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Journal of Virology, May 2007, p. 5362-5374, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.02349-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Min Deng,1 Jennifer N. Bragg,1 Steven Ruzin,1,2 Denise Schichnes,1,2 David King,3 Michael M. Goodin,4 and Andrew O. Jackson1*

Department of Plant and Microbial Biology,1 Biological Imaging Facility,2 Molecular and Cell Biology Department and HHMI Mass Spectrometry Laboratory, University of California, Berkeley, California 94720,3 Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546-00914

Received 26 October 2006/ Accepted 23 February 2007

Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). 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 colocalization 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 {alpha} homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin {alpha} binding. The P protein did not bind to the importin {alpha} 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.

* Corresponding author. Mailing address: Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720. Phone: (510) 642-3906. Fax: (510) 642-4995. E-mail: andyoj{at}berkeley.edu

{triangledown} Published ahead of print on 7 March 2007.

Journal of Virology, May 2007, p. 5362-5374, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.02349-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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