Reconstitution of reverse transcription in vitro.

• Project/Area Number: 15K15139
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Virology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: Masuda Takao 東京医科歯科大学, 大学院医歯学総合研究科, 准教授 (80219336)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2015: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: 逆転写 / 逆転写酵素 / レトロウイルス / RNA / HIV / RNaseH / 転写

Outline of Final Research Achievements

Reverse transcription of human immunodeficiency virus (HIV) was successfully reconstituted in vitro (cell-free) using recombinant HIV reverse transcriptase and in vitro synthesized HIV RNA. By using this in vitro assay, novel mechanisms to regulate HIV reverse transcription have revealed; 1) Critical role of guanosine (G) number at 5-end of virus RNA for successful 1st strand transfer, and (2) HIV has evolved to package the most efficient RNA into virus particle, providing novel mechanisms to regulate HIV reverse transcription and its coevolution history with viral genome transcription machinery.

Research Products

• [Journal Article] Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase. (2017)
  • Author(s): Takahata T, Takeda E, Tobiume M, Tokunaga K, Yokoyama M, Huang YL, Hasegawa A, Shioda T, Sato H, Kannagi M, Masuda T.
  • Journal Title: J Virol Volume: 91 Issue: 1
  • DOI: 10.1128/jvi.02003-16
  • Peer Reviewed / Open Access
• [Journal Article] Natural Single-Nucleotide Variations in the HIV-1 Genomic SA1prox Region Can Alter Viral Replication Ability by Regulating Vif Expression Levels (2016)
  • Author(s): Nomaguchi M, Doi N, Sakai Y, Ode H, Iwatani Y, Ueno T, Matsumoto Y, Miyazaki Y, Masuda T, and Adachi A
  • Journal Title: Journal of Virology Volume: 90 Issue: 9 Pages: 4563-4578
  • DOI: 10.1128/jvi.02939-15
  • Peer Reviewed
• [Journal Article] Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA. (2015)
  • Author(s): Masuda T, Sato Y, Huang Y, Koi S, Takahata T, Hasegawa A, Kawai G, Kannagi M.
  • Journal Title: Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA. Volume: 5 Issue: 1 Pages: 17680-17680
  • DOI: 10.1038/srep17680
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Role of 5’-end modification of HIV-1 RNA during reverse transcription (2016)
  • Author(s): Yu-Lun Huang, Tatsuro Takahata、Atsuhiko Hasegawa, Gota Kawai Mari Kannagi and Takao Masuda
  • Organizer: 第64回 日本ウイルス学会学術集会
  • Place of Presentation: 札幌 （札幌コンベンションセンター）
  • Year and Date: 2016-10-23
• [Presentation] HIV-1 RNA 5’ 末端配列の逆転写過程での重要性. (2015)
  • Author(s): 増田貴夫、佐藤洋子、黄渝倫、高畑辰郎、長谷川温彦、河合剛汰、神奈木真理.
  • Organizer: 第29回 日本エイズ学会学術集会
  • Place of Presentation: 東京ドームホテル、東京
  • Year and Date: 2015-11-30
• [Presentation] In vitro reconstitution of HIV-1 reverse transcription revealed an unprecedented role of 5f virus genomic RNA.or reverse transcription. (2015)
  • Author(s): Masuda T, Sato Y, Huang Y, Koi S, Takahata T, Hasegawa A, Kawai G, Kannagi M.
  • Organizer: 第63回 日本ウイルス学会学術集会
  • Place of Presentation: 福岡国際会議場、福岡
  • Year and Date: 2015-11-22