロテオーム解析、およびsiRNA screeningを行ったところ、翻訳および複製過程に関与している蛋白としてELVL1を見出した。ELVL1はHCV RNA、NS3およびNS5蛋白質と結合して、HCV RNAの翻訳と複製に関与していることが明らかとなった。ELVL1の共生発現によりHCV RNAのnuclease感受性が高まることから、ELVL1がHCV RNAの細胞内局在を制御していることが推測された。さらに“RNAを見る”ための技術開発を行った。原子間力顕微鏡下で1本鎖RNAを可視化するために、スペルミジンでコートした雲母片を基板に用いて、９Kbの1本鎖HCV RNAの構造解析を行った。AFM解析で求めたRNAの体積は塩基長に比例することから、体積測定がRNAを特定することと、特定の分子内ループの存在はAFM画像から検出できることが分かった。一方、藤田班との共同で2本鎖RNAのインターフェロン産生に必須なMDA5は、二本鎖RNAへの結合とATPase活性に依存して構造を球状に変化させること、MDA5の変異体では、RNA非存在下でも球状構造を示すことが分かった。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

HCVの複製および翻訳に重要な宿主因子を同定し、さらにその宿主因子がHCVの複製にかかわる分子機構を明らかにしてきている。また、“RNAを見る”ための技術上の問題点を克服し、技術を現実的なものとした。これまで可視化できなかった個別のウイルスゲノム構造を可視化した。この技術の応用により、ウイルスゲノムと宿主因子の相関を分子単位レベルで観察可能となることが期待できる。これまでの研究内容をすでに複数の論文として報告した。

Strategy for Future Research Activity

HCVの複製の場としての細胞内微細構造である、二重膜ベジクル形成が重要と考えられる。感染細胞における二重膜ベジクル形成機構とウイルスおよび宿主蛋白質の関係を解析する。そのために分子生物学および細胞生物学的解析を電子顕微鏡と組み合わせて解析を進める。また、感染体ゲノム-タンパク質複合体のナノメートルレベルの構造および感染体ゲノムの細胞内動態についても、HCVウイルス、インフルエンザウイルスを始めとする領域内の多様なウイルス種も対象として解析を行う。AFMによるウイルスゲノムと宿主因子の相関についても解析を進める。

Research Products

• [Journal Article] Hepatitis virus infection affects DNA methylation in mice with humanized livers (2014)
  • Author(s): Okamoto Y, Shinjo K, Shimizu Y, Sano T, Yamao K, Gao W, Fujii M, Osada H, Sekido Y, Murakami S, Tanaka Y, Joh T, Sato S, Takahashi S, Wakita T, Zhu J, Issa JP, Kondo Y
  • Journal Title: Gastroenterology Volume: 146 Pages: 562-572
  • DOI: 10.1053/j.gastro.2013.10.056
  • Peer Reviewed
• [Journal Article] Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon (2014)
  • Author(s): Suzuki R, Ishikawa T, Konishi E, Matsuda M, Watashi K, Aizaki H, Takasaki T, Wakita T
  • Journal Title: J Gen Virol Volume: 95 Pages: 60-65
  • DOI: 10.1099/vir.0.058008-0
  • Peer Reviewed
• [Journal Article] HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type I receptor (2013)
  • Author(s): Sakata K, Hara M, Terada T, Watanabe N, Takaya D, Yaguchi S, Matsumoto T, Matsuura T, Shirouzu M, Yokoyama S, Yamaguchi T, Miyazawa K, Aizaki H, Suzuki T, Wakita T, Imoto M, Kojima S
  • Journal Title: Sci Rep Volume: 22 Pages: 3243
  • DOI: 10.1038/srep03243
  • Peer Reviewed
• [Journal Article] Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction (2013)
  • Author(s): Nishimura Y, Lee H, Hafenstein S, Kataoka C, Wakita T, Bergelson JM, Shimizu H
  • Journal Title: PLoS Pathogen Volume: 9 Pages: e1003511
  • DOI: 10.1371/journal.ppat.1003511
  • Peer Reviewed
• [Journal Article] Signal Peptidase Complex Subunit 1 Participates in the Assembly of Hepatitis C Virus through an Interaction with E2 and NS2 (2013)
  • Author(s): Suzuki R, Matsuda M, Watashi K, Aizaki H, Matsuura Y, Wakita T, Suzuki T
  • Journal Title: PLoS Pathogen Volume: 9 Pages: e1003589
  • DOI: 10.1371/journal.ppat.1003589
  • Peer Reviewed
• [Journal Article] Interleukin-1 and tumor necrosis factor-alpha trigger restriction of hepatitis B virus infection via a cytidine deaminase AID (2013)
  • Author(s): Watashi K, Liang G, Iwamoto M, Marusawa H, Uchida N, Daito T, Kitamura K, Muramatsu M, Ohashi H, Kiyohara T, Suzuki R, Li J, Tong S, Tanaka Y, Murata K, Aizaki H, Wakita T
  • Journal Title: J Biol Chem Volume: 288 Pages: 31715-31727
  • DOI: 10.1074/jbc.M113.501122
  • Peer Reviewed
• [Journal Article] A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication (2013)
  • Author(s): Maehama T, Fukasawa M, Date T, Wakita T, Hanada K
  • Journal Title: Biochem Biophys Res Commun Volume: 440 Pages: 150-156
  • DOI: 10.1016/j.bbrc.2013.09.048
  • Peer Reviewed
• [Journal Article] Specific inhibition of hepatitis C virus entry into host hepatocytes by fungi-derived sulochrin and its derivatives (2013)
  • Author(s): Nakajima S, Watashi K, Kamisuki S, Tsukuda S, Takemoto K, Matsuda M, Suzuki R, Aizaki H, Sugawara F, Wakita T
  • Journal Title: Biochem Biophys Res Commun Volume: 440 Pages: 515-520
  • DOI: 10.1016/j.bbrc.2013.09.100
  • Peer Reviewed
• [Journal Article] The hepatitis E virus capsid C-terminal region is essential for the viral life cycle: implication for viral genome encapsidation and particle stabilization (2013)
  • Author(s): Shiota T, Li TC, Yoshizaki S, Kato T, Wakita T, Ishii K
  • Journal Title: J Virol. Volume: 87(10): Pages: 6031-6036
  • DOI: 10.1128/JVI.00444-13
  • Peer Reviewed
• [Journal Article] Oxysterol-binding protein family I is the target of minor enviroxime-like compounds (2013)
  • Author(s): Arita M, Kojima H, Nagano T, Okabe T, Wakita T, Shimizu H
  • Journal Title: J Virol Volume: 87 Pages: 4252-4260
  • DOI: 10.1128/JVI.03546-12
  • Peer Reviewed
• [Journal Article] Thromboxane A2 Synthase Inhibitors Prevent Production of Infectious Hepatitis C Virus in Mice with Humanized Livers (2013)
  • Author(s): Abe Y, Aly HH, Hiraga N, Imamura M, Wakita T, Shimotohno K, Chayama K, Hijikata M
  • Journal Title: Gastroenterology Volume: 145 Pages: 658-667
  • DOI: 10.1053/j.gastro.2013.05.014
  • Peer Reviewed
• [Journal Article] Antiviral Activity of Glycyrrhizin against Hepatitis C Virus In Vitro (2013)
  • Author(s): Matsumoto Y, Matsuura T, Aoyagi H, Matsuda M, Hmwe SS, Date T, Watanabe N, Watashi K, Suzuki R, Ichinose S, Wake K, Suzuki T, Miyamura T, Wakita T, Aizaki H
  • Journal Title: PLoS One Volume: 8 Pages: e68992
  • DOI: 10.1371/journal.pone.0068992
  • Peer Reviewed
• [Journal Article] Neutralizing antibodies induced by cell culture-derived hepatitis C virus protect against infection in mice (2013)
  • Author(s): Akazawa D, Moriyama M, Yokokawa H, Omi N, Watanabe N, Date T, Morikawa K, Aizaki H, Ishii K, Kato T, Mochizuki H, Nakamura N, Wakita T
  • Journal Title: Gastroenterology Volume: 145 Pages: 447-455
  • DOI: 10.1053/j.gastro.2013.05.007
  • Peer Reviewed
• [Journal Article] Human G-proteins, ObgH1 and Mtg1, associate with the large mitochondrial ribosome subunit and are involved in translation and assembly of respiratory complexes (2013)
  • Author(s): Kotani T, Akabane S, Takeyasu K, Ueda T, Takeuchi N
  • Journal Title: Nucleic Acids Res. Volume: 41 Pages: 3713-3722
  • DOI: 10.1093/nar/gkt079
  • Peer Reviewed
• [Journal Article] Visualization of structural changes accompanying activation of N-methyl-D-aspartate (NMDA) receptors using fast-scan atomic force microscopy imaging (2013)
  • Author(s): Suzuki Y, Goetze TA, Stroebel D, Balasuriya D, Yoshimura SH, Henderson RM, Paoletti P, Takeyasu K, Edwardson JM
  • Journal Title: J. Biol. Chem Volume: 288 Pages: 778-784
  • DOI: 10.1074/jbc.M112.422311
  • Peer Reviewed
• [Journal Article] Intermolecular disulfide bonds among nucleoporins regulate karyopherin-dependent nuclear transport (2013)
  • Author(s): Yoshimura SH, Otsuka S, Kumeta M, Taga M, Takeyasu K
  • Journal Title: J. Cell Sci. Volume: 126 Pages: 3141-3150
  • DOI: 10.1242/jcs.124172
  • Peer Reviewed
• [Journal Article] Nucleolar scaffold protein, WDR46, determines the granular compartmental localization of nucleolin and DDX21 (2013)
  • Author(s): Hirai Y, Louvet E, Oda T, Kumeta M, Watanabe Y, Horigome T, Takeyasu K
  • Journal Title: Genes Cells Volume: 18 Pages: 780-797
  • DOI: 10.1111/gtc.12077
  • Peer Reviewed
• [Journal Article] An alternative beads-on-a-string chromatin architecture in Thermococcus kodakarensis (2013)
  • Author(s): Maruyama H, Harwood JC, Moore KM, Paszkiewicz K, Durley SC, Fukushima H, Atomi H, Takeyasu K, Kent NA
  • Journal Title: EMBO Rep. Volume: 14 Pages: 711-717
  • DOI: 10.1038/embor.2013.94
  • Peer Reviewed
• [Journal Article] Bacillus subtilis DprA recruits RecA onto ssDNA and mediates annealing of complementary ssDNA strands coated by SsbB and SsbA (2013)
  • Author(s): Yadav T, Carrasco B, Hejna J, Suzuki Y, Takeyasu K, Alonso JC
  • Journal Title: J. Biol. Chem. Volume: 288 Pages: 22437-22450
  • DOI: 10.1074/jbc.M113.478347
  • Peer Reviewed
• [Journal Article] Early steps of double-strand break repair in Bacillus subtilis (2013)
  • Author(s): Alonso JC, Cardenas PP, Sanchez H, Hejna J, Suzuki Y, Takeyasu K
  • Journal Title: DNA Repair Volume: 12 Pages: 162-176
  • DOI: 10.1016/j.dnarep.2012.12.005
  • Peer Reviewed
• [Journal Article] AFM investigation of the organization of actin bundles formed by actin-binding proteins (2013)
  • Author(s): Gilmore JL, Kumeta M, Takeyasu K
  • Journal Title: Journal of Surface Engineered Materials and Advanced Technology (JSEMAT) Volume: 3 Pages: 13-19
  • DOI: 10.1111/gtc.12131
  • Peer Reviewed
• [Journal Article] High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events (2013)
  • Author(s): Suzuki Y, Sakai N, Yoshida A, Uekusa Y, Yagi A, Imaoka Y, Ito S, Karaki K, Takeyasu K
  • Journal Title: Sci. Rep. Volume: 3 Pages: 2131
  • DOI: 10.1038/srep02131.
  • Peer Reviewed
• [Journal Article] Antibody-based analysis reveals "filamentous vs. non-filamentous" and "cytoplasmic vs. nuclear" crosstalk of cytoskeletal proteins (2013)
  • Author(s): Kumeta M, Hirai Y, Yoshimura SH, Horigome T, Takeyasu K
  • Journal Title: Exp. Cell Res. Volume: 319 Pages: 3226-3237
  • DOI: 10.1016/j.yexcr.2013.07.021
  • Peer Reviewed
• [Presentation] C型肝炎ウイルスNS5A結合膜蛋白ELAVL1のウイルス複製・翻訳スイッチング機構の解析 (2013)
  • Author(s): 後藤耕司, 相崎英樹, 渡邉則幸, 渡士幸一, 鈴木亮介, 山越智, 四柳宏, 森屋恭爾, 小池和彦, 鈴木哲朗, 宮村達男, 脇田隆字
  • Organizer: 第61回日本ウイルス学会学術集会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 20131110-20131112
• [Presentation] Infectious Genotype 3a Hepatitis C Virus in Cell Culture (2013)
  • Author(s): S Kim, T Date, H Yokokawa, T Kono, H Aizaki, C Gondeau, P Maurel, T Wakita
  • Organizer: 20th International Meeting on Hepatitis C and Related Viruses
  • Place of Presentation: Melbourne Convention and Exhibition Centre, Melbourne, Australia
  • Year and Date: 20131006-20131010
• [Presentation] Hepatitis C virus cell culture system and antiviral development (2013)
  • Author(s): T Wakita
  • Organizer: 17th International Symposium on the Cells of the Hepatic Sinusoid
  • Place of Presentation: Osaka International Convention Center, Osaka, Japan
  • Year and Date: 20130923-20130925
  • Invited
• [Presentation] Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules (2013)
  • Author(s): H Aizaki, N Watanabe, H Aoyagi, K Watashi, R Suzuki, S Kojima, T Matsuura, K Wake, T Suzuki, T Wakita
  • Organizer: 17th International Symposium on the Cells of the Hepatic Sinusoid
  • Place of Presentation: Osaka International Convention Center, Osaka, Japan
  • Year and Date: 20130923-20130925
• [Presentation] Molecular Biology and Experimental Models of HCV (2013)
  • Author(s): T Wakita
  • Organizer: 19th Annual KASL Meeting
  • Place of Presentation: Sheraton Grande Walkerhill Hotel, Seoul, Korea
  • Year and Date: 20130613-20130615
  • Invited