Transcriptome analysis of HIV-1 latently infected cells

• Project/Area Number: 19K07591
• 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: Shirakawa Kotaro 京都大学, 医学研究科, 助教 (80728270)
• 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: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: HIV潜伏感染 / トランスクリプトーム / CRISPRスクリーニング / mTOR経路 / HIV感染症 / 潜伏感染 / 再活性化 / CRISPRスクリーン / HIV-1 / CAGE法 / CRISPR/Cas9 / トランスクリプトーム解析

Outline of Research at the Start

潜伏感染細胞を直接標識できるHIVGKOのVif欠損株を用いて、CAGE法により潜伏感染細胞分画のトランスクリプトーム解析を行う。既に取得している野生型HIVGKOとの比較を行いHIV-1 Vifの潜伏感染細胞の転写への影響を解析する。HIV-1 Vifの相互作用分子として同定されたHDAC3やその複合体の構成成分との結合様式を詳細に解析し、潜伏感染成立への影響を解析する。核内蛋白に対するsgRNAライブラリーを用いてCRISPRスクリーニングを行い潜伏感染の確立と維持および再活性化に必要な細胞性因子を同定する。以上によりHIV-1潜伏感染のメカニズムを明らかにし根治療法開発への基盤とする。

Outline of Final Research Achievements

HIVGKO enables direct identification of both productively and latently HIV-infected cells. In this study, we deep sequenced CAGE tags in non-infected and latently and productively infected cells and compared their transcriptional profiles. We detected 31 down regulated genes in latently infected cells compared to those of noninfected cells. Among these, SPP1 and APOE were downregulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have antiviral properties. Components of the mTOR pathway were significantly up regulated in productively infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV entry and integration do not trigger unique transcriptional responses when infection becomes latent and that T cells cannot recognize the integrated HIV genome when infection remains latent.

Academic Significance and Societal Importance of the Research Achievements

HIV感染症は抗ウイルス薬によりコントロール可能となったが、治療を中止すると再発してしまう。これはメモリーT細胞などのHIV潜伏感染細胞がウイルス蛋白を発現せず免疫による監視を生き残るためである。本研究ではHIV潜伏感染細胞の転写プロファイルを比較し、HIVゲノムがインテグレーションされた潜伏感染細胞に転写レベルで非感染細胞との差がほとんどないことを示された。本細胞モデルを用いてさらに潜伏感染の分子機構を明らかにできれば潜伏感染細胞を標的とした治癒を目指すための新たな治療法につながると考えられる。

Research Products

• [Int'l Joint Research] Buck Institute for Research on Aging(米国)
• [Int'l Joint Research] Buck Institute for Research on Aging(米国)
• [Int'l Joint Research] Buck Institute for Research on Aging(米国)
• [Journal Article] CAGE-Seq Reveals that HIV-1 Latent Infection Does Not Trigger Unique Cellular Responses in a Jurkat T Cell Model (2021)
  • Author(s): Matsui Hiroyuki、Shirakawa Kotaro、Konishi Yoshinobu、Hirabayashi Shigeki、Sarca Anamaria Daniela、Fukuda Hirofumi、Nomura Ryosuke、Stanford Emani、Horisawa Yoshihito、Kazuma Yasuhiro、Matsumoto Tadahiko、Yamazaki Hiroyuki、Murakawa Yasuhiro、Battivelli Emilie、Verdin Eric、Koyanagi Yoshio、Takaori-Kondo Akifumi
  • Journal Title: Journal of Virology Volume: 95 Issue: 8 Pages: 1-18
  • DOI: 10.1128/jvi.02394-20
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] APOBEC3B is preferentially expressed at the G2/M phase of cell cycle (2021)
  • Author(s): Hirabayashi Shigeki、Shirakawa Kotaro、Horisawa Yoshihito、Matsumoto Tadahiko、Matsui Hiroyuki、Yamazaki Hiroyuki、Sarca Anamaria Daniela、Kazuma Yasuhiro、Nomura Ryosuke、Konishi Yoshinobu、Takeuchi Suguru、Stanford Emani、Kawaji Hideya、Murakawa Yasuhiro、Takaori-Kondo Akifumi
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 546 Pages: 178-184
  • DOI: 10.1016/j.bbrc.2021.02.008
  • Peer Reviewed / Open Access
• [Journal Article] FRET-Based Detection and Quantification of HIV-1 Virion Maturation (2021)
  • Author(s): Sarca Anamaria D.、Sardo Luca、Fukuda Hirofumi、Matsui Hiroyuki、Shirakawa Kotaro、Horikawa Kazuki、Takaori-Kondo Akifumi、Izumi Taisuke
  • Journal Title: Frontiers in Microbiology Volume: 12 Pages: 1-13
  • DOI: 10.3389/fmicb.2021.647452
  • NAID: 120007180366
  • Peer Reviewed / Open Access
• [Journal Article] Alternative splicing of APOBEC3D generates functional diversity and its role as a DNA mutator (2020)
  • Author(s): Takei Hisashi、Fukuda Hirofumi、Pan Gilbert、Yamazaki Hiroyuki、Matsumoto Tadahiko、Kazuma Yasuhiro、Fujii Masanori、Nakayama Sohei、Kobayashi Ikei S.、Shindo Keisuke、Yamashita Riu、Shirakawa Kotaro、Takaori-Kondo Akifumi、Kobayashi Susumu S.
  • Journal Title: International Journal of Hematology Volume: 112 Issue: 3 Pages: 395-408
  • DOI: 10.1007/s12185-020-02904-y
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Critical role of PP2A-B56 family protein degradation in HIV-1 Vif mediated G2 cell cycle arrest (2020)
  • Author(s): Nagata Kayoko、Shindo Keisuke、Matsui Yusuke、Shirakawa Kotaro、Takaori-Kondo Akifumi
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 527 Issue: 1 Pages: 257-263
  • DOI: 10.1016/j.bbrc.2020.04.123
  • NAID: 120006865708
  • Peer Reviewed
• [Presentation] HIV-1潜伏感染の成立と維持に関与する分子機構 (2021)
  • Author(s): 白川康太郎
  • Organizer: 第35回日本エイズ学会学術集会・総会
• [Presentation] HIV-1 latent infection does not trigger cellular transcriptional responses in T-cells (2020)
  • Author(s): Hiroyuki Matsui, Kotaro Shirakawa, Shigeki Hirabayashi, Anamaria D. Sarca, Hirofumi Fukuda, Yasuhiro Murakawa, Emilie Battivelli, Eric Verdin, Akifumi Takaori-Kondo
  • Organizer: Keystone Symposia 2020 HIV Pathogenesis and Cure X6
  • Int'l Joint Research
• [Remarks] Verdin Lab website
  • URL: https://www.buckinstitute.org/lab/verdin-lab/