title: Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22.
abstract: Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm.
  
  
  OBJECTIVE
  Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: Jye-Chian
authors.initials: JC
authors.lastname: Hsiao
authors.affiliation: Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, Republic of China Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Chu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: Yung-Tsun
authors.initials: YT
authors.lastname: Lo
authors.affiliation: Imaging Core, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: Sue-Ping
authors.initials: SP
authors.lastname: Lee
authors.affiliation: Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Tzu-Jung
authors.initials: TJ
authors.lastname: Chen
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: Cheng-Yen
authors.initials: CY
authors.lastname: Huang
authors.affiliation: Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, Republic of China Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan, Republic of China Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan, Republic of China VYM Genome Research Center, National Yang-Ming University, Taipei, Taiwan, Republic of China Infection and Immunity Research Center, National Yang-Ming University, Taipei, Taiwan, Republic of China mbwen@ccvax.sinica.edu.tw yhping@ym.edu.tw.
authors.forename: Yueh-Hsin
authors.initials: YH
authors.lastname: Ping
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China mbwen@ccvax.sinica.edu.tw yhping@ym.edu.tw.
authors.forename: Wen
authors.initials: W
authors.lastname: Chang
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005453:Fluorescence; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000502:physiology*; D016296:Mutagenesis; D034741:RNA, Small Interfering / Q000235:genetics; D014616:Vaccinia virus / Q000502:physiology*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C484191:RAB22A protein, human; C549919:WASH protein, human