P3HR1, a Burkitt’s lymphoma cell line latently infected by EBV (Ben-Bassat et al., 1976), was cultured in RPMI1640 medium containing 10% fetal calf serum. 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. P3HR1 cells were treated with 30 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM sodium butyrate to activate the EBV lytic cycle (Luka et al., 1979; Davies et al., 1991; Chang and Liu, 2000).

Plasmids pGEX-TRIM5α and pEGFP-TRIM5α were constructed by inserting a DNA fragment, which was amplified by PCR using pLPCX-TRIM5α as a template (NIH AIDS reagent program, USA) and the primers TRIM5α-F (5′-CCGGAATTCATGGCTTCTGGAATCCTGGT) and TRIM5α-R (5′-ACGCGTCGACTCAAGAGCTTGGTGAGCACA), into the EcoRI and SalI sites in pGEX-4T1 (Amersham) and pEGFP-C2 (Clontech), respectively. DNA fragments, which encode the regions in TRIM5α from amino acids 1 to 261, 81 to 493, and 261 to 493 were amplified by PCR. These DNA fragments were then inserted into the EcoRI and SalI sites in pEGFP-C2 to generate pEGFP-TRIM5α-dC, pEGFP-TRIM5α-dN, and pEGFP-TRIM5α-dNM, respectively. The DNA fragment from amino acids 1 to 261 was also inserted into pTag2B to create pFlag-TRIM5α-dC. Plasmids that express deletion mutants of GFP-Rta, including GFP-N190, GFP-N191/415, and GFP-Rev have been described previously (Hsu et al., 2005; Chang et al., 2010; Yang and Chang, 2013). Plasmids pFlag-Ub, pFlag-Rta, and pET-Rta, which express Flag-tagged ubiquitin, Flag-tagged Rta and His-tagged Rta, were described earlier (Yang et al., 2013). For transient transfection assays, the reporter plasmid, pBMRF1, was constructed by inserting a PCR-amplified DNA fragment containing the -172 to +20 region in BMRF1 into pGL2-Basic (Chang et al., 2010). Plasmid pBMLF1-RRE containing the RRE sequence from the BMLF1 promoter and a TATA sequence was synthesized and inserted into pGL2-Basic (Chang et al., 2004). Similarly, the RRE sequence from the BMRF1 promoter and a TATA sequence was synthesized and inserted into pGL2-Basic to generate pBMRF1-RRE.

P3HR1 cells were treated with TPA and sodium butyrate for 24 h to activate the EBV lytic cycle and Rta expression. Cells were harvested by low speed centrifugation and lysed using mRIPA buffer (50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40), and proteins in the lysate were immunoprecipitated using anti-Rta antibody and protein A/G-agarose beads (Millipore). The beads were washed with mRIPA buffer for three times, and proteins on the beads were then extracted with electrophoresis sample buffer (10% glycerol, 60 mM Tris-HCl pH 6.8, 2% SDS, 2.5% β-mercaptoethanol, 2 mM EDTA) and subjected to 2-D polyacrylamide gel electrophoresis. Proteins in the gel were stained with Coomassie blue, and prospective protein spots in the gel were then excised. The proteins in the spots were digested with trypsin according to an in-gel digestion protocol (Shevchenko et al., 1996), and the resulting peptides were analyzed using a Bruker Biflex III MALDI-TOF mass spectrometer (Bruker Daltonics) (Wu et al., 2007). The m/z ratios of the digested peptides and their fragmented ions were used to search the annotated human genome in the Mass Spectrometry protein sequence Database (MSDB), using Mascot search software v1.8 (Matrix Science Inc). The search criteria used were as follows: maximum of one missed trypsin cleavage; variable modification, including carbamidomethylation; and 1 Da peptide mass tolerance. Only proteins identified as significant hits (p < 0.05) by Mascot peptide mass fingerprint search were selected.

Escherichia coli BL21(DE3)(pGEX-TRIM5α) and E. coli BL21(DE3)(pGST) were cultured to the mid-log phase and then treated with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to, respectively, induce the expression of GST-TRIM5α and GST according to a method described earlier (Chang et al., 2004). GST and GST-TRIM5α were purified from bacterial lysates using glutathione-Sepharose 4B beads (GE healthcare).

293T cells were transfected with plasmids using Turbofect (Thermo Fisher Scientific), according to the method recommended by the manufacturer. At 24–48 h after transfection, cells were harvested and lysed using mRIPA lysis buffer [50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40]. Luciferase assays were performed according to a method described earlier (Chang et al., 1998).

293T cells were transfected with pCMV-Rta and pHA-TRIM5α, and at 24 h after transfection, cells were collected and lysed in mRIPA buffer. Proteins in the lysate were immunoprecipitated with anti-Rta and anti-HA antibodies. Protein A/G-agarose beads were then added to the lysate, and proteins bound to the beads were subsequently analyzed by immunoblotting. To detect ubiquitinated proteins, 293T cells were cotransfected with pCMV-R, pTag-2B, and pLPCX-TRIM5α. At 24 h after transfection, cells were treated with 5 μM MG132 for additional 12 h. Cells were harvested according to a method described earlier (Chang et al., 2004; Yang et al., 2013) to detect ubiquitin-conjugated proteins.

Proteins were separated in SDS-polyacrylamide gels and then electrotransferred to Hybond C membranes (GE) at 90 V for 1 h, according to a method described elsewhere (Chang et al., 2004). The membrane was then probed with the appropriate antibodies, including anti-Rta (Argene), anti-TRIM5α (Santa Cruz), anti-HA (Roche), anti-GFP (Santa Cruz), anti-GST (Santa Cruz), anti-VCA (Argene), anti-BFRF3 (Wang et al., 2015), and anti-α-tubulin (Sigma) antibodies.

P3HR1 cells were treated with sodium butyrate and TPA for 24 h, harvested by centrifugation, plated on poly-L-lysine (Sigma)-coated coverslips, and fixed with 4% paraformaldehyde in PBS for 30 min. Immunostaining was conducted using anti-Rta monoclonal antibodies (Argene) and anti-TRIM5α polyclonal antibodies (Santa Cruz). Cells were then treated with Alexa Fluor^® 594 goat anti-mouse and Alexa Fluor^® 488 goat anti-rabbit antibodies (Invitrogen). Nuclei were visualized by staining with 5 mg/mL 4′-6-diamidino-2-phenylindole (DAPI). Cells were observed under a confocal laser scanning microscope (Leica TCS SP8).

TRIM5α shRNA and plasmids, including pMD2.G, pCMVDR8.91, and pLKO-shRNA, were purchased from the National RNAi Core Facility, Genomic Research Center, Academia Sinica, Taipei, Taiwan. 293T cells (2 × 10⁵) were cotransfected with plasmids expressing TRIM5α shRNA (target sequence: 5′-CCAGACATTTGTGAATTTCAA-3′; 2.25 μg), helper plasmids pMD2.G (0.25 μg) and pCMVDR8.91 (2.25 μg), using Turbofect in vitro transfection reagent (Thermo Fisher Scientific). Culture media was changed on the following day, and after an additional 24 h, viral supernatants were collected and filtered (0.22 μM), then stored at -80°C. Plasmid pLKO-shRNA was used as a negative control. For lentivirus infection, P3HR1 cells (3 × 10⁵/mL) were transduced with the generated lentiviruses, together with 5 μg/mL of polybrene. Infected P3HR1 cells were selected using 2 μg/mL puromycin in culture medium to produce stable cell lines according to the protocol¹.

P3HR1 cells were treated with TPA and sodium butyrate to induce the lytic cycle. After 5 days of culturing, virus particles released into the medium were collected by ultracentrifugation at 25,000 × g for 2.5 h. EBV copy numbers were determined according to a method described previously (Ryan et al., 2004; Chiu et al., 2007). A standard curve was established using maxi-EBV DNA purified from E. coli after qPCR analysis. The amounts of EBV DNA purified from the virus in the culture medium were similarly analyzed and compared with the standard curve.

We treated P3HR1 cells with TPA and sodium butyrate for 24 h to activate the EBV lytic cycle and allowed the expression of Rta. Proteins interacting with Rta were then coimmunoprecipitated from cell lysates, using anti-Rta antibodies. A similar immunoprecipitation experiment was conducted using anti-IgG antibody as a control. Afterward, 2-D polyacrylamide gel electrophoresis of the immunoprecipitated proteins from each experiment (Figure 1A) was conducted, and the gels were then stained with Coomassie blue and compared. The comparison results revealed 14 protein spots that appeared in the gel containing anti-Rta immunoprecipitated proteins, but not in the control gel (Figure 1A). After digesting the proteins in these spots with trypsin, the resulting peptides were analyzed by MALDI-TOF mass spectrometry. The results revealed that five protein spots had peptide fingerprints matching those in the MSDB database (Figure 1B). Among these, Spot D21 had a protein with a fingerprint matching that of TRIM5α (Figure 1C), a known E3 ubiquitin ligase that promotes protein ubiquitination. Since Rta is known to be modified by ubiquitin (Yang et al., 2013), this study further sought to investigate whether TRIM5α influences the ubiquitination status and functions of Rta.

To verify the interaction between Rta and TRIM5α, we expressed GST-TRIM5α and GST in E. coli, and bound these proteins to glutathione-Sepharose beads. GST-TRIM5α-glutathione-Sepharose beads were then added to E. coli BL21(DE3)(pET-Rta) lysates, and proteins pulled down by the beads were detected by immunoblotting with anti-Rta antibody. The results revealed that GST-TRIM5α, but not GST-glutathione-Sepharose beads, pulled down Rta (Figure 2A, lanes 4 and 5), providing in vitro evidence of a direct interaction between TRIM5α and Rta. A coimmunoprecipitation experiment using lysates from 293T cells that had been cotransfected with pCMV-Rta and pLPCX-HA-TRIM5α similarly showed that anti-TRIM5α antibody immunoprecipitated HA-TRIM5α and coimmunoprecipitated Rta (Figure 2B, lanes 4 and 8), while anti-Rta antibody immunoprecipitated Rta and coimmunoprecipitated HA-TRIM5α (Figure 2B, lanes 3 and 7).

The localization of Rta and TRIM5α in P3HR1 cells treated with TPA and sodium butyrate for 24 h was examined by indirect immunofluorescence. Under a confocal laser scanning microscope, we found that both Rta and TRIM5α formed speckles, many of which colocalized in the cell nucleus (Figures 3f–j). However, Rta was not observed if the cells were not treated with TPA and sodium butyrate (Figures 3a–e). P3HR1 cells treated with or without TPA and sodium butyrate were also stained with secondary antibodies as a control to demonstrate the specificity of anti-TRIM5α antibody (Figures 3k–t).

We cotransfected 293T cells with pFlag-TRIM5α and plasmids expressing different segments of Rta that were fused to GFP (Figure 4A). Control cells were cotransfected with plasmids expressing GFP and Flag-TRIM5α. Immunoblotting with anti-GFP antibody revealed that GFP and the GFP-Rta fusion proteins were expressed at similar levels after transfection (Figure 4B, lanes 1–5). Following immunoprecipitation with anti-Flag antibody, precipitated proteins were analyzed by immunoblotting with anti-GFP antibody. Results showed that GFP-N190 (Figure 4B, lane 8), which contains 190 amino acids from the N-terminal of Rta, was coimmunoprecipitated with Flag-TRIM5α, indicating that this is the region in Rta that interacts with TRIM5α. In addition, we also observed a weak binding effect between TRIM5α and GFP-N191-415 (Figure 4B, lane 9). However, GFP-Rev was not coimmunoprecipitated by Flag-TRIM5α (Figure 4B, lane 10). A control experiment showed that Flag-TRIM5α was not coimmunoprecipitated with GFP (Figure 4B, lane 6). These results were reproduced from at least three independent experiments. To identify the region in TRIM5α that interacts with Rta, we generated plasmids expressing GFP fused to different segments of TRIM5α (Figure 4C). Following cotransfection of these plasmids with pFlag-Rta to 293T cells, cells were subsequently lysed, and the lysates subjected to immunoprecipitation using anti-Flag antibody (Figure 4D, lanes 1–5). Immunoblot analysis with anti-GFP antibody revealed that only GFP-TRIM5α and GFP-TRIM5α-dC were coimmunoprecipitated with Flag-Rta (Figure 4D, lanes 7 and 10). In addition, 293T cells were cotransfected with plasmids encoding GFP-N190 and Flag-TRIM5α-dC, and immunoblot analysis revealed that GFP-N190 in cell lysates (Figure 4E, lane 3) was immunoprecipitated by anti-Flag antibody (Figure 4E, lane 3). However, control cells that were cotransfected with plasmids expressing GFP and Flag-TRIM5α-dC revealed that GFP was not coimmunoprecipitated with Flag-TRIM5α-dC (Figure 4E, lane 2). These results indicate that the N-terminal of Rta interacts with the N-terminal RING domain in TRIM5α.

TRIM5α has previously been shown to be an E3 ubiquitin ligase (Yamauchi et al., 2008), and the observation of Rta-TRIM5α interaction prompted us to investigate whether TRIM5α can influence Rta ubiquitination. We proceeded to transfect 293T cells with pCMV-Rta alone, or with pCMV-Rta and pFlag-Ub. At 24 h after transfection, cells were treated with MG132, a proteasome inhibitor, to prevent the degradation of Rta. When proteins from the lysates of cells transfected with pCMV-Rta alone were immunoprecipitated using anti-Flag antibody and assessed by immunoblotting with anti-Rta, a single non-specific band of about 95 kDa was detected (Figure 5A, lane 1), which was also observed in previous studies (Chang et al., 2004; Yang et al., 2013). In 293T cells cotransfected with pCMV-Rta and pFlag-Ub, low amounts of ubiquitinated Rta were detected after the proteins in the lysates were immunoprecipitated with anti-Flag antibody and assessed by immunoblotting with anti-Rta antibody (Figure 5A, lane 2). When cells were cotransfected with pCMV-Rta, pFlag-Ub, and 0.3 or 0.6 μg of pLCPX-TRIM5α, ubiquitinated Rta became more prominent (Figure 5A, lanes 3 and 4), demonstrating that TRIM5α promotes Rta ubiquitination. Subsequently, 293T cells were transfected with TRIM5α shRNA to determine whether this would reduce Rta ubiquitination. In a control experiment, immunoblotting did not detect ubiquitinated Rta in cells that were transfected with pHA-Ub (Figure 5B, lane 1). Ubiquitinated Rta was also detected in cells that were cotransfected with pFlag-Rta and pHA-Ub (Figure 5B, lane 2). However, introducing TRIM5α shRNA reduced the amounts of ubiquitinated Rta (Figure 5B, lanes 3 and 4).

It is known that Rta acts as a key immediate-early protein that transactivates viral lytic genes to move EBV into the lytic cycle. Therefore, we sought to evaluate the impact of enhanced TRIM5α expression and Rta ubiquitination on the transactivation capabilities of Rta. In a transient transfection assay, we examined how TRIM5α expression can influence Rta transactivation of the EBV BMRF1 promoter, using a luciferase reporter plasmid, pBMRF1 (Chang et al., 2004). After cotransfecting 293T cells with pCMV-Rta and pBMRF1, Rta transactivation of BMRF1 was measured by luciferase activity, and the values were taken as 100% (Figure 6A). We further included 0.1–0.4 μg of pLPCX-TRIM5α in cotransfections, and found that enhanced expression of TRIM5α gradually reduced BMRF1 promoter activation in a dose-dependent manner to just 37–80% (Figure 6A). A similar experiment also showed that cotransfection of pLPCX-TRIM5α similarly disrupted the ability of Rta to transactivate the BMRF1-RRE (Figure 6B) and BMLF1-RRE (Figure 6C) promoters, which contain Rta-responsive elements (RREs). These results show that overexpression of TRIM5α decreases Rta transactivation capability.

P3HR1 cells were infected with lentivirus expressing TRIM5α shRNA or control shRNA, and cells were then treated with TPA and sodium butyrate to activate the EBV lytic cycle. We found that, compared with cells infected with control shRNA, infection by lentivirus expressing TRIM5α shRNA caused cells to express less TRIM5α, but more Rta and EA-D (Figure 7A). The expression of TRIM5α shRNA also led to increases in the expression of two EBV capsid proteins, VCA and BFRF3 (Figure 7B). Quantitative PCR results showed that after lytic activation of P3HR1 cells infected with lentivirus expressing control shRNA, viral yield was estimated at 3 × 10⁵ EBV particles. However, for cells infected with lentivirus expressing TRIM5α shRNA, viral yields increased 400% to 1.2 × 10⁶ viral particles (Figure 7C). These results showed that TRIM5α expression serves to attenuate EBV lytic development.