Orchid fleck virus structural proteins N and P form intranuclear viroplasm like structures in the absence of viral infection

Hideki Kondo, Sotaro Chiba, Andika Ida Bagus, Kazuyuki Maruyama, Tetsuo Tamada, Nobuhiro Suzuki

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)7423-7434
Number of pages12
JournalJournal of Virology
Volume87
Issue number13
DOIs
Publication statusPublished - Jul 2013

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Orchid fleck virus
viral morphology
Viral Structural Proteins
structural proteins
Virus Diseases
Nucleorhabdovirus
infection
Proteins
proteins
Viruses
Nuclear Localization Signals
nuclear localization signals
Plant Cells
Nicotiana benthamiana
viral inclusion bodies
Flecks
importins
Karyopherins
cells
nucleocapsid proteins

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Orchid fleck virus structural proteins N and P form intranuclear viroplasm like structures in the absence of viral infection. / Kondo, Hideki; Chiba, Sotaro; Ida Bagus, Andika; Maruyama, Kazuyuki; Tamada, Tetsuo; Suzuki, Nobuhiro.

In: Journal of Virology, Vol. 87, No. 13, 07.2013, p. 7423-7434.

Research output: Contribution to journalArticle

Kondo, Hideki ; Chiba, Sotaro ; Ida Bagus, Andika ; Maruyama, Kazuyuki ; Tamada, Tetsuo ; Suzuki, Nobuhiro. / Orchid fleck virus structural proteins N and P form intranuclear viroplasm like structures in the absence of viral infection. In: Journal of Virology. 2013 ; Vol. 87, No. 13. pp. 7423-7434.
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