JVI Accepts, published online ahead of print on 7 March 2007

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J. Virol. doi:10.1128/JVI.02349-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Role of the Sonchus Yellow Net Virus N Protein in Formation of Nuclear Viroplasms

Min Deng, Jennifer N. Bragg, Steven Ruzin, Denise Schichnes, David King, Michael M. Goodin, and Andrew O. Jackson^*

Department of Plant and Microbial Biology, Biological Imaging Facility, and Molecular and Cell Biology Department, HHMI Mass Spectrometry Lab, University of California, Berkeley, CA 94720, Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0091

^* To whom correspondence should be addressed. Email: andyoj{at}berkeley.edu.

Sonchus yellow net virus (SYNV) is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (22). When expressed alone, the N protein localizes to the nuclei of plant and yeast cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (7, 8). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells, and that homologous interactions mediated by a helix-loop helix region near the amino-terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N and P protein interactions that are required for N and P co-localization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N:N and N:P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin binding. The P protein did not bind to the importin homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N protein interactions, and that P protein targeting to the viroplasm requires N:P protein interactions that occur after N and P protein import into the nucleus.

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