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
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ABSTRACT |
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Published online ahead of print on 3 February 2005 as DOI 10.1099/vir.0.80763-0.
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INTRODUCTION |
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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
. 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
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
, 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
(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-
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/
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 C8/7 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.
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METHODS |
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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 GSTC8 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 ml1] 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 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 ml1 and 100 U streptomycin ml1 (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-).
To inhibit protein synthesis, cells were treated with cycloheximide (Sigma) (stock solution, 5 mg ml1 in PBS) at a final concentration of 50 µg ml1 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- (Pharmingen/BD Biosciences) was used at a final concentration of 500 U ml1.
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 GSepharose 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 GSepharose 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/
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).
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RESULTS |
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All of the C8/7 clones represented full-length cDNAs and further analysis in yeast confirmed this interaction, not only with the alanine-substitution RBP-J
-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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DISCUSSION |
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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 C8/7 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/
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 C8/7. Specifically, this requires a domain in the C terminus of p21WAF1/CIP1 to which C8/
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
B-crystallin are also apparently unaffected by incubation with 20S proteasomes. In the case of
B-crystallin, this is despite its capacity to bind C8/
7 (Boelens et al., 2001
). Although a specific interaction with
-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
-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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ACKNOWLEDGEMENTS |
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Received 15 November 2004;
accepted 26 January 2005.
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