Epstein–Barr virus EBNA3 proteins bind to the C8/{alpha}7 subunit of the 20S proteasome and are degraded by 20S proteasomes in vitro, but are very stable in latently infected B cells

Robert Touitou, Jenny O'Nions, Judith Heaney and Martin J. Allday

Department of Virology and Ludwig Institute for Cancer Research, Wright-Fleming Institute, Faculty of Medicine, Imperial College London, Norfolk Place, London W2 1PG, UK

Correspondence
Martin J. Allday
m.allday{at}imperial.ac.uk


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A yeast two-hybrid screen using EBNA3C as bait revealed an interaction between this Epstein–Barr virus (EBV)-encoded nuclear protein and the C8 ({alpha}7) subunit of the human 20S proteasome. The interaction was confirmed by glutathione S-transferase (GST) pull-down experiments and these also revealed that the related proteins EBNA3A and EBNA3B can bind similarly to C8/{alpha}7. The interaction between these viral proteins and GST–C8/{alpha}7 was shown to be significantly more robust than the previously reported interaction between C8/{alpha}7 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Co-immunoprecipitation of the EBNA3 proteins with C8/{alpha}7 was also demonstrated after transfection of expression vectors into B cells. Consistent with this ability to bind directly to an {alpha}-subunit of the 20S proteasome, EBNAs 3A, 3B and 3C were all degraded in vitro by purified 20S proteasomes. However, surprisingly, no sign of proteasome-mediated turnover of these latent viral proteins in EBV-immortalized B cells could be detected, even in the presence of gamma interferon. In actively proliferating lymphoblastoid cell lines, EBNAs 3A, 3B and 3C appear to be remarkably stable, with no evidence of either de novo synthesis or proteasome-mediated degradation.

Published online ahead of print on 3 February 2005 as DOI 10.1099/vir.0.80763-0.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The B lymphoblastoid cell lines (LCLs) that are generated by the infection of resting human B cells with Epstein–Barr virus (EBV) in vitro have a phenotype resembling that of activated B blasts. These growth-transformed cells express only nine latency-associated EBV proteins (this is known as the growth/proliferation programme or latency III). There are six nuclear antigens [EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C and leader protein (EBNALP)] and three latent membrane proteins (LMP1, LMP2A and LMP2B); additionally, several viral RNA species of uncertain function are expressed. Together, the latent proteins induce the growth of quiescent B cells, their entry into the cell cycle and proliferation and maintenance of the viral genome as extrachromosomal episomes (reviewed by Bornkamm & Hammerschmidt, 2001).

In vivo, this ability of the virus to drive B-cell proliferation is transient but essential, because the LCL-like activated B cells retain the capacity to undergo differentiation in lymphoid tissue and thus permit latent viral genomes to enter the long-lived memory B-cell pool, the site of long-term EBV persistence. During the differentiation process, there is a progressive, regulated shutdown of latent viral gene expression until, in quiescent memory B cells, no EBV proteins can be detected (Thorley-Lawson, 2001; Thorley-Lawson & Gross, 2004). Although EBV is carried for life as a non-pathogenic infection by >90 % of the adult population, it is associated with a number of diseases. In addition to causing the self-limiting and relatively benign lymphoproliferative disorder infectious mononucleosis, EBV is associated with several B-cell tumours, including post-transplant lymphoproliferative disease, endemic Burkitt's lymphoma and Hodgkin's disease. However, the precise contribution that EBV makes to the pathogenesis of these various B-cell neoplasias remains unknown (O'Nions & Allday, 2004; Young & Rickinson, 2004). There is good evidence that EBV also plays a role in the aetiology of at least two tumours of epithelial origin – nasopharyngeal carcinoma and gastric carcinoma (reviewed by Young & Rickinson, 2004).

EBNAs 3A, 3B and 3C are three related, EBV latency-associated proteins that are expressed in the nuclei of continuously proliferating LCLs. Genetic analysis using recombinant viruses has shown that EBNAs 3A and 3C, but not EBNA3B, are essential for this activation and immortalization of B cells in vitro (reviewed by Bornkamm & Hammerschmidt, 2001; O'Nions & Allday, 2004). EBNAs 3A, 3B and 3C share limited but significant amino acid sequence similarity, have the same gene structure (a short 5' exon and a long 3' exon) and are arranged in tandem in the genome (Kieff & Rickinson, 2001). However, there is nothing to suggest that they have extensively redundant functions. All three proteins bind to a cellular DNA-binding protein known as CBF1 or RBP-J{kappa}. This factor also binds to, and targets to DNA, the EBV transactivator protein EBNA2 and also the IC-Notch effector of the Notch signalling pathway (reviewed by Zimber-Strobl & Strobl, 2001). EBNAs 3A, 3B and 3C can all repress EBNA2-mediated transactivation of the EBV LMP2 promoter (Le Roux et al., 1994) and EBNAs 3A and 3C can repress reporter plasmids containing the CBF1/RBP-J{kappa} sites derived from the EBV Cp promoter independently of EBNA2-mediated activation (Cludts & Farrell, 1998; Radkov et al., 1997). As Cp is generally the promoter for all EBNA mRNA initiation in LCLs, the EBNA3 proteins probably contribute to a negative autoregulatory control loop. In addition, both EBNA3A and EBNA3C exhibit robust repressor activity when targeted directly to DNA by fusion with the DNA-binding domain of Gal4 (Bain et al., 1996; Cludts & Farrell, 1998; Radkov et al., 1997; Waltzer et al., 1996).

EBNA3C can recruit histone deacetylases and binds to the co-repressor C-terminal binding protein (CtBP) via a PLDLS amino acid motif (Knight et al., 2003; Radkov et al., 1999; Touitou et al., 2001a). We have also shown that EBNA3A binds CtBP in vitro and in vivo via a novel, non-consensus, bipartite motif (Hickabottom et al., 2002). In this ability to bind to CtBP and CBF1/RBP-J{kappa}, EBNAs 3A and 3C resemble Hairless. Hairless is a negative regulator of the Notch pathway in Drosophila that binds to the Drosophila equivalent of CBF1/RBP-J{kappa} (Suppressor of Hairless) and recruits the co-repressor dCtBP to silence transcription (Barolo et al., 2002; Hickabottom et al., 2002). In addition, there is a very good correlation between the ability of EBNAs 3A and 3C to bind CtBP and their ability to co-operate with (Ha-)Ras in the immortalization and transformation of primary rodent fibroblasts (Hickabottom et al., 2002; Touitou et al., 2001a). This phenotype and other more direct evidence suggest that EBNAs 3A and 3C may play roles in the deregulation of cell-cycle checkpoints by EBV (reviewed by O'Nions & Allday, 2004).

Although all three EBNA3 proteins can interact with complexes of cellular proteins that are involved in transcriptional repression and gene silencing (Hickabottom et al., 2002; Touitou et al., 2001a; P. Young & M. J. Allday, unpublished data), under certain circumstances, EBNA3C (and maybe EBNA3A) can also act as a co-activator of the LMP1 gene (Lin et al., 2002; Marshall & Sample, 1995; Zhao & Sample, 2000). EBNA3C has been shown to associate with the transcription factor PU1/Spi1 and also a transactivator complex containing prothymosin-{alpha} and the histone acetyltransferase p300. However, it is not clear what, if any, role these interactions with transactivators play in the activation of LMP1 reporter plasmids or in the regulation of LMP1 expression in infected B cells (Subramanian et al., 2002; Zhao & Sample, 2000). EBNA3C also interacts with the small, ubiquitin-like proteins Sumo-1 and Sumo-3 (Lin et al., 2002); however, the significance of these interactions with EBNA3C has yet to be determined.

In the infection of humans by EBV, immunoregulation by cytotoxic T cells is very important in the maintenance of asymptomatic viral persistence and prevention of disease. The cytotoxic T-lymphocyte (CTL) response to EBV in humans is very well-defined and is largely mediated by CD8+, human leukocyte antigen class I-restricted T cells (reviewed by Moss et al., 2001). Responses are directed against antigens associated with EBV lytic replication and also the proteins expressed in latency III. In healthy immune individuals, the CTL response to latently infected cells is remarkably focused on a relatively small number of immunodominant epitopes within the EBNA3 family proteins (Khanna et al., 1992; Rickinson & Moss, 1997). The reason for the focus on these nuclear proteins is unclear, but it has been suggested that it may relate to the degree of access that they have to the antigen-processing pathways of the infected cells (Moss et al., 2001). However, although it is assumed that such processing involves targeting of these proteins to proteasomes via the ubiquitinylation system, remarkably little has been reported about the turnover of EBNA3 proteins in B cells infected latently with EBV.

During the non-lysosomal turnover of most proteins in eukaryotic cells, covalent ligation of multiple ubiquitin molecules provides the signal for recognition by the 26S proteasome. This molecular machine is a multi-subunit complex in which two 19S regulatory structures sit each end of a barrel-shaped chamber called the 20S proteasome. The 19S complexes are responsible for the recognition of polyubiquitin attached to proteins marked for degradation, and for also substrate unfolding through the action of a ring of ATPase molecules positioned adjacent to the 20S chamber. The 19S regulators are also thought to open a channel into the catalytic chamber through which unfolded substrates are then threaded into the inner chamber of the 20S complex, where proteolysis takes place (reviewed by Baumeister et al., 1998; Pickart & Cohen, 2004).

With the possible exception of ornithine decarboxylase, it has been assumed that ubiquitinylation and ATPase-mediated unfolding are essential prerequisites for proteins destined for proteasome-mediated proteolysis (Baumeister et al., 1998; Pickart & Cohen, 2004). However, it has been shown that the cyclin-dependent kinase inhibitor p21WAF1/CIP1 could be degraded by the proteasome in a ubiquitin-independent manner. Specifically, p21WAF1/CIP1 in which all the potential lysine targets for ubiquitinylation were mutated was shown to remain an effective substrate for proteasome-mediated degradation (Sheaff et al., 2000). However, it has been suggested that ubiquitin may still play a role in binding/targeting of p21WAF1/CIP1 to 26S proteasomes, as p21WAF1/CIP1 becomes tagged with polyubiquitin at its N terminus in the absence of the internal lysine residues (Bloom et al., 2003). Nevertheless, it has also been shown that p21WAF1/CIP1 can be degraded by purified 20S proteasomes and that this is dependent on an interaction between the C terminus of p21WAF1/CIP1 and the C8/{alpha}7 subunit of the 20S proteasome (Touitou et al., 2001b). These observations have led to the suggestion that p21WAF1/CIP1 – a highly unstructured polypeptide in its native state – is able to bind to the 20S proteasome in such a way that it induces the opening of a channel into the 20S chamber. In this situation, degradation is possible in the absence of 19S regulatory subunits, whether or not the substrate is ubiquitinylated (Förster & Hill, 2003; Liu et al., 2003; Orlowski & Wilk, 2003).

Here, we show that, like p21WAF1/CIP1, the EBNA3 proteins of EBV can bind to human C8/{alpha}7 in vitro and also when transfected into B cells. Consistent with this, all three viral proteins were degraded by purified 20S proteasomes in vitro but, unlike p21WAF1/CIP1, they were each found to be very stable and apparently turned over at a very low rate in B cells infected latently with EBV. This raises interesting questions about the mechanism by which the EBNA3 proteins gain access to the antigen-processing pathways in infected cells.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast two-hybrid assay.
Saccharomyces cerevisiae strain Y190 was used to screen a human B-cell cDNA library in the pACTGal-4 transcriptional activation-domain vector. Library screening was performed according to standard protocols (Clontech), using a bait plasmid encoding an RBP-J{kappa}-binding mutant of EBNA3C [pAS2-EBNA3C J{kappa}-(M)]. Plasmids carrying cDNAs encoding interacting proteins were isolated and retransformed into yeast bait strains expressing various EBNA3C polypeptides to confirm reproducibility and specificity. All plasmid DNA encoding binding partners was sequenced and compared with databases to identify the cDNA inserts.

Glutathione S-transferase (GST)-fusion pull-down assay.
GST pull-down experiments were performed essentially as described previously (Radkov et al., 1999; Touitou et al., 2001b). Briefly, bacterially expressed GST or GST–C8 fusion protein was incubated with equal amounts of [35S]methionine-labelled p21WAF1, EBNA3C, EBNA3A, EBNA3B, LMP1 or LMP2A in 200 µl EBC buffer [140 mM NaCl, 0·5 % Nonidet P-40, 50 mM Tris/HCl (pH 8·0), 100 mM NaF, 200 µM Na3VO4, 1 mg BSA ml–1] for 2 h with rotation at 4 °C. The beads were then washed four times with NETN buffer [300 mM NaCl, 1 mM EDTA, 0·5 % Nonidet P-40, 20 mM Tris/HCl (pH 8·0)], resuspended in SDS sample buffer and heated at 95 °C for 5 min. Bound proteins were resolved by SDS-PAGE and visualized by autoradiography.

In vitro degradation assay with 20S proteasomes.
p21WAF1, EBNA3A, EBNA3B, EBNA3C, pRb and {alpha}B-crystallin proteins were generated with the in vitro transcription/translation TnT-coupled reticulocyte lysate system (Promega). Each protein (5 µl per time point) was incubated at 37 °C with or without purified 20S proteasome (Affiniti) (1 µg per time point) in PBS for the indicated times. Where indicated, the peptide aldehyde inhibitor z-LLL-H (MG-132; Calbiochem) was added at a final concentration of 200 µM to inhibit 20S proteasome proteolytic activity. Proteins were resolved by SDS-PAGE and visualized by autoradiography or quantified by using a Storm 860 phosphorimager (Molecular Dynamics).

Cell culture and transfection.
DG75 is an EBV-negative human Burkitt's lymphoma-derived cell line and ED-LCL is an LCL derived from the infection of primary human B cells with the B95.8 strain of EBV. All cells were cultured at 37 °C under a 10 % CO2 humidified atmosphere in RPMI 1640 medium supplemented with 10 % (v/v) fetal calf serum, 2 mM L-glutamine, 100 U penicillin ml–1 and 100 U streptomycin ml–1 (all from Invitrogen).

DG75 cells were transfected by electroporation as described previously (Bain et al., 1996; Touitou et al., 2001b). Briefly, 10 µg pSG5-C8, pCDNA3-EBNA3A, pCDNA3-EBNA3B or pCDNA3-EBNA3C DNA was transfected into 107 DG75 cells. Electroporation was performed at 250 V, 960 µF, using a Gene Pulser (Bio-Rad). For co-transfections, 10 µg pSG5-C8 DNA was mixed with 10 µg pCDNA3-EBNA3A, pCDNA3-EBNA3B or pCDNA3-EBNA3C or pCDNA3 empty-vector DNA prior to transfection.

Inhibition of proteasome activity, inhibition of protein synthesis and treatment with gamma interferon (IFN-{gamma}).
To inhibit protein synthesis, cells were treated with cycloheximide (Sigma) (stock solution, 5 mg ml–1 in PBS) at a final concentration of 50 µg ml–1 and cells were then harvested at indicated times after treatment.

To inhibit proteasome activity, lactacystin (Calbiochem) (stock solution, 10 mM in DMSO) was added to cells at a final concentration of 2 µM before collection at the indicated times. When added to the LCL, IFN-{gamma} (Pharmingen/BD Biosciences) was used at a final concentration of 500 U ml–1.

Protein extraction, immunoprecipitation (IP) and Western blot analysis.
Protein extraction and Western blot analysis were performed essentially as described previously (Touitou et al., 2001b; Wade & Allday, 2000). Extracts were prepared either with RIPA IP buffer or by sonication and boiling in SDS sample buffer. In all cases, 30 µg total protein extract was loaded for SDS-PAGE using a mini-PROTEAN II cell (Bio-Rad).

IPs were performed essentially as described previously (Radkov et al., 1999; Touitou et al., 2001b). Briefly, 107 cells were harvested, washed once in ice-cold PBS and lysed in IP lysis buffer [50 mM Tris/HCl (pH 8·0), 150 mM NaCl, 10 % glycerol, 0·5 % Triton X-100, 2 mM PMSF, 2 mM mixture of proteinase inhibitors (Roche Molecular Biochemicals)] for 20 min at 4 °C. Cell debris was pelleted by centrifugation and the supernatant was mixed with protein G–Sepharose beads for 1 h at 4 °C. After centrifugation, complexes were precipitated from the supernatant with the required antibody by incubation at 4 °C on an orbital rotor for 2 h. To collect the immunoprecipitated complexes, the mixture was incubated with protein G–Sepharose beads at 4 °C for 1 h with rotation. The beads were washed four times in IP lysis buffer and resuspended in 30 µl SDS sample buffer. After boiling, the supernatant was resolved by SDS-PAGE. IPs and Western blots were performed with the following antibodies: mouse monoclonal anti-human C8/{alpha}7 (Affiniti), sheep polyclonal anti-EBNA3A (Exalpha), sheep anti-EBNA3B (Exalpha), mouse monoclonal anti-EBNA3C (A10, a gift from Dr Martin Rowe, University of Cardiff, UK), mouse monoclonal anti-p53 (DO1, a gift from Professor Xin Lu, Ludwig Institute for Cancer Research, London, UK), mouse monoclonal anti-cMyc (9E10, a gift from Professor Xin Lu), mouse monoclonal anti-Bcl-2 (Dako) and mouse monoclonal anti-p21WAF1 (N-20; Santa Cruz).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
EBNA3C binds human C8/{alpha}7 in a yeast two-hybrid screen
A human lymphocyte cDNA library was screened with a clone of EBNA3C containing substitutions with four alanine residues in the RBP-J{kappa}-binding site, making the protein incapable of interacting with RBP-J{kappa} (Radkov et al., 1997). From this screen, 22 interacting clones were confirmed to be genuine. Of these, three were identified as the human homologue of the SMT3B protein of S. cerevisiae and seven more were found to correspond to the human 20S proteasome subunit C8/{alpha}7. The SMT3B homologue in human cells is a small, ubiquitin-like (Sumo) protein and this result is therefore consistent with the report that EBNA3C interacts with Sumo family members (Lin et al., 2002).

All of the C8/{alpha}7 clones represented full-length cDNAs and further analysis in yeast confirmed this interaction, not only with the alanine-substitution RBP-J{kappa}-binding mutant of EBNA3C, but also with wild-type protein and several other substitution- and deletion-mutant EBNA3C proteins (Fig. 1).



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Fig. 1. EBNA3C interacts with human C8/{alpha}7 in yeast two-hybrid assays. Schematic summary of the various fusions between EBNA3C and the Gal4 DNA-binding domain (Gal4-DBD) that were used in yeast two-hybrid analysis against full-length human C8/{alpha}7 fusions with the Gal4 activation domain (Gal4-AD). All of the mutants, apart from the highly truncated mutant EBNA3C aa 250–525, bound to C8/{alpha}7 in this assay.

 
C8/{alpha}7 binds EBNA3C and also EBNAs 3A and 3B in vitro
In order to characterize further the interaction between EBNA3C and C8/{alpha}7, GST–C8/{alpha}7 fusion protein was generated and a series of ‘pull-down’ assays was performed by using in vitro-translated EBNA3C labelled with [35S]methionine. It was shown in these in vitro assays that not only did GST–C8/{alpha}7 bind EBNA3C with apparently high affinity, but also it bound the related proteins EBNA3A and EBNA3B with similar efficiency (Fig. 2a). None of the EBNA3s bound to unfused GST and, although both probably interact with the proteasome system in cells, neither EBV LMP1 nor LMP2 bound to C8/{alpha}7 in similar assays. The cellular nuclear proteins p27KIP1, p57 and p53 also failed to bind to GST–C8/{alpha}7, as shown previously (Touitou et al., 2001b; data not shown). As it has been shown that the cyclin-dependent kinase inhibitor p21WAF1/CIP1 also binds to C8/{alpha}7, a comparison was made, suggesting that all three EBNA3s bind to this proteasome subunit with far greater efficiency than does p21WAF1/CIP1 (Fig. 2b).



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Fig. 2. EBNAs 3A, 3B and 3C interact with GST–C8/{alpha}7. (a) Bacterially expressed GST or GST–C8/{alpha}7 fusion protein was incubated with approximately equal amounts of [35S]methionine-labelled EBNA3A, EBNA3B, EBNA3C, LMP1 or LMP2. Five per cent of the input protein is shown. Bound proteins were resolved by SDS-PAGE (12·5 % gel) and visualized by autoradiography. (b) Comparison of binding efficiencies. Bacterially expressed GST and GST–C8/{alpha}7 were incubated with approximately equal amounts of EBNAs 3A, 3B or 3C or p21WAF1. Various dilutions of the GST–C8/{alpha}7 fusion protein (as indicated) were used to compare the affinities of these various proteins for the C8/{alpha}7 fusion. EBNAs 3A, 3B and 3C all bound much more efficiently than the cellular protein p21WAF1.

 
EBNA3C includes multiple binding sites for C8/{alpha}7
It was possible to identify a region of 30 aa in the C terminus of the p21WAF1/CIP1 protein that was responsible for the interaction with C8/{alpha}7. This was mapped by making in-frame fusions of p21WAF1/CIP1 polypeptides with p27KIP1, as the latter is unable to bind C8/{alpha}7 (Touitou et al., 2001b). A similar strategy for making fusions with p27KIP1 was used to try to identify a site within EBNA3C that was responsible for the binding to C8/{alpha}7. The members of the series of p27KIP1–EBNA3C fusion polypeptides, represented schematically in Fig. 3, were assayed for their ability to bind GST–C8/{alpha}7. This revealed that at least four fragments spanning the length of EBNA3C could bind GST–C8/{alpha}7 and therefore no further attempt was made to map a unique binding site that could be mutated easily. Either multiple binding sites exist on the exposed surfaces of EBNA3C or the expression of short polypeptides reveals structural features that are normally concealed. Preliminary experiments with EBNAs 3A and 3B suggest that they also include multiple C8/{alpha}7 binding sites (J. Heaney & M. J. Allday, unpublished data).



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Fig. 3. EBNA3C includes at least four binding sites for C8/{alpha}7. A schematic representation of results from GST pull-down experiments using 35S-labelled fusions of p27KIP1 and fragments of EBNA3C. Bacterially expressed GST or GST–C8/{alpha}7 fusion protein was incubated with equal amounts [35S]methionine-labelled p27KIP1 or the p27KIP1–EBNA3C fusions identified by the number of the amino acids in the EBNA3C sequence. (–) indicates that no binding was detected and (+) indicates specific binding to the C8/{alpha}7 fusion protein. HDAC, Histone deacetylase.

 
EBNAs 3A, 3B and 3C all bind C8/{alpha}7 in cells
The ability of each of the EBNA3s to bind C8/{alpha}7 in a more physiological environment was determined by transfecting expression vectors for EBNAs 3A, 3B and 3C, either alone or with a C8/{alpha}7 expression vector, into DG75 B cells and performing IPs with an anti-C8/{alpha}7 specific antibody, followed by Western blotting for the different EBNA3s. Representative results, illustrated in Fig. 4, show that EBNAs 3A, 3B and 3C were all co-precipitated with modest efficiency by a monoclonal antibody (mAb) (anti-C8/{alpha}7) when exogenous C8/{alpha}7 was also expressed [similar results were obtained by using exogenous HA-tagged C8/{alpha}7 (data not shown)]. It was noted consistently that a very small but detectable amount of each EBNA was co-immunoprecipitated from extracts of cells transfected only with the EBNA3-encoding plasmids. This presumably represents the interaction of each overexpressed EBNA3 protein with endogenous C8/{alpha}7. However, several attempts to co-immunoprecipitate endogenous EBNA3A, EBNA3B or EBNA3C (with C8/{alpha}7) from LCLs infected latently with EBV were unsuccessful, even in the presence of an inhibitor of proteasome function (data not shown). This may be because there is little turnover of these viral proteins in actively proliferating LCLs and they are therefore not exposed to proteasomes (see below).



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Fig. 4. EBNAs 3A, 3B and 3C can all be co-immunoprecipitated with C8/{alpha}7 from transfected cells. DG75 B cells were transfected with 10 µg pCDNA3-EBNA3A, pCDNA3-EBNA3B or pCDNA3-EBNA3C DNA with or without 10 µg pSG5-C8/{alpha}7 (C8) DNA. Forty-eight hours after transfection, protein extracts were subjected to IP using an anti-C8/{alpha}7 mAb ({alpha}-C8) or a control antibody ({alpha}-cMyc, CAb). After resolution by SDS-PAGE (7·5 % gel), the proteins were transferred to nitrocellulose and probed with antibodies specific for EBNAs 3A, 3B or 3C.

 
EBNAs 3A, 3B and 3C are degraded in vitro by purified 20S proteasomes
Here, we have identified C8/{alpha}7, a structural component of the proteasome, with which EBNA3A, EBNA3B and EBNA3C can interact physically, both in vitro and in vivo. The cyclin-dependent kinase inhibitor p21WAF1/CIP1, which also binds to C8/{alpha}7, is degraded rapidly by purified 20S proteasomes (Liu et al., 2003; Touitou et al., 2001b), so we tested whether the EBNA3s could also be degraded by 20S proteasomes (lacking the 19S complex) in similar assays. The results showed that, in the absence of purified 20S proteasomes, in vitro-translated, [35S]methionine-labelled EBNAs 3A, 3B and 3C were each stable for at least 45 min at 37 °C. In the samples to which 20S proteasomes were added, significant degradation of all of the EBNA3 proteins occurred during a similar period of time. This proteolysis was blocked by the inclusion of the specific proteasome inhibitor MG-132 (Fig. 5a, b). In similar assays, p21WAF1/CIP1 was degraded more rapidly and more completely; however, p27KIP1 remained undegraded (Fig. 5a, b; Touitou et al., 2001b; data not shown). Two other proteins, one of which can bind to GST–C8/{alpha}7 [namely {alpha}B-crystallin (Boelens et al., 2001)] and the tumour suppressor pRb, also remained completely stable after incubation with 20S proteasomes for more than 30 min (Fig. 5c). Clearly, binding to C8/{alpha}7 is not the sole determinant of proteolysis by 20S proteasomes.



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Fig. 5. (a) EBNAs 3A, 3B and 3C are targets for ubiquitin-independent proteolysis by purified 20S proteasomes. Equal volumes (5 µl per time point) of in vitro-translated and [35S]methionine-labelled proteins (p21WAF1, EBNA3A, EBNA3B or EBNA3C) were incubated at 37 °C for the time indicated (min) in the absence (–20S) or presence (+20S) of purified 20S proteasomes (1 µg per time point). Where indicated (M), the z-LLL-H (MG-132) proteasome inhibitor was added at a final concentration of 200 µM. Proteins were resolved by SDS-PAGE (7·5 or 12·5 % gels). (b) The [35S]methionine signals shown in (a) were quantified by using a Storm 860 phosphorimager (Molecular Dynamics) and values were plotted as the percentage of the signal at t=0 (given an arbitrary value of 100 %). Multiple experiments produced similar results. {blacklozenge}, +20S; {blacksquare}, –20S. (c) Similar experiments to those described in (a) were performed by using in vitro-translated and 35S-labelled retinoblastoma protein (pRb) and {alpha}B-crystallin. Neither of these proteins showed any sign of degradation during 30 min incubation with or without 20S proteasomes.

 
In EBV-infected B cells, EBNAs 3A, 3B and 3C are very stable and show little evidence of turnover
It has been suggested that, in B cells infected latently by EBV, the EBNA3s may be processed efficiently as CTL antigens via the proteasome system (Moss et al., 2001) and it has been shown here that all three are degraded efficiently by purified 20S proteasomes in vitro. We therefore used LCLs to investigate whether the EBNA3s are degraded by the endogenous proteasome system with similar efficiency. In order to do this, the potent and highly specific proteasome inhibitor lactacystin was added to an actively proliferating LCL. Samples were analysed by Western blotting of total protein extracts of proteins solubilized in RIPA buffer and probing with antibodies directed against the EBNA3 proteins. In addition, Western blots were probed for p21WAF1/CIP1 and p53, both proteins that are known to be turned over by proteasome-mediated proteolysis and that therefore accumulate when proteasome function is ablated (Maki & Howley, 1997). Blots were also probed for Bcl-2, a protein whose level is unaffected by lactacystin. Representative results, illustrated in Fig. 6(a), show that, whilst p21WAF1/CIP1 and p53 accumulate within 12 h of adding lactacystin to the cells (p53 largely as ubiquitinylated forms), the levels of all three EBNA3 proteins remained almost unchanged. This indicates either that these viral proteins are not normally turned over by the proteasome system and/or that they are newly synthesized at an undetectably low rate. Similar experiments using total protein extracts that had been solubilized directly in SDS sample buffer produced essentially identical results (data not shown). The experiments were repeated with IFN-{gamma} also added to the cells. Although IFN-{gamma} stimulates assembly of 26S proteasomes that are particularly active in the generation of CTL epitopes (Goldberg et al., 2002), there was still no evidence of proteasome-mediated proteolysis of the EBNA3s in its presence (Fig. 6b).



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Fig. 6. In LCL cells, EBNAs 3A, 3B and 3C are very stable and show no evidence of turnover. (a) Actively proliferating LCL B cells were treated with the potent and highly specific proteasome inhibitor lactacystin at a final concentration of 2 µM. Cells were then harvested at the times indicated and proteins were extracted and resolved by SDS-PAGE (7·5 or 12·5 % gels). Proteins were then Western-blotted and probed with the antibodies indicated. (b) A similar experiment to that shown in (a) was performed but, in addition to incubating with lactacystin, IFN-{gamma} was added at a final concentration of 500 U ml–1. (c) Actively proliferating LCL B cells were treated with the protein synthesis inhibitor cycloheximide at a final concentration of 50 µg ml–1. Cells were harvested at the times indicated and protein extracts were analysed as in (a).

 
As none of the EBNA3 proteins accumulated in the presence of lactacystin, further experiments were performed in order to determine whether the EBNA3s were degraded in the absence of protein synthesis. Fig. 6(c) illustrates an experiment in which LCL cells were exposed to an inhibitor of protein synthesis, cycloheximide. At the times indicated, samples were taken and Western blots were performed on RIPA buffer-extracted proteins. Although p21WAF1/CIP1 and p53 – both proteins known to have a relatively short half-life – were undetectable in extracts within 8 h of adding cycloheximide, the levels of EBNAs 3A, 3B and 3C remained largely unaffected after this inhibition of de novo protein synthesis. As before, similar results were obtained by using total protein extracts that were solubilized directly in SDS sample buffer (data not shown). Beyond 24 h, all of these experiments became unreliable because of significant cell death in the cultures treated with cycloheximide. These data suggest that the EBNA3 proteins are very stable in LCLs and that their rate of synthesis is exceptionally low. These observations are consistent with the small amounts of EBNA3-specific mRNA that can be detected in LCLs (Kieff & Rickinson, 2001) and our repeated inability to label EBNA3C metabolically with [35S]methionine in LCLs, despite ‘pulses' of up to 16 h (R. Touitou & M. J. Allday, unpublished data). There appears to be very little turnover of these proteins in B cells infected latently by EBV.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A mutated EBNA3C protein that no longer binds CBF1/RBP-J{kappa} was used as bait in a yeast two-hybrid screen; this revealed an interaction between this viral oncoprotein and the C8/{alpha}7 subunit of human 20S proteasomes. The interaction was confirmed in yeast with additional EBNA3C polypeptides and, subsequently, by a series of GST pull-down experiments and co-IPs. Similar experiments showed that the other family members, EBNA3A and EBNA3B, are capable of binding C8/{alpha}7 with similar efficiency to that of EBNA3C. In their capacity to bind to C8/{alpha}7, the EBNA3s resemble the cyclin-dependent kinase inhibitor p21WAF1/CIP1. All of the C8/{alpha}7 clones that were recovered from the yeast two-hybrid screen represented full-length cDNAs, consistent with the previous observation that any truncation of C8/{alpha}7 abrogates its binding to p21WAF1/CIP1 (Touitou et al., 2001b). It is assumed that the small protein C8/{alpha}7 (~14 kDa) requires its complete complement of amino acid residues to adopt its correct secondary structure.

Unfortunately, attempts to identify the binding site in EBNA3C (and EBNAs 3A and B) were complicated by the discovery that at least four different regions, spread throughout the protein, are capable of binding C8/{alpha}7 in GST pull-down experiments. It is not known whether this is because there are four (or more) binding sites on the intact EBNA3C protein or whether truncation and fusion of EBNA3C to p27KIP1 reveal polypeptides that are normally concealed in the native protein. As it was unlikely that a unique, easily mutated, C8/{alpha}7-binding sequence could be identified in these EBV proteins, deletion mapping was not continued.

We have shown previously that p21WAF1/CIP1 is degraded rapidly by purified 20S proteasomes in a manner that is dependent on its interaction with C8/{alpha}7. Specifically, this requires a domain in the C terminus of p21WAF1/CIP1 to which C8/{alpha}7 binds. The domain also appears to be a major determinant of p21WAF1/CIP1 stability in cells (Touitou et al., 2001b). Although it was not possible to map similar domains in the EBNA3 proteins, it was possible to perform similar degradation assays. The results of these analyses revealed that all three viral proteins were degraded specifically in the presence of 20S proteasomes and that this proteolysis does not require polyubiquitinylation or the regulatory cap structures that are normally associated with 26S proteasomes in cells. However, not all proteins can be degraded by isolated 20S proteasomes. For instance, it was demonstrated previously that p27KIP1, lysozyme, casein and GFP are all stable in the presence of 20S proteasomes (Kisselev et al., 1999; Liu et al., 2003; Orlowski & Wilk, 2003; Touitou et al., 2001b) and we show here that the proteins pRb and {alpha}B-crystallin are also apparently unaffected by incubation with 20S proteasomes. In the case of {alpha}B-crystallin, this is despite its capacity to bind C8/{alpha}7 (Boelens et al., 2001). Although a specific interaction with {alpha}-subunits of the 20S proteasome may facilitate rapid protein degradation, it seems that a more important characteristic of proteins that are degraded by purified 20S proteasomes, such as p21WAF1/CIP1 and {alpha}-synuclein, is that they are natively unfolded (Liu et al., 2003; Orlowski & Wilk, 2003; Touitou et al., 2001b). Therefore, when these proteins are not in functional complexes that confer their structure, they have direct access to the catalytic core of the proteasome. As the EBNA3s are all degraded by 20S proteasomes (which do not include the ATP-dependent unfolding activity found in 26S proteasomes), it is reasonable to assume that these large viral proteins may also be similarly unfolded. Indeed, the predicted secondary structures of all three EBNA3s suggest that they include extensive regions of unstructured (natively unfolded) polypeptide (Fig. 7). Therefore, in the absence of binding partners and/or chaperones that may be necessary to ensure correct folding of newly synthesized proteins (Yewdell, 2002), the EBNA3s may be vulnerable to proteasome-mediated degradation, whether or not ubiquitinylation has occurred.



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Fig. 7. Schematic representation of EBNAs 3A, 3B and 3C, showing that large regions of each lack predictable structure (white regions). Black regions represent those with predicted secondary structure (helices or {beta}-strands). Analyses are based on the programs described by Baldi et al. (1999) and Pollastri et al. (2002).

 
When we determined the stability and turnover of EBNAs 3A, 3B and 3C in latently infected B cells, we were initially rather surprised to find no evidence of de novo synthesis or proteasome-mediated degradation of these proteins. Clearly, there must be a very low level of synthesis, as the cells in the population divide; however, this is undetectable by the methods used here. This result was particularly unexpected, as the EBNA3s include epitopes that apparently form the major focus of the CTL-mediated immune response against cells infected latently with EBV. Naively, one might have expected such proteins to be turned over rapidly if immunodominance was related in some way to the efficiency with which polypeptides gain access to the antigen-processing pathways. Our current view is that, in vivo, in the LCL-like infected B cells, synthesis and degradation of the EBNA3s occur at a very low maintenance level. However, after primary infection of resting B cells, there must be significant de novo synthesis of the EBNA3s and – being long and relatively unfolded – they may be degraded readily, resulting in efficient processing for antigen recognition. Therefore, early after infection, EBV-positive B blasts could be particularly sensitive to CTL-mediated killing. The hypothesis that a lack of secondary structure enhances degradation by the proteasome system is consistent with the notion that defective ribosomal products (DriPs) – generated when proteins are newly synthesized – are targeted for destruction with remarkable efficiency because they cannot adopt the appropriate conformation (Yewdell, 2002). It may well be that DriPs do not require ubiquitinylation and could be directed to proteasomes by specific interactions with 20S subunits, such as C8/{alpha}7.


   ACKNOWLEDGEMENTS
 
We are very grateful to the Wellcome Trust for financial support for this work (a programme grant to M. J. A.). We would also like to thank W. C. Boelens (Nijmegen, the Netherlands) for the {alpha}B-crystallin-encoding plasmid and Paul Freemont (Centre for Structural Biology, Imperial College, London, UK) for advice on the structural predictions.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 15 November 2004; accepted 26 January 2005.



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