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J Virol. Mar 2007; 81(5): 2459–2471.
Published online Dec 20, 2006. doi:  10.1128/JVI.02289-06
PMCID: PMC1865947

Role of the TSG101 Gene in Epstein-Barr Virus Late Gene Transcription[down-pointing small open triangle]

Abstract

Rta, an Epstein-Barr virus (EBV)-encoded immediate-early protein, governs the reactivation of the viral lytic program by transactivating a cascade of lytic gene expression. Cellular transcription factors such as Sp1, ATF2, E2F, and Akt have been demonstrated to mediate Rta transactivation of lytic genes. We report herein that Rta associates with another potent transcription factor, tumor susceptibility gene 101 (TSG101), to promote the activation of EBV late genes. Results from an EBV cDNA array reveal that depletion of TSG101 by siRNA potently inhibits the transcription of five Rta-responsive EBV late genes, BcLF1, BDLF3, BILF2, BLLF1, and BLRF2. Depletion of TSG101 impairs the Rta transactivation of these late promoters severely. Moreover, a concordant augmentation of Rta transactivating activity is observed when TSG101 is overexpressed following ectopic transfection. Mechanistically, Rta interaction with TSG101 causes the latter to accumulate principally in the nuclei, wherein the proteins colocalize and are recruited to the viral promoters. Of note, TSG101 is crucial for the efficient binding of Rta to these late promoters. As a result, cells with defective TSG101 fail to express late viral proteins, leading to a decrease in the yield of virus particles. Thus, the contribution of TSG101 to Rta-mediated late gene activation is of great importance for completion of the EBV productive lytic cycle. These observations consolidate a role for TSG101 in the replication of EBV, a DNA virus, that differs from what is observed for RNA viruses, where TSG101 aids mainly in the endosomal sorting of enveloped late viral proteins for assembly at the plasma membrane.

Switching from the latent stage to viral lytic replication is an important step in the herpesviral life cycle and leads to the production of infectious progeny (31). In the case of Epstein-Barr virus (EBV), an enveloped gammaherpesvirus with a double-stranded DNA genome, the productive lytic cycle can be induced by a variety of stimuli (31). For instance, modulation of the intracellular calcium concentration, as well as treatment with phorbol ester, sodium butyrate (SB), anti-human immunoglobulin (Ig), or transforming growth factor β, can provoke the reactivation of EBV (11, 16, 38, 46, 75). To produce mature progeny viruses, EBV needs to complete its lytic cycle, from immediate-early to early to late protein synthesis (31). Replication of viral DNA, which takes place in the host cell nucleus, occurs between early and late protein synthesis (31). Thereafter, the viral DNA is encapsidated into nucleocapsids, which egress through the nuclear membrane via a process of envelopment and deenvelopment and are eventually released into the cytoplasm (17, 31, 50). A mature virion is formed and secreted into the extracellular compartment after tegumentation and secondary envelopment of the viral nucleocapsid (20, 31, 50).

A critical role for the immediate-early proteins Zta (BZLF1 or EB1) and Rta (BRLF1) is found in the switch between latency and the lytic cycle. Both of these proteins control viral reactivation by acting as transactivators which cooperate with each other to initiate the transcription of an ordered cascade of lytic genes (13, 18, 24). Zta and Rta are sequence-specific DNA binding proteins that bind directly to their individual response elements in the promoters of EBV lytic genes (22, 69). At the onset of the lytic cascade, Zta is expressed first and activates the promoters of Rta and Zta (56, 63). Thenceforth, Rta auto-stimulates its own promoter and, vice versa, increases the transcription of Zta (41, 56). Viral early genes are then activated sequentially by these two transactivators (31). Most of the early genes encode proteins that are responsible for viral DNA replication, such as BALF5 (DNA polymerase) and BMRF1 (EA-D, polymerase accessory protein) (34, 39). The EBV late proteins are expressed during the late phase of the lytic cycle, and the majority are structural proteins that are incorporated into the viral capsid and tegument as well as the envelope (31).

The expression of most EBV lytic genes is attributable largely to transactivation by Zta and Rta, but with differential reactivities (31). Thus, several subgroups of EBV lytic genes can be distinguished based on their responses to these transactivators. The first group is activated by Zta alone, as exemplified by the early protein BMRF1, which is expressed in EBV-infected epithelial cells (6, 26). The second group is induced by Rta alone and includes the early protein BALF5 as well as the late proteins BLLF1 (gp350/220) and BLRF2 (p23 or LR2), which are expressed in some specific cell types (15, 18, 40, 58). The third group of lytic genes requires synergistic coactivation by both Zta and Rta and includes the early protein BFRF1 as well as BMRF1, which is expressed specifically in lymphocytes, in that Zta or Rta alone activates a negligible amount of BMRF1, despite the great stimulatory effect when both transactivators act together (21, 26, 55). Thus, it is clear that these transactivators cooperate to advance the lytic cycle.

To complete its lytic cycle, EBV not only needs its own proteins, Zta and Rta, to transactivate the viral promoters directly but also requires the collaboration of cellular transcription factors for the expression of several lytic genes (1, 5, 8, 14, 28, 40, 57). One major unresolved issue regarding EBV in vivo is how the cellular machinery is involved in transcribing the viral lytic genes. A recent study on this subject using microarrays for systematic screening demonstrated that EBV replication is associated with significant changes in the levels of 122 host cell RNAs (73). These include RNAs coding for transcription factors, antiapoptotic proteins, and cell signal transduction factors (73). In parallel to these findings, our previous study also demonstrated that the ERK signal pathway is important for the reactivation of EBV (8). In addition, the cellular transcription factors Egr-1 and TSG101 are significantly increased during the progression of the viral lytic cycle. The function of Egr-1 in the EBV lytic cycle has been described in detail previously (8).

To unravel further the usage of cellular machinery in EBV lytic cycle progression, we explored the probable involvement of the TSG101 protein in the present study. TSG101 mediates a variety of biological functions. For instance, it may function as a transcriptional coregulator of nuclear hormone receptors (4, 51, 64, 72); it controls the cell cycle and promotes proliferation of tumor cells (71, 74), and it is a member of the class E vacuolar protein-sorting (vps) family and involved in endosomal sorting (2, 45). Being a protein sorter, TSG101 is recruited to the endosomal membrane and interacts with early, as well as late, endosomal proteins (2, 45, 70). Thereafter, these complexes couple with the ubiquitinated protein cargo for endosomal trafficking (45). Opportunistically, human immunodeficiency virus captures this endosomal protein-sorting network, in that TSG101 is recruited by the PTAP motif within the late domain of Gag to help in the delivery of ubiquitinated Gag to the plasma membrane; this eventually promotes virion budding off (19). Likewise, the release of other pathogenic RNA virus particles, including Ebola virus and human T-cell leukemia virus type 1, also requires the endosomal sorting function of TSG101 via direct interaction with the structural proteins (3, 49). Other than assisting in viral budding, TSG101 is exploited by viruses to subvert the host immune surveillance, as illustrated by Kaposi's sarcoma-associated herpesvirus and hepatitis E virus. To disrupt the viral antigen presentation pathway, Kaposi's sarcoma-associated herpesvirus uses its K3 gene product to ubiquitinate major histocompatibility complex class I molecules and then delivers the complex to lysosome for rapid degradation by usurping the TSG101-dependent vacuolar protein-sorting pathway (25). On the other hand, hepatitis E virus utilizes its ORF3 protein to interact with TSG101 and employs TSG101 in sorting α1 microglobulin, an immunosuppressant molecule which inhibits interleukin-2 production by T cells and chemotaxis of granulocytes, for secretion (65).

Two lines of evidence from other reports imply the potential involvement of the TSG101 protein in the EBV lytic cycle. TSG101 can function as a transcriptional coregulator and can interact with the viral structural proteins to facilitate the release of viral particles (3, 4, 19, 49, 51, 64, 72). Accordingly, we systematically examined the effect of TSG101 on the EBV gene expression profile. This study demonstrates that EBV has another way of exploiting TSG101. When the endogenous TSG101 was knocked down using a small interfering RNA (siRNA) approach, the expression of several EBV late genes was impaired. The underlying mechanism was found to be Rta related and involved the transcriptional coactivation of TSG101. The functional relevance of TSG101 in Rta transactivation was ultimately shown to be crucial for efficient late protein synthesis and virion production. Thus, our finding revealed a novel strategy adopted by EBV to complete its productive lytic cycle.

MATERIALS AND METHODS

Cell culture.

NPC-TW01 is an NPC cell line which has lost its original infecting EBV after passage (37). This line was reinfected with a recombinant Akata-EBV strain carrying a neomycin-resistant gene and was named NA (10). The paired pZip-NeoSV(X)1 control plasmid transfectant (NPC-TW01-pZip) serves as an EBV-negative control. All cell lines were cultured with Dulbecco's modified Eagle's medium containing l-glutamine, penicillin-streptomycin, and 8% fetal calf serum. NA and NPC-TW01-pZip were maintained in G418-containing medium.

Plasmid construction and transfection.

Full-length Rta and green fluorescent protein (GFP)-Rta expression plasmids were generated by cloning Rta-coding sequences into the pSG5 (Stratagene) and the pEGFP-C1 vectors (BD Biosciences Clontech), respectively. In addition, Rta deletion mutants were created by ligating PCR-amplified products into the pEGFP-C1 vectors. Myc-tagged, full-length TSG101 and its deletion constructs were cloned according to a previous study (36). siTSG101 and siE7 oligonucleotides were synthesized, annealed, and cloned into the pSuper vector as described previously (19, 23). The construction of pSuper-GFP siRNA (siGFP) has been described in our previous publication (6). Regarding the cloning of reporter gene plasmids, the promoters of Zta (Zp, nucleotides [nt] 103182 to 103415 of the EBV genome), Rta (Rp, nt 106177 to 107144 of the EBV genome), BcLF1 (pBcLF1, nt 137467 to 138092 of the EBV genome), BDLF3 (pBDLF3, nt 131067 to 131246 of the EBV genome), BILF2 (pBILF2, nt 150415 to 151571 of the EBV genome), BLLF1 (pBLLF1, nt 92053 to 93153 of the EBV genome), or BLRF2 (pBLRF2, nt 88548 to 89025 of the EBV genome) were generated by PCR amplification and ligated into pGL2-basic vector (Promega). The DNA transfection was carried out using TransFast transfection reagent (Promega) according to the manufacturer's instructions.

RNA extraction and reverse transcription (RT)-PCR.

Total RNAs were isolated from cultured cells using the TRIzol reagent (Invitrogen) as specified by the manufacturer. The integrity of total RNA was determined by spectrophotometry. cDNA was generated, following the outline of the manufacturer's instructions (Invitrogen). PCR analyses of EBV gene products, as well as defender against cell death 1 (DAD-1), were carried out as described previously (9, 44).

Microarray analysis.

The oligo(dT)-primed cDNA, which was reverse transcribed from 20 μg of total RNA, was labeled with biotin-dUTP as described previously (44). A nylon membrane-based DNA array, consisting of 85 predicted open reading frames carried by the EBV genome (NTU_EBVArray), was used in this assay (44). The specific procedures of probe preparation, hybridization, and colorimetry detection were carried out as described previously (44). Ultimately, the purple chromogen on the membranes was scanned and quantified by the GenePix microarray image analysis program (Axon Instrument, Inc.).

Reporter gene assay.

Reporter plasmids were transfected transiently with the effectors (Rta and TSG101), as well as a GFP expression plasmid (pEGFP-C1, internal control), into NPC-TW01 cells. The total amounts of DNA were adjusted by adding empty vector if required. The transfected cells were trypsinized and subjected to serial analysis for GFP fluorescent intensities and Firefly luciferase activities, using a Bright-Glo luciferase assay kit (Promega). Activation was calculated by normalizing Firefly luciferase activity to GFP intensity.

Coimmunoprecipitation (co-IP).

After being precleared with 50% protein A-Sepharose (Amersham Biosciences), the protein lysates (500 μg) extracted by radioimmunoprecipitation assay (RIPA) lysis buffer were incubated with antibodies against TSG101 (GeneTex), Rta (clone 467) (27), or GFP (BD Biosciences) overnight at 4°C on a rotating rocker. Thereafter, 100 μl of 50% protein A-Sepharose was added for an additional 2 h incubation, and the Sepharose beads were then washed three times with phosphate-buffered saline (PBS). Ultimately, the bead-bound immunocomplexes were dissolved in 2× sodium dodecyl sulfate (SDS) sample buffer.

Western blot analysis.

The immunocomplexes prepared as described above or the protein lysates (lysed by lysis buffer containing 3% SDS, 2 M urea, and 2% 2-mercaptoethanol) were resolved by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to a Hybond-C membrane (Amersham Biosciences). The blots were then blocked in washing buffer (100 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.2% Tween 20) containing 4% skim milk at room temperature for 1 h. Thereafter, the blots were incubated with primary antibodies against TSG101 (GeneTex), Rta (27), EBNA1 (10), Zta (68), BMRF1 (67), BcLF1 (67), BLLF1 (67), GFP (BD Biosciences), Myc (Santa Cruz Biotechnology), lamin C (Santa Cruz Biotechnology), α-tubulin (Calbiochem), or β-actin (Sigma) at 4°C overnight. After three 10-min washes with washing buffer, the horseradish peroxidase-conjugated secondary antibodies were applied to the blots and reacted at room temperature for l h. Finally, the interested proteins were revealed by enhanced chemiluminescence (PerkinElmer), and the luminescence results were detected by exposure on X-ray films.

Immunofluorescence assay.

Cells grown on coverslips were washed with PBS and fixed with acetone at room temperature for 10 min. The cells were then reacted with anti-Rta mouse monoclonal antibodies (1:50) (Argene) and anti-TSG101 rabbit polyclonal antibody (1:100) (42) at 37°C for 1 h. After three 5-min washes with PBS, the cells were incubated with rhodamine-conjugated anti-mouse IgG and fluorescein isothiocyanate-conjugated anti-rabbit IgG (Chemicon) at 37°C for 1 h. Nuclei of the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma). Finally, images were obtained with a Zeiss LSM 510 laser scanning microscope.

Subcellular fractionation.

Cells grown in 10-cm dishes were washed twice with PBS. After addition of 1 ml hypotonic buffer (5 mM Tris [pH 7.4], 5 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride), cells were swollen on ice for 1 h. The resulting cell lysates were centrifuged at 250 × g at 4°C for 5 min, and the supernatants (cytosolic fraction) were recovered. The pellets (nuclear fraction) were washed once with PBS and resuspended in lysis buffer containing 3% SDS, 2 M urea, and 2% 2-mercaptoethanol. The nuclear fractions were sonicated before analysis by Western blotting. The purity of each fraction was assessed by probing for α-tubulin (cytosolic fraction) and lamin C (nuclear fraction).

DNA affinity protein binding assay (DAPA).

The double-stranded promoter probes were prepared by PCR, using biotin-labeled forward primers (5′-AGAGGGAGATGGGGGGAGGTCT-3′ for pBcLF1 and 5′-TTTGCAAAGGCTGTGCCACTGCT-3′ for pBLRF2) and reverse primers (5′-GTATGCTCCAGACTCTGGACTCC-3′ for pBcLF1 and 5′-GAAGAAGTACAAACAGCCGAGATT-3′ for pBLRF2). These biotin-labeled oligonucleotides were incubated with 40-μl streptavidin-agarose beads (Sigma) at 4°C for 1 h. Nuclear extracts which had been precleared with streptavidin-agarose beads were mixed with the oligonucleotide-bound beads and reacted in a binding buffer (20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, and 10% glycerol) for 1 h at 4°C. The beads were then spun down and washed four times in the binding buffer. Finally, proteins captured by the oligonucleotide-bound beads were resolved by an SDS-polyacrylamide gel and analyzed by Western blotting.

Electrophoretic mobility shift assay (EMSA).

The single-stranded oligonucleotides (5′-TCCAGTCCCACAAACGCGGCGGCGG-3′ for RRE-pBLRF2 and 5′-CCTGTGCCTTGTCCCGTGGACAATGTCCC-3′ for RRE-DR1) and their complementary oligomers were subjected to double-stranded annealing and end labeled with [γ-32P]ATP, using T4 polynucleotide kinase (New England Biolabs). The binding reaction was carried out in a total volume of 20 μl of a mixture containing 8 μg nuclear extracts, 32P-labeled oligonucleotides, and binding buffer containing 20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 10% glycerol, and 2 μg poly(dI-dC). The reaction mixtures were incubated at room temperature for 30 min and then resolved into a 6% native polyacrylamide gel which was electrophoresed in 0.5× Tris-borate-EDTA buffer. The gel was subsequently dried and exposed to autoradiography film.

Isolation of EBV particles.

Cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA; 40 ng/ml) (Sigma) plus SB (3 mM) (Sigma) for 72 h before harvesting. The culture supernatants were collected and immediately subjected to centrifugation at 10,000 × g for 30 min at 4°C to remove cell debris. To pellet viral particles, the resultant supernatant was centrifuged at 30,000 × g at 4°C for 90 min. The particles were lysed and prepared for enzyme-linked immunosorbent assays (ELISAs) and PCR assays.

Sandwich ELISA.

Plates were coated with anti-BLLF1 (gp350) antibodies (1:600 dilution) (Novus Biologicals) at 4°C overnight. After being washed three times with washing buffer (0.05% Tween 20 in PBS), the plate was blocked with PBS containing 2% skim milk and 0.2% Tween 20 at 37°C for 1 h. Each well was subsequently incubated with 20 μg cell lysate that had been lysed by RIPA (intracellular EBV) or EBV particles lysed by lysis buffer consisting of 3% SDS, 2 M urea, and 2% 2-mercaptoethanol (secreted EBV) at 4°C overnight. A 1:800 dilution of anti-BLLF1 antibodies was then added and allowed to react at 37°C for 2 h. After being washed three times, each well was incubated with a 1:4,000 dilution of horseradish peroxidase-conjugated anti-mouse IgG for 2 h at 37°C. Color development was performed using 3-3′-diaminobenzidine tetrachloride (DAB) solution, and the A450 value was measured.

Quantification of EBV copy number.

To measure the EBV DNA copy number, 1 × 106 chemically activated NA cells were lysed in 100 μl of lysis buffer (10 mM Tris-HCl [pH 8.0], 2.5 mM MgCl2, 1% Tween 20, 1% NP-40, 1 mg/ml proteinase K) and allowed to react at 50°C for at least 4 h. Proteinase K was then inactivated at 70°C for 15 min. EBNA1 was targeted in the following real-time quantitative PCR (Q-PCR) assays and detected by a model 7700 sequence detector (Applied Biosystems). The setup of the reaction condition and the calculation of the amplification data were operated as recommended by a previous publication (43). The relative EBV DNA copy numbers were determined using DNA extracted from H2B4 cells (one copy EBV/cell), which were run in parallel and in duplicate with each analysis, as a standard calibration control (7).

DNA extraction and PCR assay for secreted EBV.

The viral particles harvested by centrifugation as mentioned above were immediately resuspended in DNase I containing double-distilled H2O and reacted at room temperature for 15 min to eliminate the contaminating cellular DNA. After inactivation of the DNase I, the viral particles were lysed by lysis buffer (10 mM Tris-HCl [pH 8.0], 2.5 mM MgCl2, 1% Tween 20, 1% NP-40, 1 mg/ml proteinase K) at 50°C for at least 4 h. PCR detection of EBV DNA, in particular the BamHI W fragment, was then performed. The primers used were designed according to a previous study (44).

RESULTS

Depletion of TSG101 retards EBV late gene transcription.

Because TSG101 can regulate gene transcription (4, 51, 64, 72), we determined whether the expression profiles of EBV genes are affected by TSG101. To approach this, rapid depletion of TSG101 with siRNA (siTSG101) was achieved in an EBV-harboring NA cell prior to TPA and SB treatment. The extracted total RNA was used for microarray analysis to assess the complete set of EBV gene expression clusters (44). Of 85 EBV latent and lytic genes, only the expression of several viral late genes was found to be defective in the TSG101 knocked-down cells; however, the amounts of latent, immediate-early, and early transcripts were not altered (Fig. (Fig.1A1A and data not shown). As shown in Fig. Fig.1A,1A, the RNA levels of BcLF1, BDLF3, BILF2, BLLF1, and BLRF2 apparently were diminished in siTSG101-transfected cells compared to those of the control vector pSuper and siGFP, an irrelevant siRNA control. Simultaneously, the results were confirmed by an RT-PCR assay and supported the finding that cells with TSG101 depletion tended to have smaller amounts of BcLF1, BDLF3, BILF2, BLLF1, and BLRF2 transcripts than siGFP-transfected cells (Fig. (Fig.1B).1B). On the other hand, the expression levels of EBV immediate-early transcripts, such as Zta and Rta, as well as the early transcript BMRF1, were equivalent in siTSG101- and siGFP-transfected cells (Fig. (Fig.1).1). All these data suggest a role for TSG101 in EBV late gene expression.

FIG. 1.
Depletion of TSG101 hinders the transcription of EBV late genes. (A) Microarray analysis of EBV gene transcriptional profiles. NA cells were subcultured three times and, at each passage, transfected once with the vector plasmid pSuper or its derivatives ...

TSG101 crucially augments the transactivation ability of Rta.

Given that the expression of EBV lytic genes is controlled principally by the Zta and/or Rta transactivators (31), we questioned which transactivator directs the activation of the promoters of these five EBV late genes. In a reporter gene assay, the luciferase activities driven by the promoters of BcLF1, BDLF3, BILF2, BLLF1, and BLRF2 were detectable in response to the expression of Rta (Fig. (Fig.2A).2A). Hence, Rta alone is sufficient to stimulate the promoter activities of these late genes. Importantly, all these viral late promoters were neither activated by Zta nor enhanced by the coexpression of Zta and Rta, whereas only Zp can be transactivated synergistically by the coexistence of Zta and Rta, as expected (Fig. (Fig.2A).2A). Among these viral late promoters, pBDLF3, pBLLF1, and pBLRF2 have been documented to be activated directly by Rta, without the cooperative function of Zta, in agreement with our finding (15, 18, 33, 58).

FIG. 2.
TSG101 cooperates with Rta to transactivate EBV late genes. (A) Examination of Zta and Rta transactivating EBV late genes by a reporter assay. NPC-TW01 cells were cotransfected with 0.4 μg reporter plasmids, 0.4 μg pEGFP-C1 (internal control), ...

Having shown that the induction of these late promoters was Rta responsive, we proceeded to determine whether depletion of TSG101 affects Rta-mediated late promoter transactivation. The reporters bearing the promoters of BcLF1, BDLF3, BILF2, BLLF1, and BLRF2 were introduced into siTSG101- or siE7-harbored, EBV-negative NPC-TW01 cells, and the promoter activities were monitored in the presence of Rta. As seen in Fig. Fig.2B,2B, depletion of TSG101 greatly attenuated the ability of Rta to transactivate these late promoters, resulting in a dramatic decrease in promoter activities. To investigate further the influence of TSG101 in Rta-mediated transcription, we measured the promoter activities of BcLF1, BDLF3, BILF2, BLLF1, and BLRF2 in the cells ectopically expressing Rta and increasing amounts of TSG101. The activities of all these late promoters rose in a dose-dependent manner in response to the increasing amounts of TSG101 (Fig. (Fig.2C).2C). All these data suggest a collaborative role for TSG101 in mediating the Rta transactivation of BcLF1, BDLF3, BILF2, BLLF1, and BLRF2.

EBV Rta binds TSG101.

Several reports indicated that TSG101 may affect gene regulation through a direct interaction with transcriptional cofactors (4, 51, 64). To reveal the molecular mechanism by which TSG101 is exploited by Rta for gene activation, we first explored the possibility of physical interaction between Rta and TSG101. EBV-carrying NA cells were treated with TPA and SB to evoke the ordered cascade of the EBV lytic program so that the expression of Rta and other lytic proteins was induced (data not shown) (10). The amounts of TSG101 precipitated by the anti-TSG101 antibody were elevated in accordance with the treatment with TPA and SB in both the NA and the EBV-negative NPC-TW01-pZip cells (Fig. (Fig.3A).3A). Of note, increasing amounts of Rta were detected in the TSG101-captured immunocomplexes from the reactivated NA cells (Fig. (Fig.3A).3A). Similar results were seen when anti-Rta antibody was used to perform the co-IP assay (Fig. (Fig.3A,3A, lane 10). To determine whether the interaction of Rta and TSG101 can occur in the absence of other EBV gene products, a TSG101-specific co-IP was performed with cell lysates from Rta-transfected NPC-TW01-pZip cells. As shown in Fig. Fig.3B,3B, immunoprecipitation of TSG101 resulting in coprecipitation of ectopically expressed Rta was seen in the NPC-TW01-pZip and NA cells. Rta protein expressed in cells devoid of TSG101 was not precipitated by the antibody against TSG101, strengthening the argument that the coprecipitation of Rta was mediated through TSG101 (Fig. (Fig.3B,3B, lane 5). These results suggest that Rta proteins can interact competently with TSG101 proteins, without the help of other viral products, regardless of whether Rta is induced endogenously by chemical manipulation or expressed exogenously by ectopic transfection.

FIG. 3.
Rta interacts with TSG101. (A) Demonstration by co-IP of the interaction between TSG101 and chemically induced Rta. NA cells were treated with TPA and SB to induce the EBV lytic cycle. The EBV-negative NPC-TW01-pZip cells were treated in the same way ...

The N terminus of Rta mediates the binding of the TSG101 Ub-conjugating enzyme E2 variant (UEV) domain.

Structurally, the Rta protein possesses a DNA binding domain and a dimerization domain in the N terminus and a transactivation domain in the C terminus (48) (Fig. (Fig.4A).4A). In an attempt to map the domain of interaction between Rta and TSG101, a series of GFP-fused Rta deletion mutants, displayed in the schematic diagram in Fig. Fig.4A,4A, were coexpressed with TSG101 in NPC-TW01 cells. As revealed by the TSG101-specific co-IP and Western blot assay, we found that full-length Rta (GFP-Rta1-605), as well as the mutants GFP-Rta1-441 and GFP-Rta1-333, was able to interact with TSG101 (Fig. (Fig.4A).4A). In contrast, the fusion protein containing the Rta C terminus, GFP-Rta401-605, failed to bind TSG101 in the co-IP assay (Fig. (Fig.4A).4A). Thus, the N-terminal domain of Rta is required for interaction with TSG101.

FIG. 4.
Mapping of the Rta-TSG101 interaction domain. (A) Investigation of the Rta domain required for TSG101 interaction. A plasmid that expressed GFP-Rta and its deletion derivatives as shown in the schematic diagram (left panel) were transfected together with ...

On the other hand, TSG101 comprises an N-terminal UEV domain that is flanked by a proline-rich (Pro-rich) region, a coiled-coil (CC) domain, and a C-terminal steadiness box (S-box) (54) (Fig. (Fig.4B).4B). The reciprocal co-IP experiment showed that, among the TSG101 deletion mutants which are illustrated schematically in Fig. Fig.4B,4B, only those which retained the N-terminal UEV domain (Myc-TSG1-391, -TSG1-312, and -TSG1-160) bound Rta competently (Fig. (Fig.4B).4B). Mutants which lacked the S-box, CC, or Pro-rich domains did have the binding activities with Rta (Fig. (Fig.4B).4B). Therefore, the Rta binding region in the TSG101 protein maps to the UEV domain.

Rta colocalizes with TSG101 in the nucleus.

A confocal immunofluorescence assay was carried out to substantiate the interaction between Rta and TSG101. Within the cells transfected with the pSG5 control vector, TSG101 proteins were scattered in the cytoplasm in proximity to the nuclear membranes and displayed faintly positive reactions in the nuclei (Fig. (Fig.5A).5A). Rta was stained predominantly in the nuclei, which was indicated by counterstaining with the nuclear marker DAPI, and weakly in the cytosolic compartments (Fig. (Fig.5A).5A). Importantly, upon Rta expression, a characteristic nuclear punctate staining of TSG101 was observed in both NPC-TW01 and NA cells (Fig. (Fig.5A).5A). In addition, confocal analysis demonstrated the colocalization of Rta and TSG101 proteins, as shown by the yellow signal in the merged images (Fig. (Fig.5A,5A, right panel). To determine whether the Rta expressed by chemical induction displays a pattern of colocalization similar to that of TSG101, the cells being tested were treated with TPA and SB. Microscopically, the locations of TSG101 in the nuclei of the lytically induced cells were coincident with the site of clustering of the Rta protein and gave a yellowish colocalization signal in the merged image (Fig. (Fig.5B).5B). No Rta was detectable in the TPA- and SB-treated NPC-TW01 cells, and the nuclear TSG101 was stained negligibly (Fig. (Fig.5B).5B). This result gives further support for the physical interaction between Rta and TSG101, and these colocalization events are primarily in the nucleus.

FIG. 5.
Colocalization of Rta and TSG101. Cells grown on coverslips were transfected with pSG5 and Rta-expressing plasmids (A) or treated with TPA and SB (B). Thereafter, cells were fixed, permeabilized with acetone, and stained with the antibodies indicated ...

Rta increases the nuclear TSG101 protein level.

The cellular location of Rta is mainly the nucleus and to a lesser extent the cytoplasm (14) (Fig. (Fig.5A).5A). On the other hand, TSG101 is localized predominantly in the cytoplasm, acting as an endosomal trafficking protein, and occasionally, it is also demonstrated in the nucleus, where it regulates transcription (2, 4, 45, 51) (Fig. (Fig.5).5). Given that the subcellular location of TSG101 changed from the cytosol to the nucleus upon Rta expression, as seen in Fig. Fig.5,5, we speculated that Rta may have a role in the redistribution of TSG101 between subcellular compartments. In order to test this hypothesis, Rta-transfected cells were fractionated into nuclear and cytosolic parts and TSG101 location was determined by immunoblotting. The use of lamin C and α-tubulin as nuclear and cytosolic markers allowed monitoring of the purity and the quantity of each protein fraction. At 24 h posttransfection, Rta proteins were clearly detectable in the nuclear fraction (Fig. (Fig.6A,6A, lane 7). Meanwhile, TSG101 in the corresponding nuclear fraction showed amounts almost similar to those in the pSG5 control (Fig. (Fig.6).6). After 48 h of Rta transfection, the nuclear expression of TSG101 had evidently increased to a level higher than that of control pSG5 and was maintained at this high level at least until 72 h (Fig. (Fig.6).6). The amounts of cytosolic TSG101 were not changed visibly in any pSG5- or Rta-transfected cells (Fig. (Fig.6).6). We had observed that the amount of TSG101 in the whole-cell lysate increased when Rta was expressed (data not shown). This augmentation of TSG101 level seemed to occur mainly in the nucleus and, to a lesser extent, in the cytoplasm after quantification by IMAGEQUANT (Fig. (Fig.6B).6B). Taken together, these data indicate that the expression of Rta is attributable to the nuclear accumulation of TSG101.

FIG. 6.
Rta increases the nuclear TSG101 protein level. (A) Analysis of TSG101 subcellular location. An Rta-expressing plasmid and the control vector pSG5 were transfected into NPC-TW01 cells. The transfected cells were harvested at the times indicated and subjected ...

TSG101 facilitates Rta binding to the promoters of EBV late genes.

Considering that Rta is a DNA binding protein (22), we questioned whether the capacity of Rta to bind viral late promoters is influenced by the presence of the TSG101 protein. To address this possibility, the amounts of Rta bound to DNA promoters of BcLF1 and BLRF2 were assessed in the TSG101-depleted cells with a DAPA approach. Immunoblot assays performed on the nuclear extracts demonstrated the successful knocking down of TSG101 and equal levels of Rta expression in both siRNA-transfected cells (Fig. (Fig.7A,7A, left panel). The result indicated that Rta bound to the pBcLF1 and pBLRF2 promoters, as expected (Fig. (Fig.7A).7A). TSG101 expressed in the siGFP-transfected cells was loaded onto pBcLF1 and pBLRF2 even in the absence of Rta, and the amounts of TSG101 on these promoters did not change significantly while Rta was expressed (Fig. (Fig.7A).7A). By comparison with these siGFP control cells, the amounts of Rta bound to pBcLF1 and pBLRF2 in siTSG101 cells were decreased, which coincided with the loss of TSG101 from the promoters (Fig. (Fig.7A).7A). Detection of lamin C excluded the possibility of nonspecific binding by the biotinylated probes (Fig. (Fig.7A).7A). This finding suggested that depleting TSG101 competently diminishes the recruitment of Rta to its responsive viral late promoters.

FIG. 7.
The presence of TSG101 on EBV late promoters improves the DNA binding capacity of Rta. (A) Elucidation of the capacity of Rta to bind to pBcLF1 and pBLRF2 upon TSG101 depletion by DAPA. After two rounds of siTSG101 or siGFP transfection, the siRNA-harboring ...

Rta is known to bind directly to a cluster of EBV lytic gene promoters that contain an Rta-responsive element (RRE) (22). In addition, Rta can be targeted indirectly to the transcription factor binding sites in viral promoters by interacting with DNA-bound cellular transcription factors (5, 57). In view of the fact that the DAPA probe of pBLRF2 encompasses an RRE motif but the pBcLF1 probe does not contain an RRE, we wondered whether TSG101 may regulate Rta binding, not only with the RRE but also with the cellular transcription factor binding site. To investigate this, we used an RRE probe (RRE-pBLRF2) which corresponds to the pBLRF2 RRE sequences (12) in the consecutive EMSA experiment. Instead of full-length Rta, the GFP-Rta1-441 mutant that is truncated in the Rta C terminus but retains the DNA binding domain was used in the assay because it has better DNA binding activity (12). Data from the nuclear extracts of GFP-Rta1-441 transfection told us that GFP-Rta1-441 can bind the RRE-pBLRF2 probe in the siE7 control cells (Fig. (Fig.7B).7B). Yet, the amount of binding of Rta was markedly reduced when TSG101 was silenced, compared to that of siE7, in spite of the equivalent expression levels of GFP-Rta1-441 in both nuclear extracts (Fig. (Fig.7C).7C). A significant reduction of Rta binding efficiency was still seen when the RRE-DR1 probe, a well-defined consensus RRE from ORIlyt DR1 (22), was used in the EMSA experiment (Fig. (Fig.7B).7B). This suggests that to a large extent, TSG101 facilitates Rta recruitment to the Rta-responsive promoters in both RRE-dependent and RRE-independent manners.

TSG101 is essential for EBV late protein synthesis and virion release.

Finally, we asked whether silencing TSG101 causes a reduction in EBV late protein synthesis as a result of the deficiency in late gene transcription (Fig. (Fig.1).1). To approach this issue, the siTSG101 construct was once again used to deplete the endogenous TSG101 in EBV-harboring NA cells, and this siTSG101 was always specific and effective in knocking down TSG101 (Fig. (Fig.8A).8A). Following transfection of siRNA, the NA cells were manipulated chemically to elicit EBV lytic replication. Cells were harvested for protein extraction and analyzed for the expression profiles of viral proteins by immunoblotting. There were no differences in the expression of EBNA1, a viral latent protein, and Zta (Fig. (Fig.8A).8A). Expression of Rta and BMRF1 was slightly, but not significantly, reduced in the TSG101-depleted cells (Fig. (Fig.8A).8A). Nonetheless, expression of EBV late proteins, such as BcLF1 and BLLF1, was found to be reduced markedly in the TSG101 knocked-down cells (Fig. (Fig.8A).8A). Thus, we speculated that the impact of TSG101 depletion on late protein synthesis may prevent virion production. To pursue this possibility, a sandwich ELISA system coated with capture anti-BLLF1 (gp350) antibody was used to quantitate the intracellular virions as well as the extracellular virions that were secreted into cultured medium. From siGFP control cells without TPA and SB treatment, a moderate level of cellular BLLF1 signal was detected, suggesting that the residing EBV had undergone a spontaneous lytic cycle following the stress of siGFP transfection (Fig. (Fig.8B,8B, lane 1). Clearly, a strongly positive signal of BLLF1 was visible from the intracellular EBV particles after administering TPA and SB to the siGFP control cells (Fig. (Fig.8B,8B, lane 2). The EBV particles secreted from these TPA- and SB-treated siGFP cells also were detected concurrently, confirming the presence of virions in the culture supernatant (Fig. (Fig.8B,8B, lane 6). In contrast, the immunoreactivity for BLLF1 was undetectable in the TPA- and SB-treated, TSG101-depleted cells as well as the corresponding culture medium (Fig. (Fig.8B,8B, lanes 4 and 8), consistent with the protein level determined by immunoblotting (Fig. (Fig.8A,8A, lane 3).

FIG. 8.
TSG101 is essential for the production of EBV late proteins and viral particles. (A to D) NA cells were transfected twice with the siRNA indicated or the control plasmid pSuper. TPA and SB were then administered to these transfected cells. After 72 h ...

The quantities of intracellular EBV DNA (Fig. (Fig.8C),8C), as well as secreted viral DNA (Fig. (Fig.8D),8D), were measured in the TSG101-depleted NA cells, using Q-PCR and PCR approaches, respectively. The intracellular viral DNA content in siTSG101 transfectants was similar in amount to the pSuper- and siGFP-transfected controls (Fig. (Fig.8C),8C), suggesting that depletion of TSG101 does not impair viral DNA replication. However, no viral DNA could be detected by BamHI W-specific PCR in the culture medium of TSG101-depleted cells, in contrast to what was found for the parallel controls, pSuper and siGFP, which were positive for secreted EBV DNA (Fig. (Fig.8D).8D). All these data suggested that TSG101 depletion may hinder the viral late protein synthesis, which results in a defect in virion formation.

DISCUSSION

As a means of propagation, EBV expresses a complex network of proteins following lytic induction to produce viral progeny. During this process, the Rta protein plays a key role in initiating the lytic transcriptional cascade (24, 31). The results presented in this report suggest that Rta cooperates with a cellular transcriptional regulator, TSG101, to mediate the complete activation of viral late genes (Fig. (Fig.2).2). The functional importance of TSG101 in Rta transactivation is recognized by the results from the reporter assay showing that the transactivational activities of Rta can be diminished upon depletion of TSG101 (Fig. (Fig.2B)2B) and can be augmented in response to the ectopic expression of TSG101 (Fig. (Fig.2C).2C). The mechanistic role of TSG101 is probably through direct interaction with Rta (Fig. (Fig.3),3), and both of their N-terminal domains are necessary for this protein-protein interaction (Fig. (Fig.4).4). Based on the observation that TSG101 is accumulated in the nucleus in the presence of Rta (Fig. (Fig.5)5) and that TSG101 can affect the efficiency of the interaction between Rta and viral promoters (Fig. (Fig.7),7), we speculate that these two effects may contribute, at least to some extent, to the mechanism whereby TSG101 is a perquisite for Rta-mediated viral late gene transactivation. To our knowledge, this is the first report demonstrating the role of TSG101 in EBV lytic cycle progression.

With regard to gene regulation, TSG101 has been defined as an essential protein engaged in nuclear hormone receptor-mediated transcription (4, 51, 64, 72). Mechanistically, TSG101 is characterized as a transcriptional coregulator, and this regulatory role is dependent on its subcellular relocation from the cytoplasm to the nucleus via protein-protein interaction with cellular factors (4, 51). In fact, TSG101 binds to different regulator partners and diverse functional activities are established. For instance, coexpression of TSG101 and AATF brings in synergistic coactivation of the ligand-dependent androgen receptor activity (4), whereas cotransfection of TSG101 and Daxx causes a suppression of glucocorticoid receptor-mediated transcription (51). So, TSG101, in acting positively or negatively on gene transcriptional regulation, relies on whichever transcriptional corepressor or coactivator it interacts with and what kind of gene it targets.

In this study, we sought to analyze the phenotype of TSG101-depleted NA cells in terms of the expression of EBV lytic genes. Upon TPA and SB induction, the levels of several EBV late transcripts in virus-carrying cells lacking TSG101 are markedly reduced, and the late genes affected include BcLF1, BDLF3, BILF2, BLLF1, and BLRF2 (Fig. (Fig.1).1). BcLF1 encodes the major viral capsid antigen, which is the major structural component of the nucleocapsid (31). BDLF3 expresses a heavily N- and O-glycosylated membrane-bound protein, gp150, and this glycoprotein can be found in the envelope of purified EBV particles (29, 32). BILF2 encodes gp78/55, an N-linked glycoprotein, and also is detectable in the envelope of EBV virions (29, 47). gp350/220 are the products of BLLF1, and these N- and O-linked glycoproteins are the most abundant components of the EBV envelope and important for host cell receptor binding and host cell invasion (52, 61). BLRF2 encodes a tegument protein, p23 (60). In terms of their biological characteristics, these five late genes have several similarities. They are all viral late genes, and their transcripts are expressed after viral DNA replication (44, 73). In addition, the expression of Rta is a prerequisite for activating these gene promoters, as proved by this and other studies (Fig. (Fig.2A)2A) (12, 15, 18, 58). Of note, Zta is found to be dispensable for their activation, and even a cooperative effect of Zta and Rta on the expression of these late genes was not observed (Fig. (Fig.2A)2A) (15, 33, 58).

Several mechanisms of transactivation by which Rta regulates viral lytic gene expression have been identified. First, Rta activates promoters by binding directly to a specific GC-rich DNA sequence, RRE (12, 22). Second, Rta binds indirectly to some Rta-responsive promoters via interaction with auxiliary cellular factors, because these promoters do not contain an RRE. For instance, Rta auto-stimulates its own promoter (Rp) despite the fact that Rp does not contain an RRE (57). It is through the contribution of Sp1 binding to two Sp1 sites of Rp that the activation of Rp by Rta is mediated (57). The complex bound on these Sp1 sites has been found recently to contain MBD1-containing chromatin-associated factor 1 (MCAF1), an Sp1 interacting partner which links both Sp1 and Rta to form an Sp1-MCAF1-Rta complex, thus increasing the auto-activating activity of Rta on Rp (5). Third, a synergistic activation requiring both Rta binding to RRE and Rta interacting with a cellular factor also is found in some EBV lytic genes, as illustrated by the case of BMLF1 (Mta, EB2, or SM protein). The promoter sequence of BMLF1 contains the consensus RRE that Rta binds directly; nonetheless, the transactivating ability of Rta on BMLF1 can be enhanced further when Rta interacts with CREB binding protein (12, 30, 66). Fourth, instead of binding to RRE and/or interacting with the auxiliary transcription factors, Rta is able to activate lytic gene expression indirectly by affecting the cellular signal transduction pathway. For example, Rta activates Zp by increasing the phosphorylation of p38 and c-Jun N-terminal kinase, resulting in activation of ATF2, which binds to the ZII element of Zp (1). Alternatively, Rta can also transactivate Zp as well as the BMRF1 promoter by inducing Akt phosphorylation through a phosphatidylinositol-3 kinase-dependent pathway (14). Fifth, Rta can activate some viral lytic gene promoters mediated through DNA-bound transcriptional factors. As exemplified by the case of BALF5, Rta does not directly bind to the BALF5 promoter but transactivates this gene through USF (upstream stimulatory factor) and E2F, which bind to the promoter (40).

The participation of TSG101 in Rta-mediated activation of EBV late genes that is revealed in this study (Fig. (Fig.2)2) has provided one more instance of cellular factors facilitating Rta transactivation. Obviously, Rta interacts with TSG101 and then retains TSG101 in the nuclear compartment (Fig. (Fig.33 and and6).6). It is likely that Rta acts either together with TSG101 or indirectly through TSG101 to bind the EBV late promoters. As demonstrated by the DAPA experiment, TSG101 can be recruited to the viral promoters in an Rta-independent manner (Fig. (Fig.7A).7A). However, the levels of Rta bound to DAPA probes of pBcLF1 (without RRE) and pBLRF2 (with RRE) are decreased coincident with the depletion of TSG101 (Fig. (Fig.7A).7A). Furthermore, the consecutive EMSA experiment delineates the fact that the ability of Rta to bind to an RRE is impaired when TSG101 is silenced (Fig. (Fig.7B).7B). Hence, TSG101 seems to affect Rta binding not only to the RRE but also to the promoter elements beyond the RRE (Fig. (Fig.7).7). We suggest, therefore, that TSG101 is crucial for Rta binding to the viral late promoters through direct binding to an RRE and/or the indirect manner, by acting together with the cellular transcriptional complex.

Although the underlying mechanism is not yet well defined, some speculation may provide a hypothesis as to how TSG101 affects Rta binding to the viral promoters. First, TSG101 alone may be bound directly to the viral promoter, thereby helping in loading Rta onto the promoter region. The DNA binding ability of TSG101 was discussed in detail when the gene was discovered. Researchers predicted three separate DNA binding motifs within the TSG101 coding region (35). However, such an ability for TSG101 is controversial at present because other researchers argue that these TSG101 motifs are necessary for binding ubiquitin rather than DNA, so further experimental evidence is needed to elucidate this discrepancy (53). Second, TSG101 may alternatively complex with other DNA binding proteins to help in loading the Rta protein onto the viral promoter. According to previous publications, TSG101 seems to favor interaction with DNA binding transcription factors, such as p300 and Daxx, for modulating gene transcription (51, 64). Accordingly, to establish an active transcriptional complex for viral gene transcription, TSG101 may likely form a complex with one of these or other DNA binding factors, beyond our current understanding.

EBV is the first DNA virus for which the completion of its late lytic cycle is reported to be aided by TSG101 (Fig. (Fig.8).8). Given the fact that depletion of TSG101 perturbs the transcription, as well as translation, of EBV late genes and subsequently disrupts the synthesis of virions (Fig. (Fig.11 and and8),8), EBV seems not to resemble RNA viruses in subverting TSG101 for virion production. RNA viruses usually take advantage of the involvement of TSG101 in the protein-sorting pathway to assist in the delivery and assembly of late viral proteins, without affecting their transcription or translation (3, 19, 49). EBV may otherwise preferentially exploit the transcriptional regulatory capability of TSG101 to produce its gene products. However, the data that we present so far cannot exclude the possibility of an as-yet-undiscovered mechanism through which EBV utilizes TSG101 as a component of the viral budding machinery. Given that EBV acquires its secondary envelope at post-Golgi-derived vesicles or plasma membrane containing BLLF1, it may be that the delivery of BLLF1 needs the protein-sorting machinery, including TSG101 (2, 20, 45, 70). Additional studies may need to define whether TSG101 has a role in sorting BLLF1 for viral assembly. Perhaps EBV uses TSG101 differentially for dual purposes during its lytic cycle, first for viral late gene transcription and subsequently for late protein assembly.

In conclusion, our finding that TSG101 mediates Rta transactivation has extended the function of TSG101 in transcriptional regulation to a role in progeny virus production and provides new insight into the diversity of TSG101 usage for the budding of RNA and DNA viruses. The recent advances in antiretroviral therapy indicate that TSG101 has been emerging as a striking drug target for inhibition of human immunodeficiency virus budding (59). Our present study provides an important indication that this antiviral approach may be widely applicable.

Acknowledgments

This work was supported by the National Science Council (grants NSC 95-3112-B-002-005 and NSC 94-3112-B-002-021) and the National Health Research Institute (grants NHRI-EX 95-9419BI and NHRI-EX 94-9419BI).

We are deeply indebted to Tim J. Harrison of the UCL Institute of Hepatology, University College London, for valuable discussion and for reviewing the manuscript critically.

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 December 2006.

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