Interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection

  1. Sang Hyon Kim†,
  2. Stuart MacFarlane†,
  3. Natalia O. Kalinina†,‡,
  4. Daria V. Rakitina†,‡,
  5. Eugene V. Ryabov§,
  6. Trudi Gillespie†,
  7. Sophie Haupt†,
  8. John W. S. Brown†, and
  9. Michael Taliansky†,
  1. †Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom;
  2. ‡A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia; and
  3. §Horticulture Research International, University of Warwick, Wellesbourne, Warwick CV35 9EF, United Kingdom

  1. Communicated by Bryan D. Harrison, Scottish Crop Research Institute, Dundee, United Kingdom, May 17, 2007 (received for review April 4, 2007)

 
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Abstract

The nucleolus and specific nucleolar proteins are involved in the life cycles of some plant and animal viruses, but the functions of these proteins and of nucleolar trafficking in virus infections are largely unknown. The ORF3 protein of the plant virus, groundnut rosette virus (an umbravirus), has been shown to cycle through the nucleus, passing through Cajal bodies to the nucleolus and then exiting back into the cytoplasm. This journey is absolutely required for the formation of viral ribonucleoprotein particles (RNPs) that, themselves, are essential for the spread of the virus to noninoculated leaves of the shoot tip. Here, we show that these processes rely on the interaction of the ORF3 protein with fibrillarin, a major nucleolar protein. Silencing of the fibrillarin gene prevents long-distance movement of groundnut rosette virus but does not affect viral replication or cell-to-cell movement. Repressing fibrillarin production also localizes the ORF3 protein to multiple Cajal body-like aggregates that fail to fuse with the nucleolus. Umbraviral ORF3 protein and fibrillarin interact in vitro and, when mixed with umbravirus RNA, form an RNP complex. This complex has a filamentous structure with some regular helical features, resembling the RNP complex formed in vivo during umbravirus infection. The filaments formed in vitro are infectious when inoculated to plants, and their infectivity is resistant to RNase. These results demonstrate previously undescribed functions for fibrillarin as an essential component of translocatable viral RNPs and may have implications for other plant and animal viruses that interact with the nucleolus.

The nucleolus is a subnuclear domain and is the site of transcription and processing of rRNA and of ribosome biogenesis. In addition, the nucleolus also participates in other aspects of RNA metabolism and cell function (1, 2). The nucleolus is structurally and functionally associated with Cajal bodies (CBs), which are structures found in both animals and plants (3, 4). CBs contain different proteins including coilin, a protein essential for CB formation, and fibrillarin, a major nucleolar protein that is a core component of small nucleolar ribonucleoprotein particles (snoRNPs) and is required for rRNA processing (4–7). CBs are involved in the maturation of small nuclear RNPs (snRNPs) and snoRNPs, which traffic through CBs before accumulating in splicing speckles and the nucleolus, respectively (8, 9). Both the nucleolus and CBs have a role in RNA silencing in plants (10, 11). Finally, a number of animal and plant viruses including the RNA-containing tobacco etch virus and the DNA-containing tomato yellow leaf curl virus have a nucleolar phase in their life cycle (12, 13). Recently, we have shown that the ability of the umbravirus, groundnut rosette virus (GRV), to move long distances through the phloem, the specialized vascular system used by plants for the transport of assimilates and macromolecules, depends strictly on the interaction of one of its proteins, the ORF3 protein, with CBs and the nucleolus (14).

Umbraviruses have RNA genomes and differ from most other viruses in that they do not encode a coat protein (CP) and so do not produce conventional virus particles in infected plants (15, 16). Nevertheless, they accumulate and spread efficiently within the infected plant; their lack of a CP is compensated for by the ORF3 protein. This protein fulfils umbraviral functions that are normally provided by the CPs of other plant viruses, such as long-distance movement of viral RNA through the phloem (17, 18).

The GRV ORF3 protein interacts with viral RNA in vivo to form filamentous RNP particles, which have elements of a regular helical structure but not the uniformity typical of virus particles (16). The RNPs accumulate in cytoplasmic inclusions that are the form in which the virus is thought to move through the phloem to cause systemic infection (16). In addition to its presence in the cytoplasm, the ORF3 protein is able to traffic into the nucleus, predominantly targeting the nucleolus (19, 20). The presence of the ORF3 protein in the nucleolus was unexpected, because the entire infection cycle of GRV and other umbraviruses was previously considered to be restricted to the cytoplasm. ORF3 proteins contain two conserved domains: an arginine-rich sequence (positions 108–122; R-rich domain) and a leucine-rich region (amino acids 148–156; L-rich domain) (Fig. 1) (16, 20). The R-rich domain is involved in nuclear import, and the L149 residue, in addition to the R-rich domain, is essential for nucleolar targeting of the ORF3 protein (14, 20). The whole L-rich region also acts as a nuclear export signal (20), suggesting that the ORF3 protein traffics between the nucleus (nucleolus) and cytoplasm of infected cells.

Fig. 1.
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Fig. 1.

Correlation between the ability of the ORF3 protein to traffic through the nucleolus and the relocalization of fibrillarin, formation of viral RNPs, and long-distance movement. Wild-type and mutant ORF3 protein sequences of the R-rich and L–rich domains are shown in combination with data on nuclear (N) and nucleolar (No) localization, nuclear export (N-exp) of the ORF3 protein, relocalization of fibrillarin (Cyt. fibrillarin), RNP formation, and virus long-distance movement (LDM) (14).


The ORF3 protein is produced in the cytoplasm, enters the nucleus, and is targeted to CBs. The CBs are then reorganized into multiple smaller structures (CB-like aggregates, CBLs) that move to and fuse with the nucleolus by an unknown mechanism (14). The ORF3 protein is exported from the nucleus, leading to the formation of cytoplasmic viral RNP particles that are transported to the rest of the plant via the phloem. The integral connection between nucleolar targeting of the ORF3 protein and its biological function in virus long-distance spread has been demonstrated by the introduction of mutations in the R- and L-rich domains that block nucleolar localization and nuclear export of the ORF3 protein, respectively, resulting in failure to form viral RNPs, and of their long-distance movement (14) (Fig. 1).

In elucidating the nuclear pathway of the ORF3 protein, we also observed the partial relocalization of the nucleolar protein, fibrillarin, to the cytoplasmic inclusions containing viral RNPs, whereas normally, fibrillarin does not accumulate in cytoplasm (14). Fibrillarin is one of the major proteins of the nucleolus. It is also localized to CBs, is a core component of box C/D snoRNPs, and has methyltransferase activity directing methylation of rRNA and snRNAs (21). Interaction of some animal viruses with fibrillarin and other nucleolar proteins has been reported (12, 13, 22, 23), but their specific role in virus infections remains elusive. In this article, we demonstrate a previously uncharacterized function of fibrillarin in umbravirus systemic infection. The GRV ORF3 protein directly interacts with fibrillarin, and this interaction is essential for nucleolar localization of the ORF3 protein. Fibrillarin is also required, along with umbravirus ORF3 protein and viral RNA, for the assembly of movement-competent, infectious RNP particles. Thus, the ORF3 protein may exploit fibrillarin trafficking to reach the nucleolus and use the properties of fibrillarin to form umbraviral RNPs.

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Results

Fibrillarin Knockdown Suppresses Long-Distance Movement of GRV.

Although fibrillarin is required for essential cell functions, a significant (≈50%) decrease in levels of fibrillarin is tolerated in mice (24). We therefore examined the effect of silencing of fibrillarin expression in plants on the nucleolar localization of the ORF3 protein and its function in virus long-distance movement. A virus-induced gene-silencing construct containing a fragment of the fibrillarin gene from Nicotiana benthamiana was made by using a Tobacco rattle virus (TRV) vector, giving TRV-NbFib [supporting information (SI) Text]. As a nonsilenced control, N. benthamiana plants were infected with TRV alone. Expression of fibrillarin mRNA and production of fibrillarin were suppressed by TRV-NbFib to different levels, and plants with effectively three different phenotypes were generated: (Group I) dwarf plants with necrosis showing complete or very strong silencing (>90% decrease in the expression of fibrillarin), (Group II) symptomless plants showing a partial (50–90%) reduction of fibrillarin expression, and (Group III) symptomless plants showing no or very weak (<10%) reduction of fibrillarin expression (Fig. 2 A–C). Because of the severely affected growth or the low level of silencing of the first and third classes of plants, respectively, plants of the second type were selected for further analysis. Immunofluorescence analysis of these plants by using anti-fibrillarin antibody showed that fibrillarin levels were considerably reduced in the nucleolus and were undetectable in CBs in any of the 50 analyzed cells (Fig. 2 D). CBs were visualized in silenced and nonsilenced plants by using antibody against coilin, a standard protein marker of CBs, which detected usually one but up to three CBs per cell, as normal for these cells (Fig. 2 D). These partially silenced plants had a normal growth phenotype (Fig. 2 A) and were able to support infection by tobacco mosaic virus (TMV) and potato virus X (PVX), accumulating virus in both inoculated and uninoculated systemically infected leaves to high levels (Table 1). This suggests that reduction of fibrillarin levels in the knockdown plants (in both nucleoli and CBs) did not inhibit normal cellular functions, such as translation, that are necessary for general virus infection.

Fig. 2.
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Fig. 2.

Virus-induced silencing of the fibrillarin gene (NbFib) in N. benthamiana plants by using a TRV vector. (A–C) Expression of fibrillarin was suppressed by TRV-NbFib to different levels, and plants with effectively three different phenotypes (I, II, and III) were generated. (A) Growth phenotypes of silenced plants in comparison with control (c) nonsilenced plants. (B) Semiquantitative RT-PCR analysis of NbFib mRNA accumulation. Ethidium bromide-stained agarose gels show RT-PCR products corresponding to the fragments of NbFib (Fib) mRNA (320 bp) and ubiquitin mRNA (176 bp) used as a control, as indicated by arrows. Lanes 1–3 represent plant replicates. (C) Western blot analysis of fibrillarin (Fib) accumulation. The position of fibrillarin is shown by an arrow. Lanes 1–3 as above. (D) Immunofluorescence staining of cells of fibrillarin-silenced (group II; Fib-s) and nonsilenced (Non-s) plants with fibrillarin and coilin antibodies visualized by confocal microscopy. Fibrillarin levels are significantly reduced in the nucleolus and are undetectable in CBs, visualized by using antibody against coilin. (Scale bars, 5 μm.)


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Table 1.

Accumulation of TMV and PVX in inoculated and uninoculated leaves determined by ELISA and by infectivity assays


In contrast to TMV and PVX, GRV was unable to cause systemic infection in the fibrillarin-silenced plants. Uninoculated tip leaves of these plants did not develop infection symptoms (Fig. 3 A). Accumulation of GRV in the inoculated leaves was comparable with that in inoculated leaves of nonsilenced plants, as shown by the presence of GRV RNA (Fig. 3 B) and by infectivity tests (Table 2), but upper uninoculated leaves of the silenced plants did not contain GRV RNA or infectious viral RNP (Fig. 3 B and C). Thus, the fibrillarin deficiency in these silenced plants did not affect the ability of the virus to replicate and move locally from cell to cell but inhibited long-distance virus movement. In agreement with this, in the partially silenced Group III plants that continue to express substantial amounts of fibrillarin (Fig. 2 A–C), GRV was able to move systemically, showing a correlation between the amount of fibrillarin expressed in plants and the ability of GRV to move long distances (Table 2).

Fig. 3.
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Fig. 3.

Fibrillarin knockdown suppresses long-distance movement of GRV and nucleolar localization of ORF3 protein. (A and B) Accumulation of GRV-YB in inoculated (in) and uninoculated (u) leaves of nonsilenced (Non-s) and fibrillarin-silenced (Fib-s) N. benthamiana plants of group II (A, B, and Table 2). (A) Symptoms on systemically infected, uninoculated leaves. Nonsilenced plants show yellow blotch symptoms characteristic of the GRV-YB isolate, whereas fibrillarin-silenced plants (group II) are symptomless. (B) Northern blot analysis of RNA isolated from leaves of the infected plants. GRV RNA is shown by an arrow; rRNA bands were stained by ethidium bromide for loading control (lower blot). (C) Localization of the ORF3-GFP protein delivered by Agrobacterium into cells of the nonsilenced and silenced plants. No, nucleolus; CBL, Cajal body-like structures; N, nuclei (shown by dashed lines according to DAPI staining). (Scale bars, 5 μm.)


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Table 2.

Accumulation of GRV-YB in inoculated (In) and uninoculated (U) leaves of nonsilenced (Non-s) and fibrillarin-silenced (Fib-s) N. benthamiana plants of Group III


ORF3 Protein Does Not Localize to the Nucleolus in Fibrillarin Knockdown Plants.

We next examined the subnuclear localization of the ORF3 protein using a fusion with green fluorescent protein (ORF3-GFP) delivered by Agrobacterium into cells of fibrillarin-silenced plants (Group II). The ORF3 protein localized to the nucleolus of nonsilenced plants as expected but did not localize to the nucleolus of silenced plants (Fig. 3 C). Instead, the ORF3-GFP protein localized to multiple subnuclear structures (Fig. 3 C). These contained coilin (data not shown) and were similar to the multiple CBLs (CB-like structures) observed previously upon GRV infection or with transient expression of ORF3 (14). This cellular phenotype mimics that seen with the ORF3 protein mutant, where the single leucine L149 was replaced with alanine (L149A-GFP; ref. 14; see Fig. 1). This mutant is defective in viral long-distance movement and is unable to locate to the nucleolus but, instead, accumulates in multiple CBLs (14) (Fig. 1). These results further confirm the correlation between localization of the ORF3 protein to the nucleolus and the ability of the virus to move systemically through the plant (14) and suggest that fibrillarin is involved in the fusion of CBs with the nucleolus and, thereby, the trafficking of the ORF3 protein to the nucleolus.

Interaction of ORF3 Protein with Fibrillarin.

The relocalization of some fibrillarin to cytoplasmic inclusions along with the ORF3 protein (14) and the altered ORF3 protein localization and its lack of function in virus long-distance movement in fibrillarin-silenced plants suggest that fibrillarin is essential for the GRV infection process. To determine whether there was a direct interaction between fibrillarin and the ORF3 protein, we examined the in vitro binding of recombinant GST-tagged fibrillarin [Arabidopsis fibrillarin 2 (Fib2), Fig. 4 A] (25) by the ORF3 protein. The ORF3 protein was expressed in plants from a TMV vector, isolated, and tested by using a blot overlay (far Western) assay. Fibrillarin- and GST-containing proteins were separated by SDS/PAGE, transferred to nitrocellulose membrane, renatured, incubated with purified ORF3 protein, and protein binding was visualized by using anti-ORF3 protein antibodies. For Fib2, two protein bands of 60 and 30–35 kDa were bound by the ORF3 protein (Fig. 4 B, lane 1). The larger band corresponded to GST-fibrillarin whereas the smaller protein was a fragment of fibrillarin (Fib2*) containing the N-terminal 79 amino acid residues still tagged with GST, as determined by mass spectroscopy (data not shown; Fig. 4 A). The GST-Fib2* fragment is presumably produced by degradation of the protein or truncation during translation. GST alone did not interact with the ORF3 protein antibody (Fig. 4 B, lane 3). Removal of GST from the recombinant fibrillarin by thrombin treatment did not disrupt binding of the ORF3 protein (Fig. 4 B, lane 4). The N-terminal fragment of fibrillarin contains the glycine- and arginine-rich (GAR) domain (Fig. 4 A) (25, 26) responsible for interaction with other proteins such as spinal muscular atrophy disease protein (27). A deletion mutant, where the GAR domain was removed from GST-fibrillarin (Fig. 4 A; GST-Fib2ΔGAR), was unable to interact with the ORF3 protein (Fig. 4 B, lane 2). Taken together, these results demonstrate that the ORF3 protein directly interacts with fibrillarin in vitro and that the N-terminal GAR domain of fibrillarin is necessary for this interaction.

Fig. 4.
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Fig. 4.

Fibrillarin interacts with ORF3 protein. (A) Representation of Arabidopsis fibrillarin 2 (Fib2) showing GAR, RNA binding and α-helix domains, the GST fusion protein (GST-Fib2), GST fused with Fib2 fragment (GST-Fib2*) and the GAR deletion mutant (GST-Fib2ΔGAR). Numbers are amino acid residue positions; triangle shows position of thrombin cleavage site. (B) Far Western blot analysis of the interaction between ORF3 protein and fibrillarin, Fib2. Blots containing GST-Fib2 (also containing GST-Fib2*; lane 1), GST-Fib2ΔGAR (lane 2), GST (lane 3) and Fib2 (GST-Fib2 treated with thrombin; lane 4) were incubated with or without the ORF3 protein [+ORF3 (Left) and −ORF3 (Center)] and probed by anti-ORF3 antibody. (Right) Coomassie staining. Positions of GST-Fib2, GST-Fib2*, and Fib2 are shown on the left and those of molecular mass markers are on the right. (C) Far Western blot analysis of the interaction between the purified recombinant ORF3 protein mutants and fibrillarin. Blots containing GST-Fib2 (and GST-Fib2*) were incubated with wild-type (1) or mutant ORF3 proteins (LA, 2; L149A, 3; L153A, 4; RA, 5) and probed as above. Lane 7 represents Ponceau S staining of fibrillarin. Lane 6 corresponds to a control sample prepared from a plant infected with the TMV vector alone by using the same protocol as for the recombinant ORF3 proteins. The lower blot shows expression levels of wild-type and mutant ORF3 proteins (Coomassie staining) as indicated. M, marker of 30 kDa.


To determine the effect on the interaction of the ORF3 protein and fibrillarin of mutations in the conserved R- and L-rich domains involved in ORF3 protein nuclear trafficking (Fig. 1) mutant proteins were produced in plants after expression from TMV vectors. The mutations are shown in Fig. 1 and have been described previously (14). Briefly, replacement of all six arginine residues in the R-rich domain by alanine residues gave the RA protein (Fig. 1). Mutations to the L-rich region replaced either all of the invariant leucine residues (positions 149, 152, and 153, LA), or individual leucines, with alanine residues (Fig. 1). Of these mutants, only LA and L149A were unable to bind fibrillarin in the far Western assay (Fig. 4 C), indicating that the L-rich domain (and L149 in particular) is involved in the interaction with fibrillarin.

Fibrillarin Mediates Assembly of Umbravirus RNP Particles in Vitro.

The ORF3 protein binds to umbraviral RNA in vitro but forms RNA–protein complexes with a completely different structure from viral RNPs in infected cells (16), suggesting that other factor(s) are involved in viral RNP assembly in vivo. Because of the ORF3 protein–fibrillarin interaction and the presence of fibrillarin in umbraviral cytoplasmic inclusions, we examined the effect of fibrillarin on the interaction of the ORF3 protein with viral RNA using EM. Umbraviral RNA, transcribed from a full-length cDNA clone of pea enation mosaic virus-2 (PEMV-2), an umbravirus closely related to GRV (15), was added to fibrillarin, to the ORF3 protein, or to mixtures of both proteins. Viral RNA and ORF3 protein were mixed at a 1:400 molar ratio (previously shown to saturate RNA binding with the ORF3 protein) (16). In EM, this gave nonuniform structures (Fig. 5 A) that were clearly distinct from RNP complexes formed by the ORF3 protein in vivo (16). The ORF3 protein–viral RNA complexes consisted of two structural elements: (i) thin interwoven filaments with a diameter of ≈3–4 nm and (ii) thicker (≈6–8 nm diameter) filaments, possibly reflecting different densities of protein coating the RNA (Fig. 5 A). Increasing the relative amount of ORF3 protein did not affect the structure of the complexes. Viral RNA and fibrillarin (Fib2) (at molar ratios of 1:400 to 1:800) formed complexes similar to those formed by the ORF3 protein and viral RNA (Fig. 5 B). RNA alone did not form any visible filaments in the same EM procedure (data not shown). Mixing of the ORF3 protein and fibrillarin in the absence of viral RNA also did not give rise to filamentous structures but led to formation of disk-like complexes with a diameter of ≈18–22 nm (Fig. 5 C) possibly consisting of both ORF3 protein and fibrillarin molecules.

Fig. 5.
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Fig. 5.

EM images showing complexes generated by different mixtures of umbraviral RNA, ORF3 protein, and fibrillarin. (A) Complex of GRV ORF3 protein and PEMV-2 RNA (F, thin filaments; DC, densely coated RNA). (B) Complex of Arabidopsis fibrillarin (Fib2) and PEMV-2 RNA (F and DC as above). (C) Disk-like complexes formed by ORF3 protein and Fib2 in the absence of RNA. (D) Complexes of ORF3 protein, Fib2 and RNA (an arrow shows elements of helical structure). Insets show higher magnification. (Scale bars: A–D, 100 nm; Insets, 50 nm.)


In contrast, incubation of viral RNA with both the ORF3 protein and fibrillarin (at molar ratios of 1:400:400) led to the formation of regular filamentous structures (Fig. 5 D) similar to viral RNP particles formed in vivo after GRV infection. In EM, the particles had a diameter of ≈20 nm, and helical repeat structures were observed on some filaments (Fig. 5 D), which is in good agreement with dimensions and structure of RNP particles formed by the GRV ORF3 protein in vivo. No regular RNP particles were formed with the fibrillarin mutant lacking the GAR domain (Fib2ΔGAR), which does not interact with the ORF3 protein (data not shown). Thus, the interaction between fibrillarin and the ORF3 protein is necessary to mediate formation of RNP particles similar in structure to GRV RNP particles formed in vivo.

Viral RNP Particles Formed in Vitro Are Infectious.

In addition to structural similarities, the RNP particles formed from the ORF3 protein, fibrillarin, and viral RNA in vitro were infectious and induced numerous lesions on the test plant, Chenopodium quinoa (Table 3). The infectivity of the in vitro RNP particles was neutralized by both anti-ORF3 protein and anti-fibrillarin antibodies, whereas control anti-TMV antibody did not affect the infectivity (Table 3), suggesting that both (ORF3 and fibrillarin) proteins are present in the RNP particles. Complexes formed by the ORF3 protein and viral RNA in the absence of fibrillarin were not infectious (Table 3). Finally, infectivity of the RNP particles was not affected by RNase treatment (Table 3), showing protection of the viral RNA by the ORF3 protein and fibrillarin.

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Table 3.

Infectivity of RNP complexes formed by the ORF3 protein, fibrillarin, and PEMV-2 RNA


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Discussion

To mediate virus long-distance movement and systemic infection, the umbravirus-encoded ORF3 protein is imported from the cytoplasm into the nucleus and nucleolus and then returns to the cytoplasm. The GRV ORF3 protein traffics to the nucleolus by a process involving the reorganization of CBs into CBLs and their fusion with the nucleolus. The most likely purpose of this nuclear/nucleolar trafficking of the ORF3 protein is to recruit the major nucleolar protein, fibrillarin, and relocalize it to cytoplasm (14). Here, we show that the GRV ORF3 protein is able physically to interact with fibrillarin. Our studies also demonstrate that fibrillarin is involved in at least two stages in the GRV life cycle: (i) CBL fusion with the nucleolus is mediated by the interaction between the ORF3 protein and fibrillarin, and (ii) the ORF3 protein redistributes some fibrillarin to the cytoplasm, where it functions in the formation of viral RNP particles that are capable of long-distance movement and causing systemic viral infection. This model demonstrates previously uncharacterized functions for fibrillarin in triggering nucleolar import of the ORF3 protein by CBLs by an unknown mechanism and mediating assembly of umbraviral RNPs.

The trafficking of the ORF3 protein to CBs and their reorganization into CBLs does not require the ORF3–fibrillarin interaction, because the L149A mutant, which is unable to interact with fibrillarin, enters the nucleus and accumulates in CBLs (14). In contrast, the fusion of CBLs with the nucleolus is presumably mediated by the direct interaction between the L-rich region (L149 in particular) of the ORF3 protein and the GAR domain of fibrillarin. This interaction is likely to occur in CBs or CBLs because the L149A mutant does not interact with fibrillarin or cause CBL fusion with the nucleolus. Further support of a role for fibrillarin in this fusion is provided by the fibrillarin knockdown experiments. When no fibrillarin was detectable in CBs/CBLs, the interaction between the ORF3 protein and fibrillarin could not occur, and the ORF3 protein accumulated in multiple CBLs that did not fuse with the nucleolus. Thus, the fibrillarin-silenced plants (Group II) exhibited a phenotype equivalent to that of plants infected with the L149A or LA umbravirus ORF3 mutants: neither mutant allowed the fusion of CBLs with the nucleolus or systemic spread (14). It is also important that both TMV and PVX were able to move long distances in fibrillarin-silenced plants, suggesting that fibrillarin is involved only in the movement of plant viruses, such as umbraviruses, that have a nucleolar phase of infection. Although it is possible that the effects seen in the knockdown (Group II) plants could be due to “off-target” silencing events, taken together with results on the inability of the ORF3 L149A and LA mutant proteins to interact with fibrillarin (Fig. 4 C), our data strongly support the idea that fibrillarin is essential for both the fusion of CBLs with the nucleolus and for umbravirus systemic infection.

The physical association of the nucleolus and CBs is well documented and is controlled by molecular interactions among CB and nucleolar proteins (4, 6). The interaction of the ORF3 protein with fibrillarin may interfere with normal host protein–protein interactions or other processes affecting CB integrity, which, in turn, may cause the fusion of CBLs with the nucleolus. Given that the ORF3 protein and fibrillarin are able to interact and that both move to the nucleolus via CBLs, it is conceivable that they may move as a complex. However, it is also possible that some of the ORF3 protein pool enters the nucleolus via CBLs as uncomplexed molecules, where they can interact with fibrillarin to form ORF3-fibrillarin complexes for export to cytoplasm.

Previously, we have shown that umbraviral RNP assembly occurs in the cytoplasm (16). Fibrillarin is needed for cytoplasmic RNP formation. The redistribution of fibrillarin with the ORF3 protein, therefore, is a prerequisite for formation of umbraviral RNP particles. Here, we demonstrate in vitro that fibrillarin, in combination with the ORF3 protein and viral RNA produces filamentous RNP particles with elements of helical architecture and structural properties similar to the viral RNPs formed in vivo described by Taliansky et al. (16). Moreover, these RNP particles were infectious and protected the viral RNA from RNase treatment. This suggests that the ORF3 protein and fibrillarin are sufficient for encapsidation of the viral RNA into an infectious RNP filament capable of long-distance movement.

Fibrillarin, an RNA-binding protein, may bind the viral RNA or may act to permit or catalyze the regular assembly of proteins around viral RNA, which is unachievable by the ORF3 protein alone. When formed in phloem companion cells, the viral RNPs presumably can enter sieve elements and move through the plant to cause systemic infection. These RNP complexes may also be involved in protecting viral RNA from the plant's defensive RNA silencing response mediated by short interfering RNAs, sustaining the viral life cycle. Whether the disk-shaped particles formed by the ORF3 protein and fibrillarin are required for RNP formation and how they interact with viral RNA to organize the helical structure remains to be determined. Nevertheless, the interaction of the GRV ORF3 protein with fibrillarin in CBs/CBLs triggers all of the molecular and cellular events necessary to establish a systemic infection.

Because umbraviruses lack a CP, the main functions of CPs of other viruses (formation of virus particles, systemic spread, and aphid transmission) must be replaced somehow. The ORF3 protein–fibrillarin interaction evidently takes care of the RNP formation and long-distance movement functions. For aphid transmission, umbraviral RNA is packaged in the CP of an unrelated helper virus that is itself aphid-transmitted (15). Thus, key functions of viral CP are, in umbraviruses, replaced by these two different mechanisms.

We have recently shown that the CP of another plant virus, potato leafroll virus (PLRV, a polerovirus), targets the nucleolus (28). Our preliminary results also indicate that, like GRV, PLRV is unable to cause systemic infection in the fibrillarin-silenced Group II plants, although accumulation of PLRV in the inoculated leaves is not affected (SI Table 4). This suggests that fibrillarin involvement in virus infection could be a more general mechanism among viruses with a nucleolar phase of life cycle. Finally, some RNA silencing pathways also involve the nucleolus and CBs (10, 11) and, hence, may be similar to the virus movement pathway described here.

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Materials and Methods

Virus Strains and TMV-Based Constructs.

GRV-YB was maintained as a stock isolate. The PEMV-2 cDNA clone was described by Ryabov et al. (29). TMV(ORF3-His) expressing His-tagged ORF3 protein was described (16). Changes were introduced into ORF3 coding sequences of the construct TMV(ORF3-His) to produce mutants listed in Fig. 1 by PCR using self-complementary mutagenic primers. Preparation of in vitro RNA transcripts and inoculation of plants were as described (17).

Analysis of Fibrillarin-Silenced Plants.

RT-PCR analysis of the accumulation of fibrillarin mRNA (NbFib) was performed by using primers corresponding to a 320-nt fragment (SI Text). Amplification of a fragment of ubiquitin mRNA (176 nt) was used as a control. For Western blot analysis, proteins were fractionated on 10% SDS/PAGE and transferred onto nitrocellulose membrane. The blots were probed by anti-fibrillarin antibodies. The immune reactions were visualized by ECL system (Amersham Biosciences, Uppsala, Sweden). ELISA of TMV and PVX accumulation was carried out essentially as described (30) (for details, see SI Text). Virus infectivity in samples from inoculated and uninoculated leaves infected with TMV, PVX, and GRV was assessed by counting the mean number of lesions induced in half leaves of N. tabacum“Xanthi-nc” (TMV) and C. quinoa (PVX and GRV) plants. For Northern blot analysis of GRV RNA, RNA extracted from plants was probed with 32P-labeled cDNA probe corresponding to the clone gr21GRV (31).

Generation and Agrobacterium-Mediated Expression of ORF3-GFP Construct.

The ORF3 protein fused to the N terminus of GFP (ORF3-GFP) was generated by using the Gateway recombination system and delivered by Agrobacterium into N. benthamiana leaves as described (14).

Confocal Imaging Analysis.

Localization of GFP fused to ORF3 protein was monitored by using a TCS SP2 (Leica Microsystems, Wetzlar, Germany) confocal laser scanning microscope. To locate nuclei, the leaf tissues were infiltrated with PBS containing 4′,6′-diamidino-2-phenylindole (DAPI). Coilin and fibrillarin were localized by using primary rabbit antibody to Atcoilin and primary monoclonal antibody to human fibrillarin (Cytoskeleton, Denver, CO). The primary antibodies were visualized by Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA).

Expression and Purification of the ORF3 Protein, Fibrillarin, and Mutants of Each.

ORF3-His protein and its mutants were expressed in N. benthamiana from TMV and purified by Ni2+-affinity chromatography (16). Arabidopsis fibrillarin 2 (Fib2) coding sequence was cloned into pGEX-KG (32) to generate a construct with Fib2 tagged at the N terminus with GST (GST-Fib2). A deletion mutant where the N-terminal 77 amino acid residues (GAR domain; Fig. 4 A) were removed (GST-Fib2ΔGAR) was produced by PCR using self-complementary mutagenic primers. The GST-fused proteins were expressed in Escherichia coli and purified by glutathione-agarose chromatography, and GST was removed by thrombin treatment as described (32).

Far Western Blot Analysis of the ORF3 Protein–Fibrillarin Interaction.

GST-Fib2, GST-Fib2ΔGAR, GST, or Fib2 were fractionated on 10% SDS/PAGE and transferred onto nitrocellulose membrane (Roche Applied Science, Indianapolis, IN). The blots were renatured, blocked, incubated with the ORF3 protein or its mutants (5 μg/ml), and probed with anti-ORF3 antibody essentially as described (22). The reaction was visualized by using the ECL system.

Formation of ORF3 RNP Complexes in the Presence of Fibrillarin.

RNA transcribed from a cDNA clone of PEMV-2 (29) (75 ng) was heat-denatured at 90°C for 1 min and mixed with saturating amounts of the ORF3 protein and fibrillarin (as indicated in Results) in 15 μl of buffer A [10 mM Hepes-KOH (pH 7.6)/100 mM KCl]. For EM, the samples were stained with 2% sodium phosphotungstate and examined in a CM 10 electron microscope (Royal Philips Electronics, Amsterdam, The Netherlands).

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Acknowledgments

We thank P. Shaw (John Innes Centre, Norwich, U.K.) for providing antibodies to plant coilin. This work was supported by a grant-in-aid from the Scottish Executive Environment and Rural Affairs Department, by the Royal Society (to J.W.S.B., N.O.K., and M.T.), Biotechnology and Biological Sciences Research Council (E.V.R.), International Association for the Promotion of Cooperation with Scientists from the New Independent States of the former Soviet Union (INTAS) Fellowship (to D.V.R.), and the Russian Foundation for Basic Research (N.O.K.).

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Footnotes

  • To whom correspondence should be addressed. E-mail: mtalia{at}scri.sari.ac.uk

  • Author contributions: S.H.K., S.M., N.O.K., J.W.S.B., and M.T. designed research; S.H.K., S.M., D.V.R., E.V.R., T.G., and S.H. performed research; S.H.K., N.O.K., D.V.R., J.W.S.B., and M.T. analyzed data; and S.H.K., J.W.S.B., and M.T. wrote the paper.

  • The authors declare no conflict of interest.

  • Data deposition: The nucleotide sequence of the NbFib cDNA was deposited in GenBank (accession no. AM269909).

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0704632104/DC1.

  • Abbreviations:
    CB,
    Cajal bodies;
    CBL,
    CB-like aggregates;
    GRV,
    groundnut rosette virus;
    TMV,
    tobacco mosaic virus;
    PVX,
    potato virus X;
    TRV,
    tobacco rattle virus;
    RNP,
    ribonucleoprotein;
    Fib2,
    fibrillarin 2;
    GAR,
    glycine- and arginine-rich domain;
    CP,
    coat protein.
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