title: Orchid fleck virus structural proteins N and P form intranuclear viroplasm-like structures in the absence of viral infection.
abstract: Orchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-α homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N.
authors.affiliation: Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki, Japan. hkondo@rib.okayama-u.ac.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Kondo
authors.forename: Sotaro
authors.initials: S
authors.lastname: Chiba
authors.forename: Ida Bagus
authors.initials: IB
authors.lastname: Andika
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Maruyama
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Tamada
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Suzuki
mesh_terms: D001426:Bacterial Proteins; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D007150:Immunohistochemistry; D007181:Inclusion Bodies, Viral / Q000378:metabolism*; D007211:Indoles; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D019913:Nuclear Localization Signals / Q000235:genetics; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012328:RNA Viruses / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D014026:Nicotiana / Q000821:virology*; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007211:Indoles; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D028901:alpha Karyopherins; C054562:yellow fluorescent protein, Bacteria; C007293:DAPI