Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein

Eugene V. Ryabov1,2, Sang Hyon Kim1 and Michael Taliansky1

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


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
The 27 kDa protein encoded by ORF3 of Groundnut rosette virus (GRV) is required for viral RNA protection and movement of viral RNA through the phloem. Localization studies have revealed that this protein is located in nuclei, preferentially targeting nucleoli. We have demonstrated that amino acids (aa) 108–122 of the GRV ORF3 protein contain an arginine-rich nuclear localization signal. Arginine-to-asparagine substitutions in this region decreased the level of the ORF3 protein accumulation in nuclei. A leucine-rich nuclear export signal (NES) was located at aa 148–156 of the GRV ORF3 protein. Leucine-to-alanine substitutions in this region resulted in a dramatic increase in GRV ORF3 protein accumulation in both nuclei and nucleoli. Consistent with this, we also showed that the previously identified NES of BR1 protein of Squash leaf curl virus can functionally replace the leucine-rich region of GRV ORF3 in nuclear export.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Umbraviruses are non-segmented positive-strand RNA viruses that differ from most other plant viruses in that they do not encode a conventional capsid protein (CP). Thus no recognizable virus particles are formed in infected plants. Umbraviral genomes encode at least three proteins. Two open reading frames (ORFs) at the 5' end of the RNA are expressed by a –1 frameshift mechanism to give a single protein that appears to be an RNA-dependent RNA polymerase (Demler et al., 1993; Gibbs et al., 1996; Taliansky et al., 1996). The other two ORFs overlap each other in different reading frames. ORF4 encodes a 27–29 kDa protein, which contains stretches of similarity with several plant virus-encoded movement proteins (MPs), which mediate the movement of viral RNA from cell to cell via plasmodesmata (Taliansky et al., 1996; Gibbs et al., 1996). In gene replacement experiments, the protein encoded by ORF4 of the umbravirus Groundnut rosette virus (GRV) was shown to be able to functionally replace the MPs of unrelated plant viruses (Ryabov et al., 1998, 1999a).

Umbraviral 26–29 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.



View larger version (101K):
[in this window]
[in a new window]
 
Fig. 1. Intracellular localization of the GRV ORF3 protein in mesophyll cells of N. benthamiana. (a) Electron micrograph showing nuclear localization of the GRV ORF3 protein in a GRV-infected cell. The boxed area is shown at higher magnification in (b). Immunogold labelling of the section was carried out using polyclonal antibodies raised against a synthetic peptide corresponding to part of the sequence of the GRV ORF3 protein (aa 71–92) (Taliansky et al., 2003) and goat anti-rabbit secondary antibody conjugated to 15 nm gold particles. Nu, nucleus; No, nucleolus. Bar, 500 nm. (c–h) Intracellular localization of free GFP and GFP fusions with GRV ORF3 arginine-rich-domain and leucine-rich-domain mutants expressed from the TMV-based vector. Only areas of the cells that include the nuclei and immediate surrounding cytoplasm are shown. For GFP imaging, excitation at 450–490 nm with an emission filter of 520 nm was used (top panels). For DAPI imaging, excitation at 356 nm with an emission filter of 420 nm was used (bottom panels). The positions of nuclei in the top panels are indicated with dashed lines. Chloroplast autofluorescence appeared red with filters for GFP. All cells are shown 7 days post-inoculation with the TMV vector constructs expressing free GFP (c), GFP–GRV ORF3 (d), GFP–GRV ORF3(RN) (e), GFP–GRV ORF3(LA) (f), GFP–GRV ORF3-SqLC-NES (g) or GFP–GRV3-SqLC-NES(LA) (h). Bars, 10 µm.

 
A series of constructs was made that encoded modified GRV ORF3 protein sequences, with GFP fused to each N terminus. Changes were introduced into the GFP–GRV ORF3 coding sequence of the construct pTMV.GFP-3/4 generated previously (Ryabov et al., 1998) by overlap-extension PCR using self-complementary mutagenic primers (Higuchi et al., 1988). DNA fragments encoding the mutagenized GFP–GRV ORF3 fusion proteins were then inserted into the TMV-based vector p30B (Fig. 2). A summary of amino acid substitutions made in the GRV ORF3 region of the fusion proteins expressed by the TMV vector are shown in Table 1. Preparation of DNA templates, in vitro RNA transcription and mechanical inoculation of plants were carried out as described by Ryabov et al. (2001). Fluorescent foci of infection were visible under illumination with long-wave UV light, 3–4 days post-inoculation, in the leaves of N. benthamiana plants inoculated with TMV-based in vitro transcripts expressing the GFP-tagged GRV ORF3 mutants. The size of all foci and intensity of GFP fluorescence at the time of sampling were approximately the same for all constructs described in Table 1 and were similar in brightness to infection foci induced by TMV expressing free GFP (data not shown). To assess the subcellular localization of each GFP-tagged fusion protein, fluorescent lesions were excised from inoculated leaves of N. benthamiana plants 7 days post-inoculation. Vacuum infiltration of leaf tissues with paraformaldehyde, fixation, embedding in agarose and sectioning in a cryostat were carried out as described by van Wezel et al. (2003). To locate nuclei precisely, sections were mounted in a buffer containing 4',6'-diamidino-2-phenylindole (DAPI) and the same cells were photographed with different filters to visualize GFP and DAPI fluorescence (Fig. 1c–h). Digital images were used to quantify the levels of accumulation of GFP-tagged proteins in the cytoplasm and nucleus using ImageJ Software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). To determine the proportion of GFP-tagged GRV ORF3 proteins that had accumulated in the nucleus (Table 1), the GFP fluorescence was quantified on the images obtained with GFP-specific filters in the areas of the images corresponding to the entire cells and to the nuclei of the same cells (Fig. 1 shows only areas of cells including the nuclei and immediately surrounding regions; entire cells are not shown).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Schematic representation of the TMV-based vector expressing the GFP–GRV ORF3 fusion gene showing positions and amino acid sequences of the arginine-rich ("R") and leucine-rich ("L") domains in the GRV ORF3 sequence. Positions of amino acid residues in the GRV ORF3 protein are indicated. Boxes represent ORFs; lines represent untranslated sequences. MP, TMV movement protein gene; CP, TMV capsid protein gene; {bullet}, subgenomic promoter.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Intracellular localization of the wild-type and mutant GRV ORF3 proteins fused with GFP

 
Analysis of the predicted amino acid sequence of the GRV ORF3 protein showed the presence of several arginine- and lysine-rich clusters. Nevertheless, only one of these regions, amino acids (aa) 108–122, showed considerable arginine conservation among all known umbraviral ORF3 sequences. This region contained five arginine residues, three of which (positions 111, 115 and 122) were conserved (Taliansky & Robinson, 2003). Clusters of basic amino acids were shown to act as a ‘classical’ nuclear localization signal (NLS) typified by those of SV40 T antigen (Kalderon et al., 1984) and nucleoplasmin (Robbins et al., 1991). The sequences of ‘classical’ NLSs do not fit a tight consensus (Gorlich & Mattaj, 1996); therefore, we suggested that the region of aa 108–122 of GRV ORF3 protein may serve as a NLS. To test this hypothesis we replaced all the arginine residues (aa 108, 110, 111, 112 and 115) with asparagine to generate the construct pTMV.GFP-GRV3(RN). Basic arginine residues were replaced with polar and neutral asparagine residues rather than with hydrophobic alanine residues to ensure that the mutagenized region had a hydropathy similar to that of the wild-type GRV ORF3 protein. Indeed, similar hydropathy profiles were obtained for the wild-type GRV ORF3 protein and for GRV ORF3(RN) proteins using an algorithm described by Kyte & Doolitle (1982) (data not shown).

In N. benthamiana plants inoculated with RNA transcripts from pTMV.GFP-GRV3(RN), nuclear (and nucleolar) accumulation of the GFP–GRV3(RN) protein was dramatically reduced (or not observed at all) compared with the wild-type GFP–GRV3 fusion protein (Fig. 1d and e; Table 1). This could be the result of disruption of the nuclear import of GFP–GRV3(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 GFP–GRV 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 GFP–GRV3(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 108–122 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 10–13 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 148–156) 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 GFP–GRV 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 GFP–GRV 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 148–156) 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.GFP–GRV3-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.GFP–GRV3-SqLC-NES showed that the level of GFP–GRV3-SqLCV-NES accumulation in nuclei was similar to that of the wild-type GRV ORF3 fusion protein (Table 1 and Fig. 1d and g), while substitution of the leucine residues in the BR1 SqLCV NES sequence inserted into the GRV ORF3 protein [TMV.GFP–GRV3-SqLC-NES(LA) construct] resulted in a significant increase in GFP–GRV3-SqLCV-NES(LA) protein accumulation in nuclei (Table 1 and Fig. 1h). These results strongly supported the suggestion that the leucine-rich region (aa 148–157) 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 nuclear–cytoplasmic shuttling.


   ACKNOWLEDGEMENTS
 
We thank Ian Roberts for help with immunoelectron microscopy and Rene van Wezel for help with fluorescent microscopy. We also thank Simon Santa Cruz for providing the pTMV.GFP construct and Stuart MacFarlane and Michael Wilson for critical reading of the manuscript. We also thank Michael Wilson for his encouragement throughout this work. This work was supported by grant-in-aid from the Scottish Executive Environment and Rural Affairs Department (SEERAD, to M. T.) and by HRI core BBSRC funding (to E. V. R.).


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Demler, S. A., Rucker, D. G. & de Zoeten, G. A. (1993). The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA-2. J Gen Virol 74, 1–14.[Abstract]

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, 475–483.[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, 2936–2940.[Abstract/Free Full Text]

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, 280–289.

Gorlich, D. & Mattaj, I. A. (1996). Nucleocytoplasmic transport. Science 271, 1513–1518.[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, 695–705.[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, 7351–7367.[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, 33–38.[Medline]

Kosugi, S. & Ohashi, Y. (2002). Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation. Plant Physiol 128, 833–843.[Abstract/Free Full Text]

Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105–132.[Medline]

Nigg, E. A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779–787.[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, 615–623.[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, 303–313.[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, 98–108.[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, 1212–1217.[Abstract/Free Full Text]

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, 391–400.[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, 1185–1194.[Abstract/Free Full Text]

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, 23–33.[Abstract/Free Full Text]

Taliansky, M. E. & Robinson, D. J. (2003). Molecular biology of umbraviruses: phantom warriors. J Gen Virol 84, 1951–1960.[Abstract/Free Full Text]

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, 2335–2345.[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, 3031–3040.[Abstract/Free Full Text]

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, 696–700.[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, 1267–1276.[Abstract/Free Full Text]

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, 463–473.[Medline]

Received 26 November 2003; accepted 28 January 2004.