title: Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.
abstract: We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.
authors: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors: Shigeo
authors: S
authors: Nagashima
authors: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors: Masaharu
authors: M
authors: Takahashi
authors: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors: Suljid
authors: S
authors: Jirintai
authors: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors: Toshinori
authors: T
authors: Tanaka
authors: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors: Tsutomu
authors: T
authors: Nishizawa
authors: Department of Emerging Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki-Ken 852-8523, Japan.
authors: Jiro
authors: J
authors: Yasuda
authors: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
authors: Hiroaki
authors: H
authors: Okamoto
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D016752:Hepatitis E virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000502:physiology; D057074:Virus Release; D000099177:Tsg101 Protein
chemical_list: D000074183:ATPases Associated with Diverse Cellular Activities; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D034741:RNA, Small Interfering; D014157:Transcription Factors; D025262:Vacuolar Proton-Translocating ATPases; D014764:Viral Proteins; D000099177:Tsg101 Protein; C400973:ORF3 protein, Hepatitis E virus; C490305:VPS4A protein, human