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

      Journal of Biology
      Bacterial Proteins, Blotting, Western, Electrophoresis, Polyacrylamide Gel, Genetic Complementation Test, Immunohistochemistry, Inclusion Bodies, Viral, metabolism, Indoles, Luminescent Proteins, Microscopy, Confocal, Microscopy, Electron, Microscopy, Fluorescence, Mutagenesis, Nuclear Localization Signals, genetics, Nucleocapsid Proteins, Phosphoproteins, RNA Viruses, ultrastructure, Tobacco, virology, Two-Hybrid System Techniques, alpha Karyopherins

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          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.

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