Functional analysis of host factor participating in replication mechanism of the hepatitis E virus genome RNA

• Project/Area Number: 16K19144
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Virology
• Research Institution: Jichi Medical University
• Principal Investigator: Kobayashi Tominari 自治医科大学, 医学部, 助教 (00634164)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2016: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: E型肝炎ウイルス / レプリコン / リバースジェネティクス法 / 細胞培養 / ウイルス複製 / PSAPモチーフ / HEV / RNA複製 / PSAP / リアルタイムRT-PCR法

Outline of Final Research Achievements

In this study, in order to elucidate the replication mechanism of hepatitis E virus (HEV), we constructed an HEV replicon that can multiplicate full-length genomic RNA but cannot produce virions in transfected cells. We could obtain evidence that a 2.2-kb subgenomic RNA was transcribed, from which ORF3 proteins were translated. Obtained results suggested that we could establish a replicon-based method to analyze the replication mechanism of the HEV genomic RNA in cultured cells. In addition, since rat HEV ORF3 protein lacks an amino acid sequence motif of proline, serine, alanine and proline (PSAP) that has been demonstrated to be essential for release of human HEV particles from infected cells, various infectious rat HEV cDNA clones with mutations in proline residues were constructed. Obtained results indicated that two proline residues of the PxYP sequence play a pivotal role for egress of rat HEV particles.

Research Products

• [Journal Article] An analysis of two open reading frames (ORF3 and ORF4) of rat hepatitis E virus genome using its infectious cDNA clones with mutations in ORF3 or ORF4 (2018)
  • Author(s): Tanggis、Kobayashi Tominari、Takahashi Masaharu、Jirintai Suljid、Nishizawa Tsutomu、Nagashima Shigeo、Nishiyama Takashi、Kunita Satoshi、Hayama Emiko、Tanaka Takeshi、Mulyanto、Okamoto Hiroaki
  • Journal Title: Virus Research Volume: 249 Pages: 16-30
  • DOI: 10.1016/j.virusres.2018.02.014
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Full-length genome of a novel genotype 3 hepatitis E virus strain obtained from domestic pigs in Japan (2017)
  • Author(s): Primadharsini PP, Miyake M, Kunita S, Nishizawa T, Takahashi M, Nagashima S, Tanggis, Ohnishi H, Kobayashi T, Nishiyama T, Jirintai S, Okamoto H
  • Journal Title: Virus Research Volume: 240 Pages: 147-153
  • DOI: 10.1016/j.virusres.2017.08.003
  • Peer Reviewed
• [Journal Article] Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway (2017)
  • Author(s): Nagashima Shigeo、Takahashi Masaharu、Kobayashi Tominari、Nishizawa Tsutomu、Nishiyama Takashi、Primadharsini Putu Prathiwi、Okamoto Hiroaki、Tanggis
  • Journal Title: Journal of Virology Volume: 91 Issue: 22
  • DOI: 10.1128/jvi.00822-17
  • Peer Reviewed
• [Journal Article] Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system. (2016)
  • Author(s): Kobayashi T, Takahashi M, Tanggis, Mulyanto, Jirintai S, Nagashima S, Nishizawa T, Okamoto H.
  • Journal Title: Journal of Virological Methods Volume: 233 Pages: 78-88
  • DOI: 10.1016/j.jviromet.2016.03.004
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Enhanced pregenomic RNA levels and lowered precore mRNA transcription efficiency in a genotype A hepatitis B virus genome with C1766T and T1768A mutations obtained from a fulminant hepatitis patient. (2016)
  • Author(s): Nishizawa T, Hoshino T, Naganuma A, Kobayashi T, Nagashima S, Takahashi M, Takagi H, Okamoto H
  • Journal Title: Journal of General Virology Volume: 97 Issue: 10 Pages: 2643-2656
  • DOI: 10.1099/jgv.0.000566
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone. (2016)
  • Author(s): Nagashima S, Kobayashi T, Tanaka T, Tanggis, Jirintai S, Takahashi M, Nishizawa T, Okamoto H.
  • Journal Title: Virus Research Volume: 223 Pages: 170-180
  • DOI: 10.1016/j.virusres.2016.07.011
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles. (2016)
  • Author(s): Takahashi M, Kobayashi T, Tanggis, Jirintai S, Mulyanto, Nagashima S, Nishizawa T, Kunita S, Okamoto H
  • Journal Title: Archives of Virology Volume: 161 Issue: 12 Pages: 3391-3404
  • DOI: 10.1007/s00705-016-3047-9
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] 感染培養系を用いたE型肝炎治療薬の検討 (2017)
  • Author(s): 小林 富成、西山 尚志、長嶋 茂雄、西澤 勉、高橋 雅春、唐 吉思、Putu Prathiwi Primadharsini、岡本 宏明
  • Organizer: 第65回 日本ウイルス学会学術集会
• [Presentation] Development of a plasmid-based reverse genetics system for hepatitis E virus (2016)
  • Author(s): 小林 富成、長嶋 茂雄、西澤 勉、唐 吉思、高橋 雅春、岡本 宏明
  • Organizer: 第64回日本ウイルス学会学術集会
  • Place of Presentation: 北海道 札幌市 札幌コンベンションセンター
  • Year and Date: 2016-11-23