Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK1
Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK2
Author for correspondence: John Carr. Fax +44 1223 333953. e-mail jpc1005{at}hermes.cam.ac.uk
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Abstract |
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Introduction |
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Deletion of the 2b protein from the subgroup II Q-CMV yielded a mutant (Q-CMV2b) that was less virulent than the wild-type virus. Q-CMV
2b was unable to systemically infect cucumber (Cucumis sativus) plants but could systemically infect Nicotiana glutinosa (Ding et al., 1995a
). A chimeric virus in which the 2b protein of Q-CMV was replaced by the TAV 2b protein showed increased virulence (increased severity of symptoms and increased viral RNA accumulation) on seven different host species (Ding et al., 1996
). These findings led to the initial conclusion that the cucumoviral 2b protein functioned in the control of systemic virus movement and symptom expression (Ding et al., 1995a
, b
, 1996
). However, it is now clear that at least in part the cucumoviral 2b protein exerts its effects on viral symptom development and systemic movement by interfering with post-transcriptional gene silencing (PTGS).
PTGS is a phenomenon in which foreign, overexpressed or aberrant RNA molecules can be recognized and targeted for destruction in a sequence-specific manner (Grant, 1999 ). PTGS has been observed not only in plants but also in other eukaryotic organisms, for example in Neurospora (Cogoni & Macino, 1999
). In plants, PTGS is thought to be, among other things, a defence against viruses (Covey et al., 1997
; Ratcliff et al., 1997
; Al-Kaff et al., 1998
; Brigneti et al., 1998
; Kasschau & Carrington, 1998
). PTGS can be induced by virus infection or expression of certain viral sequences as transgenes and can be used as a means to produce virus-resistant transgenic plants (recently reviewed by van den Boogaart et al., 1998
). Remarkably, once it has been initiated in any plant tissue, PTGS can be switched on throughout the entire plant, a phenomenon called systemic gene silencing (Palauqui et al., 1997
; Voinnet et al., 1998
).
That the 2b protein is a suppresser of PTGS was demonstrated in a comparative study of the properties of the 2b protein of CMV and the HC-Pro protein of potato virus Y (Brigneti et al., 1998 ). The HC-Pro and 2b proteins are both able to suppress gene silencing, protecting the viruses against host defences (Brigneti et al., 1998
; Kasschau & Carrington, 1998
). However, the HC-Pro and 2b proteins act in different ways: HC-Pro is able to switch off gene silencing in mature leaves, while 2b is only able to prevent PTGS in developing leaves (Brigneti et al., 1998
). This suggested that CMV prevents the initiation of gene silencing, either by preventing the systemic gene silencing signal from entering the cell, or by preventing activation of signalling within the cell. This idea was supported by the observations of Béclin et al.(1998)
who found that CMV infection suppressed silencing in transgenic Arabidopsis and tobacco only in the developing leaves. Interestingly, some plants may have evolved a defence against the viruses counter-defence. When expressed ectopically from a tobacco mosaic virus vector the TAV 2b protein acts as an elicitor of the hypersensitive resistance response in tobacco (Li et al., 1999
).
Previous work on the 2b protein has been focussed on the 2b proteins of CMV subgroup II strains and TAV. Using an antibody raised against a Q-CMV 2bglutathione S-transferase fusion protein, expression of the 2b protein was detected in plants infected with Q-CMV (Ding et al., 1994 ) and TAV (Shi et al., 1997
). Unfortunately, this antibody could not detect the 2b protein from subgroup IA or B strains of CMV and could not be used to demonstrate whether or not this protein is synthesized in plants infected with subgroup IA or B strains of the virus.
Using the antibody raised against the Q-CMV 2b, Shi et al.(1997) carried out preliminary subcellular fractionation experiments and showed that in infected plant tissue the TAV 2b protein accumulates in an insoluble fraction. The 2b protein could be released from this fraction only under denaturing conditions using SDS and urea in combination (Shi et al., 1997
). These results were consistent with the 2b protein having an association with any of several different organelles including cell walls, nuclei, the cytoskeleton or membranes. However, the methodology employed by Shi et al. (1997)
could not be used to identify specifically which of these diverse subcellular locations was the authentic site(s) of 2b protein accumulation during infection with CMV or TAV. Here, we have produced antibodies to the Fny-CMV 2b protein and used them to determine (1) whether or not the 2b protein is synthesized in plants infected with a subgroup IA strain of CMV, and (2) to identify the major subcellular location(s) of 2b protein accumulation in CMV-infected plants.
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Methods |
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Inoculation of tobacco plants and extraction of protein.
Tobacco (Nicotiana tabacum L. cv. Xanthi NN) plants were inoculated with Fny-CMV (Roossinck & Palukaitis, 1990 ) at a concentration of 50 µg/ml, or mock-inoculated with water. Systemically infected leaves (showing characteristic CMV symptoms) or equivalent leaves from mock-inoculated plants were harvested 7 days post-inoculation and homogenized in a mortar and pestle with RX buffer (30 mM sodium phosphate, pH 8, 10 mM KCl, 8 mM MgCl2, 15% glycerol, 0·1 mM PMSF, 25 mM 2-mercaptoethanol) using 5 ml of buffer per gram of tissue. This extract was filtered through two layers of Miracloth (Calbiochem). Subsequent fractionation steps involved centrifugation of the extract at various g-forces (detailed in Results). All centrifugation steps were carried out at 4 °C and for 10 min in each case, unless indicated otherwise. In some cases the crude, filtered extract was centrifuged immediately at 10000 g, prior to further analysis, but in certain experiments a range of other conditions were used. Centrifugation steps using g-forces between 125 and 10000 g were carried out in a Beckman JA-20 rotor and centrifugation steps in the range 20000 to 100000 g were carried out using a Beckman SW-40 rotor. At each centrifugation or extraction step the resultant pellet was resuspended in 0·5 vols RX buffer and the protein content was measured by the Bradford (1976)
assay using a Bio-Rad kit according to the manufacturers instructions.
Serial centrifugation assay.
A homogenate from systemically CMV-infected plants was produced, filtered through two layers of Miracloth and centrifuged at 125 g for 10 min. The supernatant was recovered, and centrifuged at an increased force of 500 g. This process was continued for forces of 1000, 2000, 5000, 10000, 20000, 50000 and 100000 g. The pellet fractions and material trapped by Miracloth were all resuspended in RX buffer (500 µl) and these insoluble fractions and the 100000 g supernatant probed for 2b protein by Western blotting.
Detergent- and salt-solubility assay.
Aliquots of 10000 g pelletted material containing 200 µg of protein were treated with different concentrations of urea (2, 4, 6 and 8 M) and a range of detergents: 1% Triton X-100; 1% Triton X-114; 1% CHAPS; 1% dodecyl -d-maltoside; and 1% SDS. Samples were kept on ice for 30 min, with the exception of the SDS-treated sample which was incubated at room temperature. Each aliquot was centrifuged at 10000 g for 30 min and the pellet was resuspended in a volume equal to the supernatant. Equal volumes of pellet or supernatant proteins were probed for 2b protein by Western blotting.
Preparation of a nuclei-enriched fraction.
A crude, Miracloth-filtered extract was prepared from systemically infected tobacco tissue, layered over a cushion of 2·2 M sucrose in RX buffer and centrifuged at 4 °C for 30 min at 10000 g. The material at the interface of the extraction buffer was removed and made to the original volume of the extract with RX buffer, while the pellet was resuspended in the same volume of RX buffer. Both samples were centrifuged at 10000 g for 30 min and the pellets resuspended in 0·5 vols RX buffer. This process divides the 10000 g pellet into a light fraction (the interface) and dense fraction (the pellet). Each fraction was analysed by Western blotting using the anti-2b serum and antibodies against actin, tubulin and histone H1.
Protein analysis.
Twenty µg of protein from each fraction was run on a 17·5% acrylamideSDS (Laemmli, 1970 ) gel and transferred to nitrocellulose membrane using the method of Dunn (1986)
. The membrane was stained with Ponceau red (Sambrook et al., 1989
) to confirm equal loading and, after washing in deionized water, the membrane was dried overnight to bind proteins tightly to the nitrocellulose. To detect the CMV 2b protein the membrane was probed with a 1:15000 dilution of the anti-2b polyclonal serum in 1% BSA in PBS. Immunoblot detection of actin (ICN), tubulin (Sigma) and histones (Cambio) was performed using appropriate primary antibodies according the instructions of the suppliers. Antibody binding was detected using an anti-rabbit horseradish peroxidase conjugate as the secondary antibody and visualized by chemiluminescence using a Renaissance kit according to the instructions of the manufacturer (NEN).
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Results |
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The major polypeptide band recognized by the antiserum in Western blot analysis of extracts of CMV-inoculated tobacco leaves had an apparent molecular mass of 17 kDa (Fig. 1). This band was present in the material pelletted at 10000 g from extracts of CMV-inoculated leaves but was not present in the 10000 g pellet from mock-inoculated leaves or in the 10000 g supernatant fractions from either CMV- or mock-inoculated leaves (Fig. 1
). The 17 kDa band was not detected in extracts of tobacco mosaic virus-infected tobacco leaves, and thus was unlikely to be a virus-induced plant protein (data not shown). When the antiserum was preabsorbed with the two synthetic oligopeptides used as immunogens, antibody binding to the 17 kDa band was abolished (data not shown). Preabsorption with the synthetic oligopeptides did not affect detection of the two faint bands of 52 and 31 kDa apparent in Fig. 1
, indicating that these proteins are most likely host proteins unrelated to the 2b protein (data not shown). Thus, the antiserum was shown to be reacting with the authentic Fny-CMV 2b protein in virus-infected plant tissue.
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The data presented in Fig. 1 and the earlier results of Shi et al. (1997)
are consistent with several different locations for the 2b protein, including the cell wall, the nucleus or the cytoskeleton. As a first step in identifying which of these components is the subcellular location of the 2b protein, the sedimentation characteristics of the 2b protein were examined by a series of centrifugation steps of increasing force. A homogenate produced from Fny-CMV-infected tobacco was filtered through Miracloth and centrifuged at increasing g-forces; the pellets obtained were resuspended in RX buffer and 20 µg protein from each fraction was run on a 17·5% SDSPAGE gel, blotted to nitrocellulose and probed for 2b protein (Fig. 2
). This was also done for the material trapped by the Miracloth filtration step which, when examined by light microscopy, appeared to consist predominantly of cell wall material (data not shown).
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In order to further examine the characteristics of the 2b-containing fraction, aliquots of 10000 g pelletted material were treated with different concentrations of urea and a range of detergents. After 30 min of treatment, samples were centrifuged at 10000 g for 30 min, and the pellet resuspended in a volume of RX buffer equal to the supernatant. Equal volumes of pellet or supernatant proteins were subjected to Western blot analysis using the anti-2b antibody (Fig. 3). The 2b protein is extremely resistant to solubilization. Only relatively high concentrations of urea (4 M or greater) were able to render the 2b protein soluble (Fig. 3a
) and, of the detergents tested, only SDS, a highly denaturing ionic detergent, was able to release the 2b protein to the supernatant fraction (Fig. 3b
). The solubility experiment was repeated a number of times, and with concentrations of the detergents as high as 5% (data not shown), but still yielded results identical to those shown in Fig. 3(b)
.
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The greatest enrichment of the 2b protein was observed in the 2·2 M sucrose pellet (Fig. 4a), indicating that the 2b protein must be associated with a large, dense organelle. Although some actin and tubulin were still contaminating this fraction after the second centrifugation at 10000 g (Fig. 4b
, c
), most of the actin and tubulin in the extract were only pelletted during centrifugation at forces above 100000 g (data not shown). Histone H1 was detectable only in the 2·2 M sucrose pelletted fraction, demonstrating that this fraction was enriched in nuclei. It should be noted that the level of histone H1 in the crude 10000 g pellets from mock- and CMV-inoculated plant material was below the level of detection possible with this antibody. This experiment was repeated a further three times with similar results. Overall, these data provide strong evidence that the 2b protein is not associated with the cytoskeleton but is predominantly associated with, or located in, the cell nucleus.
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Discussion |
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Our initial sedimentation experiments indicated that the 2b protein accumulated in a cell fraction that contained large, dense components, including membranes and cytoskeleton among others. These subcellular fractions in particular were initially viewed as potential locations for the 2b protein accumulation, since both have been implicated in the replication of several plant viruses (for example, see review by Carrington et al., 1996 ). However, a possible association of the 2b protein with membranes was ruled out by the results of our experiments with detergents and urea (Fig. 3
). The possibility that the 2b protein is present in a detergent-resistant lipid fraction (as is the case for glycosylphosphatidylinositol-anchored proteins) is unlikely, because CHAPS, which is often able to partially solubilize these proteins (Brown & Rose, 1992
), had no effect on the solubility of the 2b protein (Fig. 3b
). The Triton-treated extract was centrifuged through a sucrose gradient, and the 2b-containing fraction was able to be pelleted through 2·2 M sucrose (data not shown), suggesting that the 2b protein is not associated with detergent-insoluble lipids since these would exhibit flotation in the dense sucrose (Brown & Rose, 1992
). Similar high concentrations of detergents (most commonly Triton X-100) are frequently used when preparing nuclei (Guilfoyle, 1995
) and cytoskeleton (Xu et al., 1992
) to remove contamination by membranes, mitochondria and chloroplasts. Thus, it seemed most likely that the 2b protein was co-purifying with one or both of the former organelles. Further sedimentation experiments using a sucrose cushion to separate light and dense components of the 10000 g pellet, combined with immunoblot detection of cytoskeletal and nuclear markers (Fig. 4
), indicated that in CMV-infected cells the 2b protein accumulated predominantly in or on the host cell nucleus, although secondary sites of accumulation cannot be ruled out at this time.
It is not possible to tell from this study whether the 2b protein is located inside of the nucleus or on its surface. In order to be located inside the nucleus the 2b protein would need a nuclear localization signal (NLS). Plant NLSs fall into three classes (Varagona et al., 1992 ): (1) a simian virus 40 (SV40)-like NLS consisting of short tandem stretches of basic amino acids and either a proline or glycine residue; (2) a mating type
2-like NLS consisting of a short hydrophobic region containing at least one basic residue; and (3) bipartite NLSs consisting of two basic regions separated by approximately 10 amino acids. The 2b protein does not fit neatly into any of these categories. However, its N-terminal domain is very hydrophobic and rich in basic amino acids (Fig. 5
), and these properties may be sufficient to localize the 2b protein to the nucleus. Similar lysine- and arginine-rich regions in bZIP proteins have been shown to have a dual role in nuclear localization and DNA binding (Qian et al., 1996
; Liu et al., 1997
). The arginine-rich sequences of the human immunodeficiency virus Rev and Tat proteins, which appear similar to the 2b basic region (Fig. 5
), also function as NLSs (Truant & Cullen, 1999
).
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In summary, we have shown that the solubility and sedimentation characteristics of the CMV 2b protein indicate that it is predominantly a nuclear-localized protein. A nuclear location for the 2b protein is consistent with roles in the suppression of host defences and/or prevention of the initiation of PTGS.
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Acknowledgments |
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References |
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Received 1 September 1999;
accepted 20 October 1999.