Nucleolar localization of potato leafroll virus capsid proteins

Sophie Haupt1,2,{dagger}, Tanya Stroganova1,{dagger}, Eugene Ryabov3, Sang Hyon Kim1, Gill Fraser1, George Duncan1, Mike A. Mayo1, Hugh Barker1 and Michael Taliansky1

1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 University of Dundee, Dundee DD1 4NH, UK
3 University of Warwick – HRI, Wellesbourne, Warwick CV35 9EF, UK

Correspondence
Michael Taliansky
mtalia{at}scri.sari.ac.uk


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Potato leafroll virus (PLRV) encodes two capsid proteins, major protein (CP) and minor protein (P5), an extended version of the CP produced by occasional translational ‘readthrough’ of the CP gene. Immunogold electron microscopy showed that PLRV CP is located in the cytoplasm and also localized in the nucleus, preferentially targeting the nucleolus. The nucleolar localization of PLRV CP was also confirmed when it was expressed as a fusion with green fluorescent protein (GFP) via an Agrobacterium vector. Mutational analysis identified a particular sequence within PLRV CP involved in nucleolar targeting [the nucleolar localization signal (NoLS)]. Minor protein P5 also contains the same NoLS, and was targeted to the nucleolus when it was expressed as a fusion with GFP from Agrobacterium. However, P5–GFP lost its nucleolar localization in the presence of replicating PLRV.

{dagger}These authors contributed equally to this paper.


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The RNA genome of Potato leafroll virus (PLRV; genus Polerovirus, family Luteoviridae) contains six large open reading frames (ORFs) (Fig. 1). The 5'-located ORFs encode a potential silencing suppressor protein (P0; Pfeffer et al., 2002) and RNA polymerase (ORF1 and ORF1/2) (Mayo & Ziegler-Graff, 1996). Within the 3'-located gene cluster, ORF3 encodes the major capsid protein (CP; ~23 kDa), and ORF4, which is contained within the CP gene in a different reading frame, encodes a movement protein (P4; Tacke et al., 1993). ORF5 is linked to ORF3 by a single amber termination codon and is expressed by occasional translational readthrough of this codon [P5 or ‘readthrough’ (RT) product] (Bahner et al., 1990). In addition to the major CP, PLRV particles also contain small amounts of P5 as the minor capsid protein (Bahner et al., 1990). In infected plants, PLRV is mainly restricted to cells in the vascular system (reviewed by Mayo & Ziegler-Graff, 1996; Taliansky et al., 2003). This restriction possibly occurs because PLRV movement functions do not operate in epidermis and mesophyll cells (Ryabov et al., 2001), and because PLRV cannot evade silencing-like host-defence responses in non-vascular tissues (Waterhouse et al., 1999; Voinnet et al., 1999; Barker et al., 2001; Savenkov & Valkonen, 2001).



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Fig. 1. Schematic representations of the PLRV genome and the various pGreen-based vectors. Boxes represent ORFs; lines represent untranslated sequences; t, leaky termination codon; 35S, Cauliflower mosaic virus 35S RNA promoter; poly(A) signal, nopaline synthase poly(A) sequence. (a) PLRV genome; (b) vector expressing PLRV CP-GFP; (c) a mutant version of (b), PLRV CP{Delta}(17-31)-GFP with the arginine-rich domain deleted (sequence and positions are indicated); (d) PLRV-GFP (cDNA encoding the GFP was inserted into the XhoI restriction site; Nurkiyanova et al., 2000); (e) PLRV CP-RT-GFP [in which the leaky termination codon was deleted and the RT portion truncated by insertion of GFP into the XhoI site (CP-RT-GFP)]; (f) a vector expressing free GFP; (g) PLRV CP(17-31)-GFP in which the sequence encoding the PLRV CP fragment 17PRRRRRQSLRRRANR31 was fused to the N-terminus of GFP; (h) GRV ORF3-GFP (Ryabov et al., 2004); (i) a mutant version of (h) in which the arginine-rich domain was deleted, GRV ORF3{Delta}(108-122)-GFP (sequence and positions are indicated); (j) chimeric GRV ORF3/CP(17-31)-GFP containing the 17PRRRRRQSLRRRANR31 sequence (putative NoLS) of the PLRV CP inserted in place of GRV ORF3 NoLS (ORF3 108RPRRRAGRSGGMDPR122).

 
Several reports have suggested an association of luteoviruses with the nucleus. For example, particles of Beet western yellows virus (Esau & Hoefert, 1972) and Barley yellow dwarf virus (BYDV; Gill & Chong, 1975; Nass et al., 1995) have been found in nuclei. Although no information on the nuclear association of PLRV during infection is available, expression in insect cells of the PLRV CP gene from a recombinant baculovirus results in the accumulation of CP in the nuclei (Lamb et al., 1996). These observations fit well with the prediction that luteovirus coat proteins have putative nuclear localization signals (NLS) that consist of highly basic sequences near the N-termini (Garcia-Bustos et al., 1991; Hanover, 1992; Mukherjee et al., 2003).

To explore intracellular localization of the PLRV CP in PLRV-infected plants, we used transgenic Nicotiana benthamiana plants (CW1 line) expressing the full-length PLRV genome as a transgene which is strongly silenced (Barker et al., 2001). To allow PLRV to multiply in cells, these transgenic plants were either challenged with Tobacco mosaic virus (TMV) or heat-shocked as described by Taliansky et al. (2004). Immunogold electron microscopy (IGEM) confirmed that both treatments led to a significant increase in PLRV accumulation in big groups of cells, comparable to those detected by tissue immunoprinting (Barker et al., 2001). Regardless of the means used to induce PLRV multiplication, the cytoplasm of infected cells contained large amounts of PLRV particles labelled with PLRV antibody (Fig. 2b). The nuclei, and the nucleoli in particular, were also labelled with PLRV antibody, although virus particles were not seen in these structures, suggesting that the CP accumulates in them in a form other than virus particles (Fig. 2a, c). Although in control (non-transgenic) plants, gold label was occasionally found in nuclei non-specifically associated with chromatin, it was never detected in nucleoli, indicating specific association of the PLRV CP with the nucleolus. In many cells, tubules were found that were labelled with PLRV antibodies. The tubules had a diameter of ~15–20 nm, were present in all cell types, including phloem parenchyma and companion cells, and resembled the fibrillar structures found by Shepardson et al. (1980) in potato plants aphid-infected with PLRV or by Nass et al. (1998) in BYDV-PAV-infected oat plants. These tubules were localized in the cytoplasm (Fig. 2a, d) and nucleus, specifically targeting and often disturbing the integrity of the nucleolus (Fig. 2a, c). In another approach, we used Pea enation mosaic virus-2 (PEMV-2) to help PLRV invade mesophyll tissues, as described by Ryabov et al. (2001). Intracellular distribution of the PLRV CP in doubly infected (PLRV+PEMV-2) wild-type N. benthamiana plants was similar to that described for CW1 transgenic plants (data not shown) and therefore was not affected by PEMV-2. The fact that the localization of PLRV CP did not depend on a factor used for development of PLRV multiplication strongly suggests that the nucleolar localization of the PLRV CP is intrinsic to the protein itself rather than being induced by other factors used for the induction of PLRV multiplication. Using transgenic Nicotiana tabacum plants expressing the full-length PLRV genome (Franco-Lara et al., 1999), similar results were obtained, thus demonstrating that nucleolar targeting is not restricted to N. benthamiana (data not shown).



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Fig. 2. Intracellular localization of PLRV CP in N. benthamiana transgenic (CW1) plants. Electron photomicrographs showing: (a) a general view of a cell infected with PLRV [note that the outlined areas are shown in (c) and (d) at a higher magnification]; (b) another cell showing a large number of PLRV particles labelled with PLRV antibody in the cytoplasm; (c) the nucleus and nucleolus [in the cell shown in (a)] also labelled with PLRV antibody and containing tubules but no virus particles; (d) cytoplasmic tubules [in the cell shown in (a)] labelled with PLRV antibody. Immunogold labelling of sections was done using polyclonal antibody raised against PLRV and goat anti-rabbit secondary antibody conjugated to gold particles. Bars, 1 µm (a, c) and 250 nm (b, d). N, nucleus; No, nucleolus; Tu, tubules detected in the nucleus and cytoplasm.

 
To investigate whether the PLRV CP could transport an exogenous green fluorescent protein (GFP) to the nucleolus, we generated a cDNA construct encoding PLRV CP fused to the N-terminus of GFP (PLRV CP-GFP) (Fig. 1). This construct included the Cauliflower mosaic virus 35S promoter and a nopaline synthase poly(A) signal, and was inserted into the binary plant expression vector pGreen 0029 (Hellens et al., 2000) to form pGR.PLRV CP-GFP. It was electroporated into Agrobacterium tumefaciens carrying plasmid pSoup as a helper (Hellens et al., 2000). Transient expression of PLRV CP-GFP was achieved by infiltration of the Agrobacterium to the underside of a leaf using a syringe without a needle. Confocal laser scanning microscopy (CLSM) was used to monitor the intracellular localization of the PLRV CP-GFP. To locate nuclei precisely, leaf tissues were mounted in a buffer containing 4',6'-diamidino-2-phenylindole (DAPI) and the same cells were excited at different wavelengths to visualize GFP and DAPI fluorescence. The green fluorescence was present mainly in nuclei, preferentially targeting nucleoli of all cell types (including phloem parenchyma and companion cells), although it was also found in cytoplasm (Fig. 3a). In contrast, free GFP expressed from the pGreen 0029 vector (pGR.GFP) was clearly visible in the cytoplasm and nucleoplasm, but was never detected in nucleoli (Fig. 3b).



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Fig. 3. Intracellular localization of PLRV CP derivatives fused to GFP. Confocal images of wild-type (a–h) and transgenic (CW1; i, j) N. benthamiana plants 5 days after agroinfiltration with constructs expressing PLRV CP-GFP (a, j), free GFP (b), PLRV CP{Delta}(17-31)-GFP (c), PLRV CP(17-31)-GFP (d), ORF3/CP(17-31)-GFP (e), GRV ORF3{Delta}(108-122)-GFP (f) and PLRV CP-RT-GFP (g, i), and infected with PLRV-GFP (h). For GFP imaging, excitation at 488 nm and collection between 500 and 513 nm were used. In (a) and (b), panels iv represent images of whole cells, while panels i represent higher magnification. For DAPI imaging, excitation at 405 nm and collection between 449 and 461 nm were used (panels ii). Panels iii represent overlay images. N, nucleus; No, nucleolus. Positions of the nucleoli are indicated by arrows (GFP images) or an arrowhead (DAPI images). Bars, 5 µm for panels i (a–j) and 25 µm for panels iv (a, b). Bright fluorescent spots within the nucleus in GFP images in (c) and (f) are not associated with the nucleolus and possibly represent aggregates of the corresponding GFP fusion proteins in the nucleoplasm.

 
Analysis of the predicted amino acid sequence of different luteoviruses showed the presence of the conserved putative arginine-rich NLS near the N-termini (Garcia-Bustos et al., 1991; Hanover, 1992; Mukherjee et al., 2003). To determine if this region is also linked to nucleolar targeting of the PLRV CP, a nucleotide sequence corresponding to its 17PRRRRRQSLRRRANR31 fragment was deleted from the construct pGR.CP-GFP by overlap-extension PCR (Higuchi et al., 1988) to give pGR.CP{Delta}(17-31)-GFP (Fig. 1). CLSM showed that the nucleolar accumulation of the mutagenized protein [PLRV CP{Delta}(17-31)-GFP] was substantially reduced (or not observed at all) compared to the wild-type PLRV CP-GFP (Fig. 3c), although its localization in the nucleoplasm remained, possibly due to the well-known affinity of GFP for the nucleoplasm.

To confirm that the arginine-rich region of PLRV CP (17PRRRRRQSLRRRANR31) can direct the nucleolar localization of a protein, we prepared a pGreen 0029 vector-based construct [pGR.CP(17-31)-GFP; Fig. 1] in which fragment 17PRRRRRQSLRRRANR31 was fused to the N-terminus of GFP. CLSM demonstrated a clear nucleolar localization of this chimeric protein (Fig. 3d). We also investigated the ability of the 17PRRRRRQSLRRRANR31 sequence of the PLRV CP to functionally replace the NoLS of another plant virus protein localized to the nucleolus. As with the PLRV CP, the ORF3 protein encoded by Groundnut rosette virus (GRV) has been shown to target the nucleolus (Ryabov et al., 1998, 2004; Taliansky & Robinson, 2003; Kim et al., 2004). Arginine-to-asparagine substitutions in the region 108RPRRRAGRSGGMDPR122 of this protein abolished its accumulation in nucleoli (Ryabov et al., 2004), suggesting a role for this domain in nucleolar localization. Therefore, we prepared a construct [pGR.ORF3/CP(17-31)-GFP; Fig. 1] which expressed a GFP-tagged GRV ORF3 protein containing the PLRV CP 17PRRRRRQSLRRRANR31 sequence (putative NoLS) inserted in place of the GRV ORF3 NoLS (ORF3 108RPRRRAGRSGGMDPR122). CLSM showed that the ORF3/CP(17-31)-GFP protein generated as a result of such a replacement accumulated in the nucleolus (Fig. 3e). In contrast, the GRV ORF3-GFP protein with deletion of the ORF3 NoLS [GRV ORF3{Delta}(108-122); Fig. 1] used as a control was detected throughout the nucleoplasm and in some bodies associated with the nucleus, but not in the nucleolus (Fig. 3f). Collectively, these results add further support to the suggestion that the 17PRRRRRQSLRRRANR31 region of the PLRV CP is directly involved in the nucleolar localization of the protein as a NoLS.

Taking into account the presence of NoLS in the CP portion of the PLRV P5, it could be expected that the P5 would also be localized in the nucleolus. Indeed, when the cDNA construct encoding the P5–GFP fusion, with the leaky AUG termination codon deleted and a slightly truncated RT portion (CP-RT-GFP; Fig. 1), was expressed in plant tissues from the pGreen vector (pGR.CP-RT-GFP), green fluorescence was detected in the nucleolus (Fig. 3g). However, these results seemingly contradict our previous observations, in which no fluorescence was detected in nucleoli of plants expressing the same CP-RT-GFP protein from the full-length PLRV genome (PLRV-GFP; Nurkiyanova et al., 2000; see also Fig. 1). In this situation, fluorescence was observed throughout the nucleoplasm, but not in the nucleolus (Nurkiyanova et al., 2000; see also Fig. 3h), suggesting that the P5 can be retained outside of, or redirected from, the nucleolus in the presence of the replicating virus. To test this idea, the CP-RT-GFP protein was produced in transgenic plants expressing a full-length PLRV genome (line CW1) using an Agrobacterium-mediated delivery system. In contrast to non-transgenic (control) N. benthamiana plants (Fig. 3g), in CW1 transgenic plants green fluorescence was not targeted to the nucleolus (Fig. 3i), confirming the ability of the replicating PLRV to prevent CP-RT-GFP accumulation in the nucleolus. In contrast to CP-RT-GFP, the nucleolar localization of PLRV CP-GFP was not affected by replicating PLRV (Fig. 3j).

The nucleolus, a prominent subnuclear compartment, is regarded as the site of transcription and modification of rRNA and biogenesis of ribosomal subunits, and also participates in other aspects of cell function, including regulation of the cell cycle, cell growth, and ageing (Reviewed by Lamond & Earnshaw, 1998; Pederson, 1998; Cockell & Gasser, 1999; Carmo-Fonseca et al., 2000; Olson et al., 2000).

In this work we have shown that the PLRV CP region 17PRRRRRQSLRRRANR31 operates as a NoLS and directs the protein to the nucleolus. Because of the ability of GFP (used as a reporter protein fused to PLRV CP) to target nucleoplasm it is difficult to conclude whether the NoLS can also act as the NLS that targets the PLRV CP from the cytoplasm into the nucleus through the nuclear pore complex, or whether other PLRV CP sequences are involved in this process.

A specific role of nucleolar targeting of PLRV CP may be the modulation of host gene expression (for example by interacting with ribosomal components) to adapt it for the needs of virus infection. It is also possible that PLRV has developed the ability to ‘recruit’ a nucleolar component(s) to exploit its function(s) in virus replication, assembly or movement.

The NoLS of the PLRV CP is located near the N-terminus of the protein. Based on known structural protein sequences of different icosahedral viruses, the N-terminal arginine-rich domain is likely to be involved in the interaction with viral RNA (Dolja & Koonin, 1991). Thus, the RNA binding domain and the NoLS may overlap and compete with each other to modulate the involvement of the CP in different virus functions.

As with the major CP, the extended P5 version also containing the NoLS is directed to the nucleolus when it is individually expressed in plant tissues using an Agrobacterium delivery system. However, in the presence of replicating PLRV, only the major CP localizes to the nucleolus. We suggest that P5 protein does not accumulate in the nucleolus in the presence of PLRV infection because PLRV RNA or PLRV-encoded or -induced factors retain this protein outside the nucleolus.

The involvement of the nucleolus in virus infections could also be a feature of other viruses. Indeed, a number of animal viruses interact with the nucleolus and its proteins (reviewed by Hiscox, 2002). Many of these interactions are not restricted to any particular type of virus, with examples from retroviruses, DNA viruses and RNA viruses. Furthermore, the plant virus GRV encodes a long-distance movement protein that is also targeted to the nucleolus (Taliansky & Robinson, 2003; Kim et al., 2004; Ryabov et al., 2004).


   ACKNOWLEDGEMENTS
 
The work was supported by a grant-in-aid from the Scottish Executive Environment and Rural Affairs Department (SEERAD), by the Royal Society (Post-Doctoral NATO/Royal Society Fellowship for T. S.) and by HRI core Biotechnology and Biological Sciences Research Council (BBSRC) funding (to E. V. R.). We thank Dr Phil Mullineaux and Dr Roger Hellens (John Innes Centre) for pGreen 0029.


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Received 11 April 2005; accepted 27 June 2005.



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