ABSTRACT
The binding of Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) to the latent replication origin (oriP) triggers multiple downstream events to support virus-induced pathogenesis and tumorigenesis. Although EBV is widely recognized as a B-lymphotropic infectious agent, little is known about how tissue-specific factors are involved in the establishment of latency. Here, we showed that EBNA1 binds B cell activator PAX5 to promote EBNA1/oriP-dependent binding and transcription. In addition to showing that short hairpin RNA (shRNA)-mediated PAX5 knockdown substantially abrogated the above EBNA1-dependent functions, two mini-EBV reporter plasmids were used to perform nonlytic nano-luciferase (nLuc) activity and chromatin immunoprecipitation (ChIP) assays to show how EBNA1 cooperates with PAX5 to activate the transcription at the oriP site. The expression plasmids of two PAX5 mutants, V26G (EBNA1 binding mutant) and P80R (which remained EBNA1 associated), were used to assess their capability to restore the defects caused by PAX5 depletion in EBNA1/oriP-mediated binding, transcription, and maintenance of the genome copy number of the mini-EBV episome reporter in BJAB cells stably expressing EBNA1 or that of the EBV genome in EBV-infected BJAB cells. Since p300 is known to be associated with PAX5, we showed that the loss of function of the P80R mutant in support of EBNA1/oriP-mediated transcription under PAX5 depletion conditions was linked to its defective binding to p300. ChIP-quantitative PCR (qPCR) confirmed that P80R indeed failed to recruit p300 to the oriP DNA. Our discovery suggests that EBV has evolved an exquisite strategy to take advantage of tissue-specific factors to enable the establishment of viral latency.
IMPORTANCE Although B cells are known to be the primary target for EBV infection, there is limited knowledge regarding the mechanism that determines this preferable tissue tropism. An in-depth understanding of the potential link of tissue-specific factors with the viral genes and their functioning is key to deciphering how EBV induces persistent infection in the distinct types of host cells. In this study, a substantial protein-protein interaction mediated by the B cell-specific activator PAX5 and EBNA1 was identified as the general requirement for the binding of EBNA1 to the latent replication origin and for downstream events. Of importance, the EBNA1-PAX5-p300 network is directly linked to EBNA1-dependent transcription. These findings suggest that targeting the viral gene-associated tissue-specific factors may lead to new therapeutic strategies for EBV-associated malignancies.
INTRODUCTION
Although Epstein-Barr Virus (EBV) has been implicated in several types of lymphotropic and epitheliotropic malignancies, studies reveal that B lymphocytes are the primary targets for EBV to establish a lifelong infection in the host (1, 2). An EBV-driven B cell transformation platform has been widely employed to study EBV-induced pathogenesis and tumorigenesis in vitro and in humanized mouse models (3–5). Deciphering the mechanistic insights into virus-host interactions mediated by the viral genes and B cell-specific factors is the key to understanding how EBV determines tissue tropism. EBV infection converts B cells into indefinitely growing lymphoblastoid cell lines (LCLs), the maintenance of which relies on the expression of a subset of latency-associated genes and noncoding RNAs (2). EBV nuclear antigen 2 (EBNA2) and leader protein (LP) drive the transcription of both cellular and viral promoters during the early phase of infection (6), whereas EBNA1 and EBNA3A to EBNA3C participate in the different modes of transcription regulation to modulate target gene expression (7, 8). Furthermore, latency-associated membrane proteins (LMP) trigger antiapoptotic responses to sustain long-term viral infection (9). The causal associations of EBV with human malignancies are partially attributed to its prevalence in more than 90% of the adult population worldwide (10). Eradication of EBV-associated cancers has become one of the major challenges in anticancer research. EBNA1 is noted as the only viral gene expressed in almost all EBV-positive neoplasms; hence, gaining in-depth knowledge of how EBNA1 exploits cellular factors is essential to understanding the general role of EBV in tumorigenesis.
The DNA damage-dependent antiviral defense response, which is tightly coordinated with the appearance of linear virion DNA and the formation of a functional extrachromosomal replicon, is a key determinant of the establishment of EBV latency in host cells (11). Circularization of the viral genome provides a template to allow the transcription of LMP2A and LMP2B from the terminal repeats (TR), and subsequently, both viral products prevent the reactivation of the lytic cycle (12). EBNA1 participates in all episome-dependent events through its binding to the cis elements residing within the latent replication origin (oriP), which consist of two components, a dyad symmetry (DS) element and a family of repeats (FR) (2). Although the EBNA1 DNA binding domain (DBD) has been structurally characterized (13, 14), mechanistic insights into EBNA1/oriP-mediated episome-dependent events have been derived mainly from genetic studies using recombinant viruses and biochemical studies examining the interactions of EBNA1 with cellular factors (15, 16). EBNA1 recruits both the origin of recognition complex (ORC) and minichromosome maintenance (MCM) proteins to tether the viral genome to the chromosome (11). Consequently, a persistent and stable copy number of the EBV epigenome is maintained after host cell division.
B lymphocytes are the most permissive for the establishment of latent EBV infection, which is expected to exploit tissue-specific factors to build up its pathogenesis. EBV infection is initiated through binding with its receptor, CD21, which induces the concomitant expression of both CD23 and the B cell early activation antigen (Bac-1) to facilitate its latent infection (4). Two B cell-lineage-specific factors, PAX5 and early B cell factor 1 (EBF1), have been noted for their involvement in B cell commitment, development, and leukemogenesis (17, 18). EBF1 binds to and stabilizes EBNA2 chromatin binding to facilitate the transcriptional regulation of target genes (19, 20). PAX5 has been implicated in the modulation of LMP2A and LMP2B transcription from the TRs, activation of the W promoter (Wp), and the prevention of BZLF1 transcription (21–23). The discovery of PAX5 binding sequences within the TRs suggests that PAX5 probably participates in TR-dependent events, which warrants in-depth study (21, 22). Thus far, none of the EBV latent genes have been reported to be associated with PAX5. Here, we show the occurrence of protein-protein interactions (PPI) mediated by EBNA1 and PAX5 both in vitro and in vivo. We also mechanistically characterized the critical role of PAX5 for EBNA1/oriP-mediated binding, transcription, and maintenance of the genome copy number of the oriP plasmid and EBV epigenome in specified cellular contexts.
RESULTS
Identification of the physical interaction between EBNA1 and PAX5 in vitro and in vivo.Although binding of PAX5 to the Wp or TRs of viral DNA has been implicated in EBV-mediated B cell transformation (24, 25), none of the viral proteins have been reported to be associated with PAX5. EBNA1 and EBNA2 are key players, with the capability to regulate viral and cellular gene expression through their association with the cognate elements or transcription factors. We first attempted to explore the potential physical interaction between plasmid-expressed EBNA1 or EBNA2 and Flag epitope-tagged PAX5 (FPAX5) by performing a transfection-mediated coimmunoprecipitation (co-IP) assay under DNase/RNase treatment conditions. M2-conjugated agarose was found to coprecipitate EBNA1 but not EBNA2, suggesting the existence of a nucleic acid-independent interaction between EBNA1 and FPAX5 (Fig. 1A). In contrast, EBNA1 was not coprecipitated by FPAX6 (Fig. 1B). In LCLs, the endogenously expressed PAX5 was found to coimmunoprecipitate with EBNA1 and vice versa, whereas the known EBNA1-interacting protein nucleolin (NCL) was not associated with PAX5 (26) (Fig. 1C). Similarly, the bacterially expressed histidine-tagged PAX5 (HPAX5) recombinant protein pulled down approximately 5% of the endogenous EBNA1 from the LCL lysate, whereas neither EBNA2 nor NCL was associated with HPAX5 (Fig. 1D). The nuclear colocalization of PAX5 and EBNA1 in the LCLs or PAX5 and Flag-tagged EBNA1 (FEBNA1) in the BJAB cells stably expressing FEBNA1 was further demonstrated by immunofluorescence (IF) confocal microscopy (Fig. 1E). Our data indicate that EBNA1 and PAX5 mediate a genuine PPI in the B-lineage cells latently infected with EBV.
PPI mediated by EBNA1 and PAX5 in vitro and in vivo. (A) The expression plasmids of FPAX5, EBNA1, or EBNA2 were used for transfection-mediated co-IP assays. To avoid nucleic acid-dependent PPI occurring, all of the cells were collected in lysis buffer supplemented with DNase I and RNase A prior to conducting the immunoprecipitation assay for this and all of the following co-IP experiments. M2 agarose-precipitated matrices were blotted for FPAX5, EBNA1, or EBNA2 with the use of 2% input of the lysate as a loading control. (B) The expression plasmids of EBNA1, FPAX5, or FPAX6 were included in the co-IP assay. The immune blots for the indicated proteins are shown. (C) LCL lysate was used to perform co-IP assays using antibodies for EBNA1 (E1), PAX5 (P5), nucleolin (NCL), or IgG control. Western blotting was performed with 2% of the input as loading control. (D) Ni- nitrilotriacetic acid (NTA) Sepharose-bound His-tagged PAX5 (HPAX5) was used as the protein bait to pull down cell lysates derived from LCL or BJAB cells. The HPAX5-bound proteins were identified by immune blot analysis. Five percent input of each protein from the lysates is shown. (E) LCL, BJAB stably expressing Flag-tagged EBNA1 (FEBNA1) (BJAB-FEBNA1), or BJAB cells were used to perform immunofluorescence confocal microscopy. PAX5-stained cells were visualized by a rhodamine-conjugated secondary antibody, whereas the primary EBNA1 antibody was fluorescein isothiocyanate (FITC) conjugated. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).
PAX5 depletion impairs both EBNA1/oriP-dependent transcription and EBV-dependent cell growth of EBV-positive neoplastic B cells.The existence of PPI between EBNA1 and PAX5 inspired us to further assess the potential involvement of PAX5 in EBNA1/oriP-dependent events. Previous studies have shown that transfection-mediated EBNA1-dependent transcription of oriP-Luc reporter plasmids produced an approximately 10-fold activation in B cell lymphoma BJAB cells (27), and ectopic FPAX5 further augmented oriP-Luc activity by approximately 2.6-fold but did not affect EBNA2/LMP1-Luc-mediated transcription (28) (Fig. 2A). EBNA1-induced activation of oriP-Luc was almost entirely abrogated by lentivirus-expressed PAX5-3′ UTR-specific short hairpin RNA 1 (shRNA1) (shPAX5#1) or by shPAX5#2, whereas scramble shRNA control (shScr) exhibited no effect (Fig. 2B). Each PAX5 shRNA depleted more than 90% of the endogenously expressed target protein. Plasmid-expressed FPAX5 fully restored the defects of EBNA1/oriP-Luc-mediated transcription in two independent PAX5-depleted BJAB cell lines. The effects of PAX5 depletion on cell proliferation were next assessed in four EBV-positive and two EBV-negative cell lines (Fig. 2C). PAX5 knockdown resulted in a 15 to 20% reduction, or an over 90% blocking effect, on cell growth in EBV-negative B cell lymphoma BJAB/or Burkitt’s lymphoma (BL) Akata cells compared to that in other EBV-positive cell lines, including three LCLs and one BL EBV-positive Akata (Akata+) cell line (Fig. 2D). The above data suggest that the critical requirement of PAX5 in EBNA1/oriP-mediated transcription is crucially linked to EBV-dependent cell proliferation.
PAX5 has a critical role in EBNA1/oriP-dependent functions. (A) FPAX5 expression plasmids (10 or 30 μg) were assessed for their effects on either EBNA1/oriP-Luc- or EBNA2/LMP1-Luc-mediated transcription. A cytomegalovirus (CMV)–β-galactosidase (β-Gal) expression vector was used as an internal control. The degree of EBNA1- or EBNA2-dependent transcription was determined as fold activation, where the resulting luciferase activity is corrected by β-Gal activity. The final results were presented as mean ± standard deviation (SD) for data from three experiments for this and all of the following luciferase activity assays. The statistical analysis for the indicated comparison groups to the control was determined by paired Student’s t test, with P < 0.05 (*) versus P > 0.05 (†). (B) BJAB cells were transduced with lentivirus-expressed shPAX5#1 or shPAX5#2 or with shScr control. The immune blots for endogenous PAX5 and actin control are shown. Cells were cotransfected with EBNA1 or FPAX5 and oriP-Luc, as well as with internal control CMV–β-Gal reporter plasmids. Plasmid-expressed EBNA1, FPAX5, and actin control was determined by Western blotting. The EBNA1/oriP-Luc-induced transcription in each sample is presented and compared. (C) EBV-positive Akata BL cells (Akata+), LCL1, LCL2, or IB4 cells (5 × 106) transduced for 72 h with green fluorescent protein (GFP) expression vector, shPAX5#1, shPAX5#2, or shScr control were collected to perform Western blot assays and for the following indicated experiments. (D) Each selected cell line (5 × 103 cells per 100 μl) was aliquoted into a 96-well microtiter plate in triplicate and subjected to a proliferation assay using the trypan blue exclusion method. The effects of PAX5 depletion on the cell growth of two EBV-negative versus four EBV-positive cells lines were compared. The number of viable cells at the indicated time point was determined by cell counting with a hemocytometer. The results from three independent experiments are represented as mean ± SD for each time point.
EBNA1 requires PAX5 to associate with either oriP DNA or TR-DNA.The involvement of PAX5 in EBNA1/oriP-dependent transcription could likely be attributed to its effects on EBNA1 binding to the cognate elements residing within the oriP. A chromatin immunoprecipitation (ChIP) assay was performed on this line to quantify the enrichment of either EBNA1 or PAX5 at their target sites, oriP versus TRs, residing in the EBV genomic DNA derived from Akata+, LCL1-2, and IB4 cells, with or without PAX5 depletion (Fig. 3A and B). In addition to verifying the enrichment of both EBNA1 and PAX5 at their cognate EBV DNA fragments, ChIP-quantitative PCR (qPCR) also identified EBNA1 enrichment at the TR-DNA or PAX5 enrichment at the oriP DNA (Fig. 3C to E). It should be noted that protein enrichment at the oriP was quantified by ChIP-qPCR using DS (26) and FR (29) scanning primers. Two primer sets amplified similar amounts of targeted DNA from each chromatin-immunoprecipitated sample, suggesting that PAX5 knockdown had the same general impact on protein enrichment at either the DS or FR repeats within the oriP, including that of EBNA1, PAX5, and NCL. Instead of DS or FR, oriP DNA is used as the common term for all of the following ChIP assays. The previously identified EBNA1-associated protein, NCL, was also detected at both the oriP and TR-DNA. In PAX5-depleted Akata+ or LCL1-2 cells, a 70 to 95% reduction in EBNA1 enrichment or a 50 to 95% reduction in NCL enrichment at the oriP or TR DNA, respectively, was observed. Neither EBNA1 nor NCL was shown to associate with the PAX5 target site residing within the CD79a promoter (22). In addition, none of the proteins were observed at the EBV BamHI C promoter (Cp), and the enrichment of H3Ac on the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was not altered by PAX5 depletion (Fig. 3G and H). Surprisingly, PAX5 depletion did not affect the enrichment of EBNA1 or NCL at the oriP DNA of an integrated EBV genome in IB4 cells (30) (Fig. 3D and E). These findings indicate that PAX5 is a general requirement for EBNA1 to localize to either the oriP or TR-DNA of the EBV epigenome.
EBNA1 requires PAX5 to associate with either the oriP or TR-DNA. (A) Schematic diagram of the EBV linear genome. The EBNA1 binding sites within the oriP and the PAX5 binding sites in the TRs are shown. FR, family of repeats; DS, dyad symmetry repeats. The areas targeted by ChIP-quantitative PCR (qPCR) are marked by a pair of arrowheads. (B) Schematic diagrams illustrating two types of the EBV genome derived from Akata +, LCL, and IB4 are shown. (C to H) A series of ChIP assays were performed on the four cell lines with the same treatments as those described in the Fig. 2C legend, using antibodies for PAX5, EBNA1, NCL, or IgG control. The amounts of coimmunoprecipitated TR-DNA, DS-DNA, FR-DNA, CD79a promoter (22), C promoter (Cp), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter from three independent experiments were quantified by qPCR and determined as percentage of input DNA (mean ± SD) for this and all of the following described ChIP assays.
EBNA1 DNA binding domain and PAX5-V26 are important for PPI.Since the PPI mediated by EBNA1 and PAX5 had been demonstrated, we next sought to identify the specific domains that directly participate in protein binding. The biomolecular fluorescence complement (BiFC) assay was used to screen the PAX5 binding-domain of EBNA1 following confirmation of proper protein expression by each transfected BiFC plasmid (Fig. 4A, left). Cotransfection of yellow fluorescent protein (YFP)-N terminus-fused Flag-tagged PAX5 (YN-FPAX5) with YFP-C terminus-fused Flag-tagged EBNA1 (YC-FE1) provided prominent BiFC images (Fig. 4A, middle and right). Moreover, the effects were also observed by the coexpression of YC-FE1 Δ100-377, Δ1-377, or DBD and YN-FPAX5, whereas YC-FE1 ΔDBD or YC-Flag-tagged NCL (YC-FNCL) exhibited no effect. A co-IP assay further confirmed that endogenous PAX5 was not associated with FEBNA1-ΔDBD (Fig. 4B). FEBNA1 mutants with a partial N-terminal or C-terminal deletion of DBD, FE1Δ459-530, and FE1Δ531-607 remained bound to the endogenous PAX5.
The PPI induced by EBNA1 and PAX5 is required for EBNA1/oriP-mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into 293T cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V26G and P80R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.
The co-IP assay showed that among the eight PAX5 mutants, the truncated deletion mutants of the pair domain (PD) and the homeodomain (HD) failed to bind EBNA1, whereas none of the remaining FPAX5 deletion mutations affected EBNA1 binding compared to that with FPAX5 (Fig. 4C). The two previously identified DNA-binding null mutants of PAX5 (23), V26G and P80R, exhibited distinct binding phenotypes to EBNA1. The former completely lost its association with EBNA1, while the latter remained effectively bound to EBNA1 (Fig. 4D).
p300 and H3K4Me3 have previously been implicated in PAX5-mediated transcription (22, 31), and the co-IP assay showed that FPAX5 or V26G efficiently bound with the above two endogenous proteins, while P80R did not (Fig. 4D). In an EBNA1-dependent transcription platform, the transfected FPAX5 enhanced oriP-Luc transcription from 10-fold to 30-fold, thus demonstrating a 3-fold upregulation. In addition, P80R caused an 80% reduction in the EBNA1-dependent transcription of oriP-Luc, whereas V26G showed no effect (Fig. 4E). Interestingly, we observed that P80R caused downregulation in the EBNA1-induced luciferase activity of oriP-Luc, suggesting that this mutant may block the endogenous function of PAX5. Our data reveal that EBNA1-DBD and PAX5-V26 are the two important components participating in the PPI.
PAX5 recruits cellular transcription enhancers to both oriP DNA and TR-DNA.The PAX5 binding sites have been previously identified in the TRs of EBV DNA (21, 22); thus, the further observation of PAX5 occupancy at the oriP DNA likely relies on its binding to EBNA1. To assess this possibility, a transfection-mediated ChIP assay was performed using BJAB cells transfected with an oriP DNA plasmid and the EBNA1 expression vector. EBNA1 enrichment at the oriP DNA was determined by ChIP-qPCR using DS scanning primers to be ∼0.4% of the input DNA, while the abundance of PAX5 was ∼0.2% when EBNA1 was presented (Fig. 5A). Here, we only used the DS primer set to quantify the protein enrichment at the oriP DNA in ChIP assays. In the absence of EBNA1, no significant enrichment of PAX5 was observed at the oriP DNA. The extensive ChIP assay further identified the coenrichment of the transfected FPAX5 or P80R but not the EBNA1-binding mutant V26G at the oriP DNA in the presence of EBNA1, indicating that FPAX5 or P80R is recruited to the oriP site through their PPI with EBNA1 (Fig. 5B). The TR plasmid DNA was used next to perform a reciprocal ChIP assay to show that EBNA1 enrichment at the TR-DNA is a PAX5-dependent process. We found that in the absence of EBNA1, endogenous PAX5 was sufficient to localize to the TRs in BJAB-shScr cells, while PAX5 depletion abrogated the enrichment of both EBNA1 and PAX5 (Fig. 5C). Since the PAX5 binding sequences within the TRs are different from its classical cognate elements (22, 23), transfected V26G and P80R were used next to assess their relative binding activity to the TRs compared to that of FPAX5. ChIP-qPCR identified FPAX5 enrichment at the TR-DNA as ∼0.3% of the input, while V26G caused a 45% reduction, and P80R failed to localize to the TRs (Fig. 5D). Apart from showing that P80R was not bound to p300 or H3K4Me3 (Fig. 4 D), this study further identified P80R as a TR binding mutant (Fig. 5E). Our data reflect that the PPI mediated by EBNA1 and PAX5 is essential for EBNA1 to create dual links with the oriP and TR-DNA.
The p300 recruitment to the oriP DNA is PAX5 dependent. (A) BJAB cells cotransfected with EBNA1 or control vector (−), and the oriP plasmid were subjected to a ChIP analysis using antibodies for EBNA1 (E1), PAX5, or IgG control. The abundance of coimmunoprecipitated oriP DNA by each antibody is shown. The statistical analysis for the indicated comparison groups was determined by paired Student’s t test for this and all of the following ChIP assays; P < 0.05 (*) versus P > 0.05 (†). (B) BJAB cells cotransfected with FPAX5 or each of its point mutants along with the oriP plasmid were coimmunoprecipitated for M2 or IgG control. The binding activity of EBNA1 or the association of FPAX5 or its point mutants with the oriP was determined by qPCR as their enrichment at the oriP DNA. The immune blots for plasmid-expressed proteins or the internal GAPDH control are shown. (C) BJAB-shPAX5#1 or BJAB-shScr cells were cotransfected with EBNA1 or control vector and TR plasmid. The abundance of TR-DNA coimmunoprecipitated by each antibody is shown. (D) BJAB cells cotransfected with FPAX5 or its point mutants were coimmunoprecipitated for M2 or IgG control. The binding activity of FPAX5 or its point mutants to the TRs was determined by qPCR as their enrichment at the TR-DNA. (E) The phenotypes of FPAX5 and its mutants in binding to EBNA1, p300, H3K4Me3, and TRs are summarized. (F) Aliquots (5 × 106 cells) of an LCL were used to perform immunoprecipitation assays for EBNA1, PAX5, and IgG control, respectively. The cell lysates were pretreated with DNase/RNase prior to performing the experiment. The precipitated proteins were identified by immunoblotting analysis using antibodies for EBNA1 (E1), PAX5, p300, H3K4Me2, and H3K4Me3, respectively. (G) Aliquots (5 × 106 cells) of an LCL were used to perform a ChIP assay using the indicated antibodies. The amounts of coimmunoprecipitated oriP DNA or TR-DNA were determined as the percentage of input DNA ± SD. The enrichment of each protein at the oriP DNA or TR-DNA in PAX5-deplected cells versus the control was compared by statistical analysis.
In Fig. 4D, we demonstrate the potency of PAX5 to additionally create a protein link with the transcription enhancers p300 and H3K4Me3, which could likely guide them to localize to the oriP, thus promoting EBNA1-mediated transcription. The interactions between endogenous PAX5 and p300/or H3K4Me3 were first verified by a co-IP assay in the LCLs treated with DNase/RNase (Fig. 5F). In contrast, EBNA1 exhibited null binding activity for both p300 and H3K4Me3. In addition to confirming the enrichment of both EBNA1 and PAX5 at the oriP/or TR-DNA of the LCL EBV genome, ChIP-qPCR further identified the enrichment of p300 and H3K4Me2 at the oriP DNA, as well as at the TR-DNA (Fig. 5G). Apart from impairing EBNA1 recruitment to both the oriP and TR-DNA, we noted that PAX5 depletion caused a robust dissociation of p300 from the oriP DNA and of both p300 and H3K4Me3 from the TR DNA. These findings highlight the critical role of PAX5 in promoting p300 recruitment to the oriP DNA and the recruitment of both p300 and H3K4Me3 to the TR-DNA.
PAX5-mediated recruitment of p300 is essential for EBNA1/oriP-mediated transcription.Two reporter plasmids encompassing an upstream oriP, zero or four copies of the TR, and a downstream SV40 promoter-driven nano-luciferase (nLuc) expression cassette were designated oriP-nLuc or oriP-TRs-nLuc, respectively, and were used to confirm that the production of secreted nLuc from each reporter plasmid was indeed EBNA1 dependent (Fig. 6A). Transfected EBNA1 or cotransfected EBNA1/FPAX5 induced a 6.5-fold versus ∼13.3-fold activation of oriP-nLuc, and an 11.8-fold versus 23.2-fold activation of oriP-TRs-nLuc. These findings indicate that the TR-DNA is not essential but that its presence can further augment EBNA1/oriP-mediated transcription by approximately 2-fold. Since the oriP-TRs-nLuc reporter plasmid encompasses both oriP and TR sites, it mimics a mini-EBV epigenome that allows us to monitor how EBNA1 and PAX5 cooperate to perform episome-dependent events. The transfected expression plasmid of EBNA1, FPAX5, or FPAX5 mutants were expressed at similar levels in both PAX5-depleted BJAB cells and controls (Fig. 6B). In agreement with the findings described in Fig. 2B, PAX5 depletion abolished EBNA1-induced transcription from the oriP-nLuc and oriP-TRs-nLuc reporter plasmids. These were found to be fully restored by transfected FPAX5, whereas neither V26G nor P80R had an effect.
PAX5 recruits p300 to support EBNA1/oriP-mediated transcription. (A) The schematic diagrams of two mini-EBV episome reporter plasmids, oriP-nLuc and oriP-TRs-nLuc. Each reporter plasmid was cotransfected with the expression plasmids of EBNA1 and FPAX5 or control into BJAB cells. EBNA1-dependent transcription was verified by the detected nonlytic luciferase activity produced by the reporter plasmid. (B) BJAB cells depleted for PAX5 or shScr control were cotransfected with the expression vectors of EBNA1, FPAX5, or FPAX5 point mutants with the indicated reporter plasmid. The resulting nonlytic nLuc activity was assayed at 24 h posttransfection. (C) BJAB-shPAX5#1 transfected with EBNA1, FPAX5 or its point mutants, and the oriP-nLuc plasmid were subjected to ChIP assays using the indicated antibody. The amounts of coimmunoprecipitated oriP DNA or TR-DNA were quantified by qPCR. The abundance of coimmunoprecipitated-DNA was determined as the percentage of input ± SD. (D) The same experimental procedure described above was carried out, except the oriP-nLuc was replaced by oriP-TRs-nLuc. (E) BJAB cells depleted for PAX5 or shScr control were used to perform a transfection-mediated ChIP assay. The enrichment of EBNA1, endogenous PAX5, transfected FPAX5 and/or its mutants, p300, H3K4Me2, and H3K4Me3 at the oriP DNA or TR-DNA of the oriP-TRs-nLuc reporter plasmid was quantified by qPCR following the same protocols described elsewhere.
Two mini-EBV reporter plasmids, described in Fig. 6A, were next used to explore how EBNA1 and FPAX5 or the mutants coordinate to make a direct/or indirect physical link to the oriP or TR-DNA by performing a transfection-mediated ChIP assay in PAX5-depleted BJAB cells. Following PAX5 depletion, EBNA1 alone could not localize to the oriP or TR-DNA before being cotransfected with FPAX5 or its mutants (Fig. 6C and D). ChIP-qPCR identified the abundance of EBNA1 with cotransfected FPAX5 or P80R at the oriP DNA as 0.75 to 1% versus 0.35 to 0.5% of the input DNA, while V26G had no effect, indicating that P80R restored ∼50% of EBNA1 enrichment compared to FPAX5. In PAX5-depleted conditions, transfected FPAX5 readily recruited EBNA1 to the TR-DNA of oriP-TRs-nLuc, whereas neither V26G nor P80R had an effect. Taken together, our data showed that the PPI mediated by EBNA1 and PAX5 indeed holds two proteins to simultaneously make proper associations with both oriP and TRs.
An extensive ChIP assay was next conducted to monitor the PAX5-dependent recruitment of the transcription enhancer components to the oriP or TRs of the oriP-TRs-nLuc reporter plasmid. In control BJAB-shScr cells, the enrichment of EBNA1, PAX5, H3K4Me2, and p300 was identified at the oriP DNA, while an abundance of EBNA1, PAX5, H3K4Me3, and p300 was observed at the TR-DNA (Fig. 6E). PAX5 knockdown caused the dissociation of EBNA1 and p300 from the oriP DNA and of EBNA1, p300, and H3K4Me3 from the TR-DNA, while H3K4Me2 remained consistently bound to the oriP DNA. Transfected FPAX5 readily restored the enrichment of each protein at the oriP DNA or TR DNA in the PAX5-depleted BJAB cells. Although V26G remained bound to p300, H3K4Me3, and TRs, it failed to recruit any of the proteins to the oriP DNA due to loss of its association to EBNA1. On the contrary, V26G was able to recruit both H3K4Me3 and p300 to the TR DNA by serving as a bridge to connect the enhancer components and TR DNA. Since P80R only retained its binding to EBNA1 but no longer associated with p300 and H3K4Me3, it simply restored EBNA1 enrichment to the oriP DNA. Our data reveal that PAX5-mediated recruitment of p300 to the oriP is central to EBNA1/oriP-mediated transcription.
PAX5 is required for the maintenance of the genome copy number of a mini-EBV episome reporter plasmid or EBV genome in the context of BJAB B-lymphoma cells.Binding of EBNA1 to the oriP is a prerequisite for EBNA1/oriP-mediated episome-dependent events; thus, PAX5 depletion is expected to debilitate the persistence of EBV genome copy number. Nevertheless, the critical PAX5 requirement for preventing lytic reaction (23) and EBV-dependent cell growth hampers investigation of the precise role of PAX5 in the maintenance of EBV genome copy number in LCLs. BJAB cells exhibited a much lower PAX5 dependence on cell growth compared to that of LCL and Akata+ cells (Fig. 2D); thus, the use of BJAB derivatives could largely reduce the side effects caused by the decline in dividing cells following PAX5 depletion. The oriP-TRs-nLuc plasmid exhibits a mini-EBV episome profile, which is expected to allow the copy number of EBNA1 to stably persist in BJAB cells stably expressing FEBNA1/oriP-TRs-nLuc (BJAB-FE1/oriP-TRs-nLuc). Moreover, EBV-infected BJAB cells (BJAB-B95.8) (31) could be used to study the effect of PAX5 depletion on the maintenance of a stable copy number of the EBV genome. BJAB-FE1/oriP-TRs-nLuc and BJAB-B95.8 cells were transduced with shRPAX5#1 or ShScr control, and the genome copy number of the oriP-TRs-nLuc plasmid or the EBV epigenome was assessed by qPCR at 0 to 120 h posttransduction. An approximately 50% reduction of the genome copy number of oriP-TRs-nLuc or that of the EBV genome after 96 to 120 h of shPAX5#1 transduction was observed compared to that of the control. Of importance, transfected FPAX5 fully rescued the genome copy number of oriP-TRs-nLuc plasmids in PAX5-depleted BJAB-FE1/oriP-TRs-nLuc cells, whereas neither V26G nor P80R had an effect. Together, our data suggest that PAX5 could contribute to the persistence of copy number maintenance of oriP-TRs-nLuc or the EBV genome via its link to EBNA1.
DISCUSSION
The DNA damage response (DDR) induced by the linear viral genome is a typical host antiviral defense in which herpesviruses convert their virion DNA into a circular minichromosome after entry into host cells to ensure the tight regulation of viral gene expression and viral DNA replication (32). Although the mechanisms of DNA circularization are poorly understood, the TRs are involved in DNA circularization, as they contain the essential elements for DNA end processing and homologous recombination (31, 33, 34). Kaposi’s sarcoma-associated herpesviruses (KSHV) require TRs to mediate viral episome formation and maintenance, since their latent origin of viral replication lies within the TRs (33–35). EBV TRs are not required for the replication and maintenance of oriP plasmids, since the plasmids contain the oriP sequences in EBNA1-expressing cells. Nevertheless, the TRs are involved in DNA circularization, since they contain essential elements for DNA end processing and homologous recombination (35–37). The identification of two PAX5 binding sites in each TR suggests that PAX5 may assist in the maintenance of a stable copy number of the viral genome when EBNA1 and PAX5 are linked through a PPI (21, 22). In this study, we showed a virtual contribution of PAX5 to EBNA1-mediated binding to the oriP, and the transcription was tightly linked to their protein networks. The fact that PAX5 did not associate with oriP DNA in the absence of EBNA1 further suggested that the oriP did not contain the cognate sequences for PAX5 binding. In particular, the PAX5-DBD was shown to form simultaneous dual linkages to the EBV-TRs and EBNA1. This unique biochemical feature could likely enable PAX5 to promote the persistence of EBV latent infection in multiple ways.
The tethering of viral episomes to the metaphase chromosomes is a common strategy that allows the herpesviruses to sustain a stable genome copy number during host cell division (11, 38). The episome maintenance of KSHV requires direct binding of the latency-associated antigen (LANA) to the TRs and histones (33, 34). In addition to knowing that its N-terminal RGG-like motif can recruit CCCTC-binding factor (CTCF) and cohesin (39), our study suggests that the EBNA1 C-terminal DBD may induce a high-order chromatin structure at the TRs via its interaction with PAX5 (40, 41). Although binding to the oriP is central to the downstream EBNA1/oriP-dependent events; the EBNA1-bound PAX5 mutant P80R only restores EBNA1/oriP-mediated binding, while it fails to support the EBNA1-dependent transcription and genome maintenance of a mini-EBV episome reporter, oriP-TRs-nLuc. In addition to binding to the cognate site properly, EBNA1 must develop distinct mechanisms to fulfill the diverse requirements from the downstream episome-dependent events.
Accordingly, PAX5 has been shown to be a master player for EBV-dependent cell proliferation and was previously identified as an inhibitor for lytic EBV reactivation (23), making it challenging to decipher the precise role of this B cell-specific factor in the maintenance of EBV genome copy number. Nevertheless, we were able to show that PAX5 indeed assists in the EBNA1-dependent genome copy number maintenance of the mini-EBV episome or EBV genome in two BJAB derivative cell lines because they exhibit a low dependence on PAX5 for cell proliferation. The use of the stable clone BJAB-FE1/oriP-TRs-nLuc could avoid the precipitous loss of oriP plasmids after several days of transfection (42). The inability of P80R to support the persistence of a stable copy number of the mini-EBV episome reporter could be partially due to its defects in binding to the TRs or to loss of interactions to unknown chromatin modifiers, such as MCM or ORC. Moreover, the dependence of EBNA1 on PAX5 to localize to the oriP implies that binding of PAX5 to EBNA1 could be the driving force itself or that such PPI could potentially recruit chromatin modifiers to stabilize the association of EBNA1 with the oriP. Our study suggests that EBV could have evolved PAX5-dependent strategies to carry out multiple redundant mechanisms to establish and maintain viral latency in the B cells.
Since neither V26G (EBNA1 binding mutant) nor P80R (EBNA1 bound) were able to restore the defects in EBNA1/oriP-Luc-induced transcription levels of oriP-TRs-nLuc caused by PAX5 depletion, it implies that multiple PAX5-driven events are crucially involved in EBNA1/oriP-mediated transcription. Apart from knowing the EBNA1/PAX5-mediated PPI is a basic requirement, the inability of P80R to restore EBNA1/oriP-dependent transcription in a PAX5-depleted condition could be due to the loss of its interaction with p300 or to the partial loss of function of PAX5 on its target genes (43). Although the TRs are not essential, the PAX5-mediated recruitment of p300 and H3K4Me3 to the TRs may further enhance the EBNA1/oriP-mediated transcription via the PAX5-EBNA1-oriP axis. The EBNA1-induced transcription from oriP-TRs-nLuc is approximately 2-fold higher than that from oriP-nLuc, suggesting that the addition of TRs indeed promotes EBNA1/oriP-dependent transcription. The discovery of the involvement of the global transcription enhancer p300 in EBNA1-mediated transcription sheds light on the current understanding of this novel transcription event. Lacking the classical activation domain in its encoded amino acids could be partially compensated by EBNA1’s associations with the cellular transcription factors or regulators, such as OCT2, NPA1, and Brd4 (44–46). Our study identified PAX5-TAD as another important EBNA1 binding domain, suggesting that this activation sequence may guide EBNA1 to create a link with the general transcription machinery. Although EBNA1 did not directly bind p300, the ChIP study demonstrated that PAX5 could recruit p300 to the oriP through PPI, suggesting PAX5 could simultaneously link to EBNA1 and p300, thus mediating EBNA1-PAX5-p300 complex formation at the oriP. The EBNA1-PAX5-p300 network may offer another route to support the EBNA1-mediated transcription of cellular genes (Fig. 7D). We infer that EBNA1 likely exploits the PAX5-dependent recruitment of p300 and H3K4Me3 to modulate the cellular gene expression at its global target sites in the B cell-lineage cells.
PAX5 is implicated in the maintenance of the genome copy number of the mini-EBV episome and EBV genome in specified BJAB derivative cell lines. (A) BJAB-FE1/oriP-TRs-nLuc or (B) BJAB-B95.8 cells were transduced with lentivirus-expressed shPAX5#1. The growth curve for each cell line with shPAX5#1 or control shScr transduction from 0 to 120 h is shown. The stable copy number of the oriP-TRs-nLuc plasmid and the EBV genome at 0 to120 h posttransduction were quantified by qPCR. The immune blots for EBNA1, PAX5, and GAPDH control are shown. (C) The BJAB-FE1/oriP-TRs-nLuc stable clone was transduced with shPAX5#1 for 72 h. Cells were then collected and transfected with FPAX5 or its mutants and incubated for another 24 h. The genome copy number of oriP-TRs-nLuc was determined by qPCR as the relative amount versus the genomic GAPDH DNA. The obtained genome copy number for oriP-TRs-nLuc in shScr transduced cells was set to 1 for the subsequent comparative analyses. (D) The proposed model depicting EBNA1/PAX5-mediated transcription from the EBV epigenome is shown.
Recent advances in EBNA1 research have elevated the critical role of some nucleolus factors, including NPM1, NCL, and RPL4, in EBNA1/oriP-dependent functions (16, 26, 47). Since PAX5 depletion had no effects on the physical interaction mediated by EBNA1 and NCL or RPL4, PAX5 is expected to carry out unique mechanisms to support EBNA1/oriP-dependent events. The capability of PAX5 to control gene expression by recruiting chromatin remodeling, histone modification, and basal transcription machinery could surely expedite EBNA1/oriP-mediated transcription enhancement (40, 41). Although further studies are required, the EBNA1/PAX5-induced protein complex at the oriP can be considered tissue-specific viral regulators that restrict B cells as the most favorable targets for latent infection with EBV. The virtual requirement of PAX5 for EBNA1 functioning reflects that PAX5 may appear as a global requirement for EBV-induced pathogenesis and/or tumorigenesis for certain types of neoplastic-B cell diseases. On the other hand, PAX5 by itself has been considered a biomarker for the differential diagnosis of B cell leukemias, as well as that of lymphomas (48). Further extensive studies of Pax5, along with the viral genes and/or immune genes, are necessary for conclusive results. The mechanism by which EBV establishes latency in non-B cell lineages remains unknown. It can be inferred that EBNA1 employs similar strategies to exploit other tissue-specific factors to sustain the persistence of the EBV genome in the non-B cell-lineage cellular environments. Our discovery provides a breakthrough for both fundamental and clinical EBV researchers by demonstrating the virtual involvement of a B cell-specific factor in EBV episome-mediated events. Understanding how EBV develops tissue-specific strategies for viral genome maintenance may provide new insights into the mechanisms driving cellular genomic instability and antitumor cancer drug discovery.
MATERIALS AND METHODS
Plasmids, DNA recombination, and mutagenesis.The Flag-tagged PAX5 and PAX5-BiFC plasmids were generated by subcloning full-length PAX5 cDNA into the BamHI and XhoI sites of the pSG5-Flag and BiFC expression vectors, respectively (26, 49). The template for PAX5-flanking DNA amplification was purchased from OriGene (catalog no. MR222617). The PAX5 mutants were created using a Q5 site-directed mutagenesis kit (catalog no. E0554; New England Biolabs [NEB]). The expression plasmids for EBNA1-dependent BiFC and/or oriP-Luc reporter assays have been described previously, whereas YN-FPAX5 was created by subcloning the PAX5 cDNA into the BamHI and XhoI sites of the BiFC YFP N terminus expression vector (YN) (26). The non-lytically based mini-EBV reporter plasmids were generated by subcloning the synthetic flanking sequences for oriP, TR, or both, as well as for nano-luciferase, into the BamHI and HindIII sites of the pGL3 control vector (Promega). The oriP DNA- or TR-DNA-containing plasmids, p-oriP and p-TRs, were produced by subcloning the indicated synthetic flanking sequences into the XhoI and HindIII sites of the pGL3 vector. All of the resulting recombinant clones were verified by DNA sequencing (Genomics Taiwan).
Lentivirus production and transduction.The lentivirus-expressed shRNA vectors (TRCN16058 and TRCN432567) for PAX5 were purchased from the National RNAi Core Facility, Academia Sinica Taiwan. For virus production, ∼2 × 106 to 5 × 106 293T cells in a 10-cm culture dish were transfected with lentiviral DNA mix (16 μg/μl pCMV-ΔR8.91, 0.7 μg/μl pMD.G, and 7 μg/μl shRNA) by a calcium phosphate transfection procedure. The medium was replaced with fresh 293 culture medium after 12 to 16 h of transfection, and 40 to 48 h after transfection, the supernatant was harvested to pass through a 0.45-μm membrane (catalog no. HAWG03700; Merck). To silence PAX5, 5 × 105 cells per ml of each selected cell line were transduced with 1 ml of lentivirus supernatant in the presence of 8 μg/ml of Polybrene and cultured in a 6-well plate for 72 h. Each transfectant was cultured in new medium supplemented with 5 ng/ml of puromycin prior to performing the desired experimental procedures.
Production of His-tagged PAX5 recombinant protein.The His-tagged PAX5 expression vector was produced by subcloning the PAX5 cDNA into the BamHI and HindIII sites of the pTRC vector (catalog no. V360-20; Thermo Fisher Scientific). The His-tagged PAX5 recombinant protein (HPAX5) was expressed in Escherichia coli BL21(DE3) and purified by Ni Sepharose high-performance affinity resin (catalog no. 302188; Amersham Biosciences). Briefly, 5 ml of bacterial culture was grown overnight to the saturated stage and inoculated into 200 ml of LB-ampicillin (Amp) medium. The culture was grown until the optical density (OD) reached 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 M, and 6× His-tagged PAX5 protein production was induced for 4 h. Cells were collected in 10 ml lysis buffer (100 mM NaH2PO4 and 20 mM Tris-HCl [pH = 8.0]) supplemented with 1× protease inhibitors (catalog no. 78429; Thermo Fisher). Sonication was performed on ice using a Microson XL2000 instrument at scale 4 for 1 min (3 times). Prewashed 10% Ni-nitrilotriacetic acid (NTA) slurry (0.5 ml) was added to 10 ml lysate and mixed gently by rotating for 60 min at 4°C. The resin beads were collected and washed twice with 4 ml wash buffer (100 mM NaH2PO4, 20 mM Tris-HCl, and 20 mM imidazole [pH = 8.0]). HPAX5 was eluted with 0.5 ml elution buffer (100 mM NaH2PO4, 20 mM Tris-HCl, and 200 mM imidazole [pH = 8.0]) by rotating at 4°C for 1 h. Fractions were collected and then analyzed by SDS-PAGE.
Cell lines and cell culture.BJAB is an EBV-negative B cell lymphoma cell line (50). Lymphoblastoid cell lines (LCL) LCL1 (51) and LCL2 (16) were previously established in the laboratory. LCL3 is an LCL that was newly established in this study. LCL1 is referred as lymphoblastoid cells or as LCL when only one cell line was used for the indicated study. IB4 is another LCL in which the EBV genome has been integrated into the chromosome (30). Akata is an EBV-negative Burkitt’s lymphoma cell line, whereas EBV-positive Akata (Akata+) is a counterpart of Akata (52). BJAB-B95.8 is an EBV-reinfected BJAB cell line (31). BJAB cell lines stably expressing FEBNA1 and the oriP-TRs-nLuc reporter plasmid were established by transfection of the above expression plasmids into BJAB cells with the selection of 2 μg/ml puromycin (catalog no. P8833; Sigma-Aldrich). 293T is a human embryonic kidney cell line (ATCC CRL-3216). All suspension cells were cultured in RPMI 1640, while 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (catalog no. 25030081; Thermo Fisher Scientific), and penicillin (100 U/ml)-streptomycin (100 μg/ml) (catalog no. 15140122; Thermo Fisher Scientific). All of the cell lines were verified as mycoplasma negative by a mycoplasma detection kit (catalog no. 4460626; Thermo Fisher Scientific).
Immunoprecipitation and immunoblot analyses.For each immunoprecipitation assay, 5 × 106 LCL or BJAB cells were transfected with the indicated plasmids using a BTX830 electroporator (settings, 2 impulses at an intensity of 180 V/cm, 20-ms duration per pulse). After 24 h of transfection, cells were collected in lysis buffer (1% NP-40, 10% glycerol, 2 mM EDTA, 50 mM Tris-HCl [pH 7.0], and 150 mM NaCl) supplemented with 1× protease inhibitor cocktail (catalog no. 78440; Thermo Fisher Scientific) and 2.5 μl of 5 mg/ml DNase I and 0.5 μl of 10 mg/ml RNase A per 0.5 ml of cell lysate. M2 beads (catalog no. A2220; Sigma) or protein G agarose beads (catalog no. 10054549; GE Healthcare) bound by each indicated antibody were prepared as 10% slurry in 0.5 ml lysis buffer and used to perform an immunoprecipitation assay. For the immunoblot assays, cells were washed twice with phosphate-buffered saline (PBS) and lysed in ice-cold sample buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 6% (vol/vol) glycerol, 2 mM dithiothreitol (DTT), and 0.01% bromophenol blue (wt/vol). Samples were briefly sonicated 5 times for 2 s on a Microson XL2000 ultrasonic processor prior to SDS-PAGE.
ChIP-qPCR and quantitation for EBV genome copy number.The ChIP assay was performed according to the instruction manual for the EZ-ChIP chromatin immunoprecipitation kit (catalog no. 17-371; Merck Millipore). Briefly, 5 × 106 cells were collected for each immunoprecipitation assay. The cross-linking of protein and the DNA complex was achieved by incubating the collected cells under 1% formaldehyde fixation conditions for 15 min at room temperature. Cells were resuspended in sonication buffer (20 mM Na3PO4 and 500 mM NaCl), and DNA shearing by sonication was performed three times on a Microson XL2000 ultrasonic processor (scale, 3) for 30 s each time. A real-time quantitative PCR (qPCR) assay for EBV genome copy or coimmunoprecipitated DNA was performed using an Applied Biosystems 7500 real-time PCR system. The H3Ac/GAPDH-promoter ChIP-qPCR kit used was purchased from Sigma-Aldrich (catalog no. 17-615). The EBV copy number in each cell line was determined relative to the level of the genomic GAPDH control, whereas the coimmunoprecipitated DNA was determined as the percentage of input DNA ± standard deviation (SD). The antibodies for the immunoprecipitation and Western blot assays and the primers for the ChIP-qPCR and oriP-TRs-nLuc DNA qPCR are described in Table 1.
Antibodies and primers used in this study
BiFC images and immunofluorescence confocal microscopy.293T cells (5 × 104) were transfected with 2 μg of each BiFC plasmid in a six-well plate by performing a calcium phosphate transfection procedure. The BiFC images were taken on a Zeiss Axioplan fluorescence microscope mounted with a Spot Insight 2-megapixel color mosaic digital camera after 48 h of transfection. BJAB, BJAB-FEBNA1 stable clone, LCL1, or B cells with EBV infection at the indicated number of days postinfection were used to perform an immunofluorescence (IF) assay. Cells were washed twice with PBS and incubated in intracellular (IC) fixation buffer (catalog no. 00-8222-49; Thermo Fisher Scientific) for 20 min. Cells were then collected for incubation in 1× permeabilization buffer (catalog no. 00-8333-56; Thermo Fisher Scientific) at room temperature for 5 min. EBNA1-fluorescein isothiocyanate (FITC) (catalog no. orb4774; Biorbyt) or IH9 for PAX5 (Santa Cruz Biotechnology) was used for immunostaining, which was followed by a PBS wash procedure. PAX5 was visualized by a rhodamine-conjugated secondary antibody (catalog no. 115-025-062; Jackson ImmunoResearch). The nucleus was counterstained with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies catalog no. P36935). Fluorescence confocal microscopy was performed using a confocal microscope set (Carv II confocal imager equipped with an inverted-system optical microscope consisting of an Olympus IX71S8F3 microscope and a high-quantum-efficiency charge-coupled device [CCD] camera). The confocal images were processed with VisiView imaging software.
Transfection-mediated firefly luciferase or secretion-based nano-luciferase (nLuc) reporter and cell proliferation assays.BJAB, BJAB-shScr, or BJAB-shPAX5 cells (107) were cotransfected with 10 μg of FEBNA1 expression vector, 5 μg of oriP-Luc or 1 μg oriP-nLuc or oriP-TRs-nLuc reporter plasmid, and 1 μg of cytomegalovirus (CMV)–β-galactosidase (β-Gal) or p-SEAP2 (catalog no. PT3057-1; TaKaRa) internal control reporter plasmid. The EBNA1/oriP-Luc-mediated transcription reporter assay has been described previously (16, 26), whereas the nLuc activity assay (catalog no. E1500 and N1110; Promega) was performed on a Tecan Spark 10M hybrid microplate reader. Cell proliferation assays were conducted using the trypan blue exclusion method to determine the number of viable cells present in a cell suspension every 24 h for five consecutive days. Aliquots of 5 × 103 cells per 100 μl of each selected cell line were transduced with lentivirus PAX5 3′ UTR-expressed green fluorescent protein (GFP), shRNAs, shPAX5#1 or shPAX5#2, or scramble shRNA (shScr) control, and seeded in 96-well plates.
Statistical analysis.All of the described experiments were done at least in triplicate, and the statistical data were subsequently expressed as the mean ± standard deviation (SD). After the quantitative data were obtained from the comparative analysis, the statistical analysis was performed using Student’s t test. A P value of <0.05 (*) represents a significant observed difference in the study group compared to the control or reference group, whereas a P value of >0.05 (†) means that no effect was observed.
ACKNOWLEDGMENTS
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
The research work is supported by grants TCMMP105-07-01 and 106-03-01 from the Buddhist Tzu Chi Medical Foundation and by MOST 105-2320-B-320-013-MY3 and 106-2320-B-320-004-MY3 from the Ministry of Science and Technology to C.-W.P. C.-D.L. was supported by MOST 107-2811-B320-506 and 108-2811-B320-501 from the Ministry of Science and Technology. The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.
C.-D. Liu, H.-L. Lee, and C.-W. Peng conceived the idea and developed the theory. C.-D. Liu and H.-L. Lee performed the experiments and verified the analytical methods. C.-W. Peng wrote the paper, and all authors discussed the results and contributed to the final manuscript.
FOOTNOTES
- Received 30 November 2019.
- Accepted 23 December 2019.
- Accepted manuscript posted online 15 January 2020.
- Copyright © 2020 American Society for Microbiology.
