title: Nucleocapsid protein from fig mosaic virus forms cytoplasmic agglomerates that are hauled by endoplasmic reticulum streaming.
abstract: Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming.
  
  
  OBJECTIVE
  Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Miura
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Kensaku
authors.initials: K
authors.lastname: Maejima
authors.affiliation: Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tomomitsu
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Misato
authors.initials: M
authors.lastname: Fukuoka
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Yusa
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Yamaji
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan anamba@mail.ecc.u-tokyo.ac.jp.
authors.forename: Shigetou
authors.initials: S
authors.lastname: Namba
mesh_terms: D003593:Cytoplasm / Q000821:virology*; D004721:Endoplasmic Reticulum / Q000821:virology*; D030681:Ficus; D016253:Microscopy, Immunoelectron; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D010942:Plant Viruses / Q000502:physiology*; D055503:Protein Multimerization; D021381:Protein Transport; D012328:RNA Viruses / Q000502:physiology*; D014026:Nicotiana
chemical_list: D019590:Nucleocapsid Proteins