Visualization of replicating genome of hepatitis B virus: development of the method for alpha-Cre complementation and frame overlap

• Project/Area Number: 26670221
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Virology
• Research Institution: The University of Tokyo
• Principal Investigator: Saito Izumu 東京大学, 医科学研究所, 教授 (70158913)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: B型肝炎ウイルス / split Cre / α相補 / 蛍光スクリーニング / HBV創薬 / Ｂ型肝炎ウイルス / preS / ウイルスゲノム複製

Outline of Final Research Achievements

We constructed genomes of hepatitis B virus (HBV), in which PreS region were replaced by split Cre’s (α-Cre andβ-Cre for first and latter halves, respectively), while overlapping Pol frame is maintained. Simultaneous expression of both the split Cre’s resulted in the Cre activity. The replacement does not exceed the genome size that can be replicated. The HBV genome described here expressed split Cre and, when the other split Cre was supplied from outside the genome, functional Cre activity was observed and turn on the GFP expression present outside. Because the replication of HBV genomes constructed here have not sufficiently been examined within the term of this study, further experiments are needed to confirm replication of these genomes quantitatively. Moreover, more information could be available if adenovirus vector systems are used instead of transfection. When they are confirmed, these HBV genomes must be useful for fluorescent screening of ant-HBV medicine.

Research Products

• [Journal Article] Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice (2016)
  • Author(s): Hirotake Ichise, Akiko Hori, Seiji Shiozawa, Saki Kondo, Yumi Kanegae, Izumu Saito, Taeko Ichise, and Nobuaki Yoshida
  • Journal Title: Experimental Animals Volume: 65 Issue: 3 Pages: 231-244
  • DOI: 10.1538/expanim.15-0126
  • NAID: 130006882370
  • ISSN: 0007-5124, 1341-1357, 1881-7122
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Single-domain Intrabodies against HCV Core Inhibit Viral Propagation and Core-induced NF-κB Activation (2016)
  • Author(s): Suzuki R, Saito K, Matsuda M, Sato M, Kanegae Y, Shi G, Watashi K, Aizaki H, Chiba J, Saito I, Wakita T, Suzuki T
  • Journal Title: J Gen Virol Volume: 印刷中 Issue: 4 Pages: 887-892
  • DOI: 10.1099/jgv.0.000423
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Preferable sites and orientations of transgene inserted in the adenovirus vector genome : The E3 site may be unfavorable for transgene position (2015)
  • Author(s): M Suzuki, S Kondo, Z Pei, A Maekawa, I Saito and Y Kanegae
  • Journal Title: Gene Therapy Volume: 1 Issue: 5 Pages: 1-9
  • DOI: 10.1038/gt.2014.124
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures. (2014)
  • Author(s): Lystad, AH., Ichimura, Y., Takagi, K., Yang, Y., Pankiv, S., Kanegae, Y., Kageyama, S., Suzuki, M., Saito, I., Mizushima,T., *Komatsu, M., *Simonsen, A.
  • Journal Title: EMBO Rep. Volume: 15 Issue: 5 Pages: 557-565
  • DOI: 10.1002/embr.201338003
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication. (2014)
  • Author(s): Kondo S, Yoshida K, Suzuki M, Saito I, Kanegae Y
  • Journal Title: PLoS One Volume: 9 Issue: 10 Pages: e108627-e108627
  • DOI: 10.1371/journal.pone.0108627
  • Peer Reviewed / Open Access
• [Journal Article] Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles (2014)
  • Author(s): Fukuhara T, Wada M, Nakamura S, Ono C, Shiokawa M, Yamamoto S, Motomura T, Okamoto T, Okuzaki D, Yamamoto M, Saito I, Wakita T, Koike K, Matsuura Y
  • Journal Title: PLoS Pathog Volume: 10(12) Issue: 12 Pages: e1004534-e1004534
  • DOI: 10.1371/journal.ppat.1004534
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Efficient production of the replicating HBV genome using "HBV-AdV System" (2015)
  • Author(s): Suzuki M., Kondo S., Yamasaki M., Saito I., Shibasaki M. and Kanegae Y.
  • Organizer: 2015 International Meeting Molecular Biology of Hepatitis B Viruses
  • Place of Presentation: Bad Nauheim Germany
  • Year and Date: 2015-10-04
  • Int'l Joint Research
• [Presentation] Multiplex guide RNAs targeting HBV genome expressed using adenovirus vector improve the efficiency of CRISPR/Cas9 system (2015)
  • Author(s): Maekawa A., Kondo S., Suzuki M., Saito I. and Kanegae Y.
  • Organizer: 2015 International Meeting Molecular Biology of Hepatitis B Viruses
  • Place of Presentation: Bad Nauheim Germany
  • Year and Date: 2015-10-04
  • Int'l Joint Research
• [Presentation] アデノウイルスベクターを用いた定量的HBV複製ccc及びrcゲノム検出法の開発 (2014)
  • Author(s): 近藤小貴、鈴木まりこ、山崎学、鐘ヶ江裕美、野本明男、斎藤泉
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] アデノウイルス感染初期におけるvirus-associated RNAの役割 (2014)
  • Author(s): 近藤小貴、鈴木まりこ、前川文、斎藤泉、鐘ヶ江裕美
  • Organizer: 第62回日本ウイルス学会学術総会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2014-11-10 – 2014-11-12
• [Presentation] アデノウイルスベクターを用いた効率的なHBVゲノム複製解析システムの開発：cocalently closed circular DNA (CCC)の検出 (2014)
  • Author(s): 鈴木まりこ、近藤小貴、山崎学、鐘ヶ江裕美、野本明男、斎藤泉
  • Organizer: 第62回日本ウイルス学会学術総会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2014-11-10 – 2014-11-12
• [Presentation] Development of new methods to detect the replication HBV genome in the cells infected with adenovirus vector expressing pregenome RNA. (2014)
  • Author(s): Suzuki M, Kondo S, Yamasaki M, Kanegae Y, Nomoto A, and Saito I.
  • Organizer: 2014 International Meetin on Molecular Biology of Hepatitis B Virus
  • Place of Presentation: UCLA , Los Angeles, California, USA
  • Year and Date: 2014-09-03 – 2014-09-06
• [Presentation] Development of a novel adenovirus vector for cancer-specific and stable expression: mini-adenovirus vector (mini-AdV). (2014)
  • Author(s): Yoshioka T, Maekawa A, Suzuki M, Kondo S, Kanegae Y, and Saito I.
  • Organizer: The 20th Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 東京慈恵会医科大学
  • Year and Date: 2014-08-06 – 2014-08-08
• [Presentation] Dually safer adenovirus vector lacking virus-associated RNA genes with significantly low immune responses. (2014)
  • Author(s): Kondo S, Maekawa A, Suzuki M, Saito I, and Kanegae Y.
  • Organizer: he 20th Annual Meeting of Japan Society of Gene Therapy
  • Place of Presentation: 東京慈恵会医科大学
  • Year and Date: 2014-08-06 – 2014-08-08
• [Remarks] 東京大学医科学研究所遺伝子解析施設
  • URL: http://www.ims.u-tokyo.ac.jp/idenshi/pg154.html
• [Patent(Industrial Property Rights)] 複数のユニットが多重に連結したDNAカセットおよび該カセットをを含むベクターの製造方法 (2014)
  • Inventor(s): 斎藤 泉
  • Industrial Property Rights Holder: 国立大学法人東京大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2014-242914
  • Filing Date: 2014-12-01