1 UMR 1095 ASP (INRA-Université Blaise Pascal), Campus des Cézeaux, 24 Avenue des Landais, 63177 Aubière Cedex, France
2 UMR 385 BGPI, CIRAD-INRA-ENSAM, TA 41/K, Campus de Baillarguet, 34398 Montpellier Cedex 5, France
3 CPBS, CNRS UMR 5160, Faculté de Pharmacie, 15 Av. Charles Flahault, 34093 Montpellier Cedex 5, France
4 UMR GDPP (INRA-UVSB2), IBVM, BP 81, 33883 Villenave d'Ornon Cedex, France
Correspondence
Saloua Badaoui
Saloua.Badaoui{at}univ-bpclermont.fr
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ABSTRACT |
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INTRODUCTION |
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The proteasome plays a central role in many cellular processes (Coux et al., 1996). It can also interfere in vitro with protein synthesis from viral RNAs and mRNAs from virus-infected cells (Homma et al., 1994
), in relation to an intrinsic proteasome-associated endonuclease activity targeting viral RNAs and some cellular mRNAs containing tRNA-like structures (Gautier-Bert et al., 2003
). The presence of residual fragments of the RNA substrate in purified proteasome as well as the reconstruction in vitro of complexes between proteasome and RNA substrates confirmed the 20S endonuclease activity (Ballut et al., 2003
; Gautier-Bert et al., 2003
). Recently, the 20S endonuclease activity was found to be targeted specifically against the RNA genome of two plant viruses, Tobacco mosaic virus (TMV) and Lettuce mosaic virus (LMV), suggesting that this 20S RNase could have an antiviral function in vivo (Ballut et al., 2003
).
The 20S proteolytic activities are involved in the specific turnover of several viral proteins in mammals (Hu et al., 1999; Reinstein et al., 2000
) and plants (Drugeon & Jupin, 2002
; Karsies et al., 2002
; Reichel & Beachy, 2000
). In turn, some viruses have developed strategies to interfere with this proteolytic machinery through interaction with the 20S core or the 19S regulatory complex (Apcher et al., 2003
; Seeger et al., 1997
; Zhang et al., 2000
). However, to date there is no description of a plant virus interfering with the proteasome protease, and no description of a virus protein interfering with the 20S endonuclease activity, despite its probable role in constitutive antiviral defence.
The RNA genome of plant viruses in the genus Potyvirus (family Potyviridae) is translated into a polyprotein that is further processed by three virus-encoded proteinases (Carrington et al., 1990). One of these proteinases, HcPro, is a multifunctional protein (Maia et al., 1996
): as a strictly cis-acting proteinase it is responsible for its self-cleavage from the polyprotein precursor (Carrington & Herndon, 1992
) and, besides, it is also involved in a number of infectious processes as diverse as aphid transmission (Govier et al., 1977
), cell-to-cell (Rojas et al., 1997
) and long-distance movement (Saenz et al., 2002
), suppression of gene silencing (Llave et al., 2000
), synergism between co-infecting viruses (Pruss et al., 1997
) and symptom development (Redondo et al., 2001
). Some, but not all, of these biological functions might be related, for instance the inhibition of silencing-based host defence and the implication in virus synergism and as a virulence determinant. LMV HcPro is a 52 kDa protein with two structural domains (Plisson et al., 2003
). The proteinase is C-terminal and in several potyviruses the N-terminal one-fifth of HcPro, included in the largest structural domain, is required for aphid transmission but dispensable for replication (Dolja et al., 1993
; German-Retana et al., 2000
). HcPro binds RNA in a sequence non-specific manner (Maia & Bernardi, 1996
). In addition, it interacts with various virus-encoded proteins such as capsid protein (CP) (Roudet-Tavert et al., 2002
), P1 (Merits et al., 1999
) and VPg (Yambao et al., 2003
), as well as with itself (Urcuqui-Inchima et al., 1999
), perhaps in relation with its ability to oligomerize in vivo (Thornbury et al., 1985
) and in vitro (Plisson et al., 2003
). HcPro also interacts with various host proteins (Guo et al., 2003
), including a calmodulin-related protein involved in gene silencing (Anandalakshmi et al., 2000
).
Since LMV RNA was shown to be a target of the proteasome RNase activity in an earlier report (Ballut et al., 2003), it was hypothesized that this RNase activity could have an antiviral function in vivo. In this scenario, an inhibitory effect by viral proteins could be anticipated, and more specifically of proteins involved in virus pathogenicity, such as HcPro. Therefore, the effect of LMV infection on the proteasome was investigated and, more specifically, the possibility that HcPro interacts physically with the proteasome 20S core was evaluated, as well as the possible effects of such an interaction on the 20S enzymic activities, both proteinase and RNase.
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METHODS |
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Production of recombinant LMV HcPro.
Three different forms of recombinant HcPro were produced from plants infected with recombinant LMV constructs. In hisHcPro (Plisson et al., 2003), a poly histidine tag (His6) was inserted between HcPro aa 3 and 4. In his
HcPro, aa 4102 were replaced with His6. In strepHcPro, a Strep-tag II (Voss & Skerra, 1997
) followed by a thrombin cleavage site replaced the His6 of hisHcPro.
Pea or lettuce plantlets were inoculated as described previously (German-Retana et al., 2000). Leaves were harvested 23 weeks after inoculation and ground in 2 vols of ST buffer (100 mM Tris pH 8·0, 20 mM MgSO4, 500 µM EGTA) with 500 mM NaCl, 0·2 % Na2SO3 and 0·1 % polyvinylpyrrolidone. The homogenate was filtered through four layers of cheesecloth and one layer of miracloth, and centrifuged (60 min at 100 000 g). To concentrate the soluble fraction, differential precipitation with 20 % followed by 40 % (NH4)2SO4 was carried out and the pellet was resuspended in ST with 0·5 or 1 M NaCl and 1 mM Pefabloc. Such partially purified HcPro was stored at 70 °C and centrifuged (5 min at 5000 g) after thawing, prior to further HcPro purification.
For strepHcPro, the supernatant was applied on a phenylSepharose column (Sigma) at room temperature. The column was washed with 5 vols ST with 1 M NaCl. HcPro was eluted with 5 vols ST and concentrated by precipitation with 60 % (NH4)2SO4. The pellets were resuspended in ST with 100 mM NaCl, and applied onto a 1 ml streptactin column (IBA). After rinsing with 5 ml ST with 100 mM NaCl, strepHcPro was eluted with 5 ml ST with 100 mM NaCl and 10 mM desthiobiotin.
For His6-tagged HcPro, the supernatant was mixed with 1 ml Ni-NTA resin and 10 % methanol was added. After 30 min incubation on ice, the resin was rinsed twice for 30 min in a batch procedure with 50 ml ST with 500 mM NaCl and 10 % methanol, followed by two washes with 50 ml ST with 100 mM NaCl and the resin was applied on a column. The proteins eluted with 5 ml ST with 100 mM NaCl and 500 mM imidazole were precipitated with 60 % (NH4)2SO4 and resuspended in ST with 100 mM NaCl.
Protein separation by gel filtration.
Infected leaves were homogenized in 20 mM Tris/HCl pH 7·4, 5 mM MgCl2, 1 mM DTT, 100 µM EDTA pH 7·4, 50 mM KCl, 6 % sucrose, and filtered through four layers of cheesecloth. The homogenate was centrifuged for 10 min at 4000 r.p.m. (Sorvall SS34 rotor) and the supernatant was further centrifuged for 1 h at 15 000 r.p.m. (Sorvall SS34 rotor) and then again for 2 h at 42 000 r.p.m. (Beckman Ti45 rotor) on a 30 % sucrose cushion.
The supernatant was loaded onto a gel filtration column (Superose 6; Amersham). The proteins from 1 ml fractions were precipitated with 10 % trichloroacetic acid and separated by 12·5 % SDS-PAGE (Laemmli, 1970) for analysis. After electrophoresis, the protein fractions were analysed for the presence of 20S proteasome by Western blotting using polyclonal rabbit antibodies raised against 20S proteasome purified from cauliflower. When these antibodies were tested against proteasomes from calf liver, cauliflower or sunflower in preliminary Western blot experiments, a positive reaction was observed only with the plant proteasome. When these antibodies were tested in Western blot against a post-ribosomal supernatant from pea, lettuce or sunflower, the only positive signal observed was against subunits from the 20S proteasome.
In vitro analysis of protein interactions on Ni-NTA agarose.
Purified hisHcPro (150 µg) was retained (30 min at room temperature with shaking) in HC buffer (100 mM Tris/HCl pH 8·0, 100 mM NaCl, 2 mM EGTA) on Ni-NTA agarose beads (200 µl previously equilibrated in HC). After washing excess protein with HC, the beads were equilibrated in TBK240 (20 mM Tris/HCl pH 7·4, 2·5 mM MgCl2, 3·5 mM DTT, 240 mM KCl) and incubated with shaking overnight at 4 °C with 150 µg of 20S proteasome. After washing with TBK240 to eliminate excess 20S proteasome, hisHcPro and the proteins retained were eluted with 250 mM imidazole and analysed by SDS-PAGE.
Biacore analysis.
Surface plasmon resonance (SPR) allows real-time analysis of molecular interactions (Pollard-Knight et al., 1990). SPR was performed at 25 °C using a Biacore 3000 (Biacore AB). Purified 20S or hisHcPro were covalently immobilized on carboxymethylated dextran sensor chips (CM5) via their amino groups (EDC/NHS activation) according to the manufacturer's instructions. A control surface was prepared with the same treatment but without proteins. The surface densities of hisHcPro and 20S proteasome were 7041 RU and 6548 RU (a resonance unit, RU, is equivalent to 1 pg protein immobilized per mm2). The running buffer was HBS-EP (10 mM HEPES pH 7·4, 150 mM NaCl, 3 mM EDTA, 0·005 % P20) with a 50 µl min1 flow. To study the physical interaction between proteasome and HcPro, 50 µg min1 20S proteasome was simultaneously injected on immobilized hisHcPro and on a control surface and, conversely, various concentrations of hisHcPro were simultaneously injected on immobilized 20S proteasome and on a control surface. The same protocol was performed for his
HcPro and strepHcPro. Between each run, the sensor surfaces were regenerated with a pulse of 5 µl 0·03 % SDS.
In vitro transcription.
A full-length TMV cDNA cloned in pGEM-4Z (Promega) was obtained from Dr W.O. Dawson (Lake Alfred, Florida). Transcription with the T7 RNA polymerase (RiboMAX Large-Scale RNA Production Systems-T7; Promega) yielded TMV RNA.
Peptidase and proteinase assays.
The chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase activities of 20S were measured by quantitating methylcoumarin amide (MCA) released from peptides substrates (Dahlmann et al., 1991). Fluorescence was measured with a spectrofluorimeter (JY3D; Jobin-Yvon Instruments) at 442 nm; excitation was at 352 nm. Briefly, 20S proteasome was incubated (15 min at 37 °C) in the presence or absence of hisHcPro prior to addition of 200 µM substrate. A 1 : 2 (20S : hisHcPro) molar ratio was used, in agreement with the symmetrical structure of the 20S core. The assays (30 min at 37 °C) were carried out with 200 µM substrate in 100 µl 250 mM Tris/HCl, 10 mM DTT (pH 8·5).
The 20S proteolytic activity was assayed by measuring the breakdown of FITC-labelled casein. The assays (30 min at 37 °C) were carried out with 10 µM casein in 50 mM Tris/HCl pH 7·4. Purified 20S proteasome (15 µg) was incubated (15 min at 37 °C) in the presence or absence of hisHcPro at the same ratio as above, prior to adding casein. The fluorescent products were visualized under UV after 12·5 % SDS-PAGE. The degradation rate was deduced from the band intensities using Molecular Analyst (Bio-Rad). In control experiments, the same assays were performed in the same conditions in the presence of hisHcPro without 20S proteasome.
RNase assay.
This procedure was described in detail previously (Ballut et al., 2003). Briefly, TMV RNA (6 µg) was incubated (30 min at 37 °C) in 200 µl TBK240 with 20 µg 20S proteasome previously incubated (15 min at 37 °C) or not with purified hisHcPro at the same molar ratio as above. The products were analysed by gel filtration on a Superose 6 column equilibrated with TBK240. The RNA degradation rate was deduced from the difference in absorbance with the control.
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RESULTS |
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Purified 20S proteasome was applied to hisHcPro immobilized onto Ni-NTA agarose beads. After washing with TBK240, the proteins were eluted with imidazole and analysed by SDS-PAGE. The 20S proteasome and hisHcPro co-eluted from the column (Fig. 2). The 20S proteasome was not retained in the absence of hisHcPro and no co-elution was detected for thyroglobulin, a protein approximately the same size as the 20S complex. These results suggest a specific interaction in vitro between hisHcPro and 20S proteasome.
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DISCUSSION |
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Affinity chromatography and SPR independently revealed that HcPro binds specifically to the proteasome 20S complex in vitro. In addition, SPR indicated that the binding was strong and not significantly affected by the nature of the N-terminal tag sequence of HcPro. The deletion of the N-terminal region of HcPro increased the association rate without affecting as much the dissociation rate, suggesting that the interaction surface is located in the remaining part of HcPro, and is partly blocked or masked by the N-terminal portion of the protein. In HcPro, the N terminus (aa 1100 deleted in HcPro) appears structurally independent of the rest of the molecule, which is made up of two major structural domains: one spanning approximately from aa 100 to 230 and the other comprising the C-terminal ca. 150 aa (Plisson et al., 2003
). The HcPro N terminus is strictly required for aphid transmission but dispensable for the other known functions of HcPro, including gene silencing inhibition and viral accumulation (Dolja et al., 1993
; German-Retana et al., 2000
; Plisson et al., 2003
). It now also appears to be dispensable for interaction with the proteasome.
Human immunodeficiency virus-1 (HIV-1) Tat associates with seven and
20S proteasome-subunits (Apcher et al., 2003
). Similarly, binding of HcPro to several proteasome sites could explain the drastic proteasome mobility shift observed upon viral infection. This shift, from 650 to about 2000 kDa (a sedimentation range of about 30S), could possibly be related to an association of the proteasome with several copies of HcPro. Alternatively, other cellular or viral proteins, specifically expressed upon viral infection, could also bind to the proteasome or to the proteasomeHcPro complex. Finally, proteasome aggregation in the presence of HcPro, possibly in relation with the ability of the latter to oligomerize (Plisson et al., 2003
; Thornbury et al., 1985
), could also cause the observed shift through the formation of a high molecular mass network.
In several models, virus-encoded proteins interfere with the proteasome enzymic activities. Generally, this concerns the proteolytic activity of the 20S complex in relation to its 19S and 11S regulators. HIV-1 Tat inhibits both the binding of the 11S regulator to the 20S core and the 20S peptidase activities (Apcher et al., 2003; Seeger et al., 1997
). In plants, a role of the ubiquitin degradation pathway and therefore of the proteasome has been demonstrated for the turnover of several RNA virus proteins (Drugeon & Jupin, 2002
; Hazelwood & Zaitlin, 1990
; Hericourt et al., 2000
; Jockusch & Wiegand, 2003
). In this context, the stimulation of the proteasome 20S proteolytic activity in the presence of HcPro suggests that potyviruses could modulate the proteasome-based protein degradation pathway for their own purposes. The hypothesis that the observed stimulation of the protease activity is related to the endogenous activity of HcPro is unlikely since the HcPro proteinase is inactive in trans (Carrington et al., 1989
), and in addition no endogenous activity of hisHcPro was observed in control experiments in this study. A role of the stimulation of the 20S protease activity through the virus-encoded protein HcPro could be, for example, to increase the turnover of host proteins involved in defence and/or of some of the virus proteins necessary in the earlier stages of its cycle. This would directly favour virus accumulation, and could also interfere with the cell cycle. The interaction of Clink, a plant DNA virus protein, with SKP1, a component of the ubiquitin-based protein degradation pathway, was tentatively associated with the virus ability to alter the cell cycle (Aronson et al., 2000
).
Another characteristic of the interaction between HcPro and the proteasome with no equivalent in other systems described so far is the inhibition of the 20S proteasome RNase activity in the presence of HcPro. The mode of action of this inhibition is still unclear, since the experimental data presented here do not discriminate between a direct inhibition by binding to an RNase subunit of the proteasome and protection of the RNA target through the RNA-binding ability of HcPro (Maia & Bernardi, 1996). However, the demonstration of an interaction between HcPro and the proteasome as well as the need for prior incubation of HcPro with the proteasome for the RNase assays to detect an inhibition argues for a direct effect of HcPro on the RNase activity rather than through protection of the template.
Taken together, the association of the proteasome with an RNase activity specifically aimed at virus genomic RNAs (Ballut et al., 2003) and the ability of HcPro to target proteasome and interfere with this RNase activity, clearly open perspectives regarding the functions of HcPro during viral infection. Indeed, this suggests that the proteasome RNase activity could be involved in a novel plant anti-viral defence system that potyviruses have evolved to counteract. The proteasome RNase activity interferes in mammals and plants with the translation of viral RNAs with a 5' cap structure and no 3' polyadenylate, like TMV, by preventing the formation of initiation complexes (Homma et al., 1994
). It also interferes with the translation of viral RNAs with no 5' cap but with a 3' polyadenylate, like Cowpea mosaic virus (Petit et al., 2000
). Therefore, neither the 5' cap structure nor the 3' polyadenylate of plant viral RNAs are required for mRNA recognition and translation inhibition. Proteasome interference with the formation of initiation complexes could be mediated by direct interactions between proteins of these two complexes (Dunand-Sauthier et al., 2002
) or from template mRNA cleavage by the proteasome endonuclease, resulting in RNAs unable to circularize and therefore to promote assembly of a functional translation initiation complex (Wells et al., 1998
). Therefore, the inhibition of the proteasome RNase by HcPro could be directly linked to keeping high levels of viral RNA translation.
In addition, the proteasome endonuclease could also target virus RNA outside the translation context. Inhibition of the proteasome 20S RNase by HcPro is reminiscent of the ability of HcPro to inhibit RNA silencing, a plant antiviral defence (Voinnet, 2001). While the proteasome as a structure and RNA silencing as a mechanism are two conserved features among eukaryotes, several lines of evidence suggest that the proteasome RNase activity is probably not directly related to RNA silencing. One of the most striking features of RNA silencing is a sequence-specificity related to previous exposure to a sequence identical to at least a portion of the target (Tang et al., 2003
), while the proteasome RNase specificity depends on intrinsic properties of the target RNA molecules, such as the presence of secondary structures, rather than being acquired (Gautier-Bert et al., 2003
). The selective degradation of viral RNAs by the 20S complex could represent an alternative pathway, parallel to RNA silencing and reinforcing the cellular antiviral defence in plants.
Such a scheme would be essentially similar to the dual antimicrobial defence of vertebrates, where an innate and an adaptive immunity pathway cross-talk to ensure both an early broad-spectrum protection and a finer-tuned but delayed response (Janeway, 2001; Yoo & Desiderio, 2003
). Similarly, plants could have two pathways of antiviral defence, both based on RNA breakdown. The first component of this dual strategy would be innate and target non-host RNAs in a specific but broad-spectrum fashion, mediated by the proteasome RNase activity. The second component, adaptive and based on RNA silencing, would result in a somewhat delayed but fine-tuned degradation of the virus genome and include a systemic trigger. In this scenario, HcPro would be a dual-function pathogenicity effector inhibiting both pathways and therefore effectively linking them. Further experiments are clearly needed to investigate the potential contribution of the proteasome to the plant antiviral defence in vivo, to understand the role of the interference of HcPro with the proteasome enzymic activities during the infection process, and to establish whether this is a specificity of the potyvirus system or a feature also found in other plant RNA viruses.
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ACKNOWLEDGEMENTS |
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Received 14 April 2005;
accepted 4 June 2005.
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