1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Correspondence
Eugene V. Ryabov
eugene.ryabov{at}hri.ac.uk
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ABSTRACT |
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MAIN TEXT |
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Umbraviral 2629 kDa ORF3 proteins show no significant similarity with any recorded or predicted proteins other than each other (Taliansky et al., 1996). ORF3 proteins have been shown to stabilize Tobacco mosaic virus (TMV) RNA and facilitate its long-distance movement within infected plants, replacing TMV CP, which is normally essential for both these functions (Ryabov et al., 1999b
, 2001
). These properties suggest that ORF3 proteins can interact with viral RNA to protect it and transport it to and through the phloem. In support of this hypothesis, it has been shown that GRV-encoded ORF3 protein is able to interact with viral RNA to form filamentous ribonucleoprotein (RNP) particles, which have elements of regular helical structure but not the uniformity typical of virus particles (Taliansky et al., 2003
).
In infected cells, GRV ORF3 protein expressed as a fusion protein with green fluorescent protein (GFP) from a TMV vector was located in cytoplasmic granules, some of which were associated with TMV-specific amorphous X-body inclusions, and also in nuclei, preferentially targeting nucleoli (Ryabov et al., 1998). To confirm nuclear localization of the GRV ORF3 protein in the case of native GRV infection, Nicotiana benthamiana leaf tissue systemically infected with GRV (YB isolate) was analysed by immunoelectron microscopy using antibodies against the ORF3 protein, as described by Ryabov et al. (1999a)
. Although in healthy (control) tissues gold label was occasionally found in nuclei non-specifically associated with chromatin, it was never detected in nucleoli. In contrast, in GRV-infected cells the label was found not only in the nucleus but also the nucleolus, indicating specific incorporation of the ORF3 protein in the nucleolus and therefore in the nucleus (Fig. 1
a, b). Localization of GRV ORF3 in the nucleus indicates that this protein can be transported between cytoplasm and nucleus during the course of virus infection. As a first step towards understanding the functions of GRV ORF3 protein in nuclei and nucleoli, we mapped regions of the GRV ORF3 protein that are involved in nucleocytoplasmic shuttling.
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In N. benthamiana plants inoculated with RNA transcripts from pTMV.GFP-GRV3(RN), nuclear (and nucleolar) accumulation of the GFPGRV3(RN) protein was dramatically reduced (or not observed at all) compared with the wild-type GFPGRV3 fusion protein (Fig. 1d and e; Table 1
). This could be the result of disruption of the nuclear import of GFPGRV3(RN). In contrast, free GFP expressed from the TMV vector was clearly visible in the nucleoplasm without specific association with nucleoli (Fig. 1c
). The molecular mass of the GFPGRV ORF3 fusion proteins (approximately 58 kDa) exceeds the size exclusion limit of the nuclear pore and thus cannot diffuse passively into the nucleus (Nigg, 1997
). Therefore, the lack (or reduced rate) of nuclear import of the GFPGRV3(RN) protein is the most likely cause of the reduced, or absence of, accumulation of the fusion protein in the nucleoplasm, thus confirming the suggestion that the region of aa 108122 of GRV ORF3 protein acts as a NLS.
Inspection of the predicted amino acid sequence of the GRV ORF3 protein revealed that the region between residues 148 and 156 contains five leucine residues, three of which (positions 150, 153 and 154) are conserved among all umbraviral ORF3 proteins (Taliansky & Robinson, 2003). Leucine-rich hydrophobic sequences of 1013 aa have been identified as an essential nuclear export signal (NES) (Wen et al., 1995
; Gorlich & Mattaj, 1996
; Nigg, 1997
; Haasen et al., 1999
) in a wide range of proteins that shuttle between the nucleus and cytoplasm, including the Rev protein of Human immunodeficiency virus 1 (Fischer et al., 1995
), transcription factor TFIIIA from Xenopus (Fridell et al., 1996
) and the BR1 protein of Squash leaf curl virus (SqLCV) (Ward & Lazarowitz, 1999
).
To test the possible involvement of the leucine-rich region of the GRV ORF3 protein (aa 148156) in nuclear export, we substituted GRV ORF3 protein leucine residues 148, 149, 152, 153 and 156 with alanine residues to produce the construct pTMV.GFP-GRV3(LA). N. benthamiana plants inoculated with this construct showed the presence of aggregates of the GFPGRV ORF3(LA) protein in nuclei at much higher levels compared with accumulation of the GFP fusion with wild-type GRV ORF3 (Table 1; Fig. 1d and f
). This higher level of GFPGRV ORF3(LA) protein accumulation in nuclei (Table 1
) compared with GFP fused to the wild-type GRV ORF3 protein suggested that the leucine-rich region of GRV ORF3 act as a NES. Nevertheless, it cannot be completely ruled out that leucine-to-alanine substitutions may contribute to better nuclear import of the protein. To confirm that the leucine-rich region of GRV ORF3 protein (aa 148156) is a NES, we used an approach that exploits the ability of NESs derived from different proteins functionally to replace one another (Wen et al., 1995
; Haasen et al., 1999
; Ward & Lazarowitz, 1999
). For example, the NES derived from Xenopus TFIIIS protein can functionally replace the NES of BR1 protein of SqLCV in nuclear export (Ward & Lazarowitz, 1999
). We prepared a TMV vector construct, pTMV.GFP-GRV3-SqLC-NES, that expressed GFP-tagged GRV ORF3 proteins containing the NES sequence of the BR1 protein of SqLCV (the region between aa 184 and 194; Ward & Lazarowitz, 1999
) inserted in place of the putative GRV ORF3 NES (the region between aa 147 and 157). In addition, the construct pTMV.GFPGRV3-SqLC-NES(LA), which contained the NES of SqLCV BR1 in which leucine residues essential for nuclear export (Ward & Lazarowitz, 1999
) were substituted by alanine residues (see Table 1
and Fig. 2
), was used as a control. Fluorescence microscopy of infection foci induced by the viral construct TMV.GFPGRV3-SqLC-NES showed that the level of GFPGRV3-SqLCV-NES accumulation in nuclei was similar to that of the wild-type GRV ORF3 fusion protein (Table 1
and Fig. 1
d and g), while substitution of the leucine residues in the BR1 SqLCV NES sequence inserted into the GRV ORF3 protein [TMV.GFPGRV3-SqLC-NES(LA) construct] resulted in a significant increase in GFPGRV3-SqLCV-NES(LA) protein accumulation in nuclei (Table 1
and Fig. 1h
). These results strongly supported the suggestion that the leucine-rich region (aa 148157) of GRV ORF3 is essential for nuclear export.
Regulation of bidirectional trafficking of macromolecules between the nucleus and cytoplasm through the nuclear pore complex lies at the core of many fundamental cellular processes. Nucleocytoplasmic shuttling in plant cells has been reported for proteins such as transcription factors that function in the nucleus and proteins of DNA viruses that replicate in the nucleus (Kosugi & Ohashi, 2002; Sanderfoot & Lazarowitz, 1995
; Sanderfoot et al., 1996
). Replication of GRV occurs in the cytoplasm, where the GRV ORF3 protein forms RNP complexes that presumably take part in viral RNA protection and its long-distance phloem-associated movement. The importance of nuclear import and export of GRV ORF3 protein is supported by the observation that the NLS and NES of the GRV ORF3 protein, which account for approximately 10 % of the umbraviral ORF3 protein sequence, were the most conserved regions (data not shown).
Characterization of the NES and NLS of the GRV ORF3 protein provides the basis for further research aimed at elucidating the role of nuclear involvement of the ORF3 protein in the development of umbravirus infection and the biological significance of its nuclearcytoplasmic shuttling.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Fischer, U., Huber, J., Boelens, W. C., Mattaj, I. W. & Luhrmann, R. (1995). The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475483.[Medline]
Fridell, R. A., Fischer, U., Luhrmann, R., Meyer, B. E., Meinkoth, J. L., Malim, M. H. & Cullen, B. R. (1996). Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev. Proc Natl Acad Sci U S A 93, 29362940.
Gibbs, M. G., Cooper, J. I. & Waterhouse, P. M. (1996). The genome organization and affinities of an Australian isolate of carrot mottle umbravirus. Virology 224, 280289.
Gorlich, D. & Mattaj, I. A. (1996). Nucleocytoplasmic transport. Science 271, 15131518.[Abstract]
Haasen, D., Kohler, C., Neuhaus, G. & Merkle, T. (1999). Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana. Plant J 20, 695705.[CrossRef][Medline]
Higuchi, R., Krummel, B. & Saaki, R. K. (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16, 73517367.[Abstract]
Kalderon, D., Richardson, W. D., Markham, A. F. & Smith, A. E. (1984). Sequence requirements for nuclear localisation of SV40 large T antigen. Nature 311, 3338.[Medline]
Kosugi, S. & Ohashi, Y. (2002). Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation. Plant Physiol 128, 833843.
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105132.[Medline]
Nigg, E. A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779787.[CrossRef][Medline]
Robbins, J., Dilworth, S. M., Laskey, R. A. & Dingwall, C. (1991). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615623.[Medline]
Ryabov, E. V., Oparka, K. J., Santa Cruz, S., Robinson, D. J. & Taliansky, M. E. (1998). Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors. Virology 242, 303313.[CrossRef][Medline]
Ryabov, E. V., Roberts, I. M., Palukaitis, P. & Taliansky, M. E. (1999a). Host-specific cell-to-cell and long-distance movements of cucumber mosaic virus are facilitated by the movement protein of groundnut rosette virus. Virology 260, 98108.[CrossRef][Medline]
Ryabov, E. V., Robinson, D. J. & Taliansky, M. E. (1999b). A plant virus-encoded protein facilitates long-distance movement of heterologous viral RNA. Proc Natl Acad Sci U S A 96, 12121217.
Ryabov, E. V., Robinson, D. J. & Taliansky, M. (2001). Umbravirus-encoded proteins that both stabilise heterologous viral RNA in vivo and mediate its systemic movement in some plant species. Virology 288, 391400.[CrossRef][Medline]
Sanderfoot, A. A. & Lazarowitz, S. G. (1995). Cooperation in viral movement: the geminivirus BL1 movement protein interacts with BR1 and redirects it from the nucleus to cell periphery. Plant Cell 7, 11851194.
Sanderfoot, A. A., Ingram, D. J. & Lazarowitz, S. G. (1996). A viral movement protein as a nuclear shuttle: the geminivirus BR1 movement protein contains domains essential for interaction with BL1 and nuclear localization. Plant Physiol 110, 2333.
Taliansky, M. E. & Robinson, D. J. (2003). Molecular biology of umbraviruses: phantom warriors. J Gen Virol 84, 19511960.
Taliansky, M. E., Robinson, D. J. & Murant, A. F. (1996). Complete nucleotide sequence and organisation of the RNA genome of groundnut rosette umbravirus. J Gen Virol 77, 23352345.[Abstract]
Taliansky, M., Roberts, I. M., Kalinina, N., Ryabov, E. V., Raj, S. K., Robinson, D. J. & Oparka, K. J. (2003). An umbraviral protein, involved in long-distance RNA movement, binds viral RNA and forms unique, protective ribonucleoprotein complexes. J Virol 77, 30313040.
Van Wezel, R., Liu, H., Wu, Z., Stanley, J. & Hong, Y. (2003). Contribution of the zinc finger and DNA binding by suppressor of post-transcriptional gene silencing. J Virol 77, 696700.[CrossRef][Medline]
Ward, B. M. & Lazarowitz, S. G. (1999). Nuclear export in plants: use of geminivirus movement proteins for a cell-based export assay. Plant Cell 11, 12671276.
Wen, W., Mienkoth, J. L., Tsien, R. Y. & Taylor, S. S. (1995). Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463473.[Medline]
Received 26 November 2003;
accepted 28 January 2004.