The F-box E3 ligase protein FBXO11 regulates EBNA3C-associated degradation of BCL6

ABSTRACT Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt’s lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50–100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.


IMPORTANCE
The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction.It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation.Nonetheless, our findings carry significance for the develop ment of emerging strategies aimed at addressing EBV-associated cancers.KEYWORDS Epstein-Barr virus, EBNA3C, FBXO11, E3 ligase, ubiquitin E pstein-Barr virus (EBV), the first human oncogenic virus to be isolated, infects over 95% of adults worldwide and establishes persistent infection throughout life (1,2).EBV-associated malignancies contribute to approximately 2% of all cancer-related deaths (3).During latent EBV infection, the expression of EBV genes depends on the specific program of latency, which dictates the expression pattern required to drive cell proliferation.Previous studies indicate that during latency III, which is observed in lymphoblastoid cell lines (LCLs), all six Epstein-Barr nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and EBNA-LP) as well as latent membrane proteins LMP1 and LMP2 (LMP2A and LMP2B) are expressed (4).This also expressed the EBV small RNAs, which include the EBERs and miRNAs seen in most latency programs (5).
The B-cell lymphoma 6 (BCL6) protein is an important transcriptional repressor that plays a vital role in B-cell development and is involved in regulating immune responses.It is primarily expressed in germinal center (GC) B cells, a specialized region within the secondary lymphoid organs, where B cells undergo intense clonal expansion, somatic hypermutation, and affinity maturation in response to antigens (6).BCL6 prevents premature differentiation of B cells by recruiting co-repressor complexes and chromatinmodifying enzymes to target gene promoters, leading to the suppression of transcrip tion.Importantly, genetic alterations or dysregulation of BCL6 is associated with the development of B-cell lymphomas.
Previously, we reported that EBNA3C regulates BCL6 through two distinct mecha nisms.First, it interacts with BCL6, leading to its degradation via the ubiquitin-protea some pathway (7).Additionally, EBNA3C hinders the transcriptional activity of the BCL6 promoter, resulting in the suppression of BCL6 mRNA expression (8).BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas (9).Recent research demonstrated that BCL6 is a Skp Cullin Fbox E3 ligase substrate and is targeted for ubiquitination and degradation by the SCF complex (9,10).SCF complexes contain four essential components: Skp1, Cullin, Rbx1/Roc1/Hrt1, and an F-box protein for its E3 ligase activities (11).SCF complexes facilitate interaction between substrates and ubiquitin-conjugating enzymes, which covalently transfer ubiquitin to substrates, which are subsequently degraded.The F-box protein is the subunit of the SCF complex that binds specific substrates, linking it to the complex by binding Skp1 through the F-box (12).The F-box proteins are essential in regulating SCF activity during the cell cycle.The affinity of the F-box protein for protein substrates is regulated through Cdk/ cyclin-mediated phosphorylation of target proteins.In normal germinal center B cells, the expression of BCL6 is reduced.However, this signaling pathway is obstructed in a specific subgroup of diffuse large B-cell lymphomas (DLBCLs) due to genetic changes or modifications in the BCL6 gene (13).A subset of DLBCLs demonstrate chromosomal translocations or mutations that disrupt the IRF-responsive region in the BCL6 promoter and block its downregulation by CD40 regulation (14).
F-box proteins are characterized by an amino-terminal 40-residue F-box motif that binds Skp1, followed by protein-protein interaction modules such as leucine-rich repeats or WD (Trp-Asp) repeats that bind substrates (15).There are three classes of F-box proteins: FBXL, FBXW, and FBXO (15).FBXL are proteins containing an F-box and leucine-rich repeats, FBXW are proteins containing an F-box and WD (Trp-Asp) repeats, and FBXO are proteins containing an F-box and either another motif or no other motif (15).FBXO11 is an F-box protein that belongs to the FBXO class, and its dysregulation is linked to oncogenesis (15).
In DLBCL, BCL6 overexpression is observed because of the deletion of FBXO11 (9).When reconstituted, ubiquitination and degradation were promoted and BCL6 levels decreased (9).In EBV-positive DLBCL, BCL6 expression is observed to be frequently decreased (16).Therefore, it was important to investigate whether FBXO11 is responsible for its downregulation due to the pathological differences between EBV-positive and negative DLBCLs.The objective of our present study was to elucidate the regulatory mechanisms involving FBXO11, BCL6, and EBNA3C in contributing to EBV-mediated oncogenesis.

The expression of BCL6 is dramatically decreased in the presence of EBNA3C and FBXO11
Previously, we reported that during primary EBV infection, the levels of BCL6 were downregulated (8).We also observed that mutant EBV that were devoid of EBNA3C were expressing significantly more BCL6 as compared to the cells infected with wild-type EBV (8).In order to further validate our previous findings and establish a connection between the latency program of EBV and BCL6 expression, we conducted western blot assays to assess the protein levels of BCL6 in various cell lines, both EBV negative and EBV positive.We now show that BCL6 protein levels were dramatically decreased in EBV-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines Mutu III, Sav III, and Kem III, which exhibit the latency III program when compared to the same isogenic cells that express the latency I program namely Mutu I, Sav I, and Kem I and the EBV-negative BJAB (Fig. 1A).Densitometric analysis confirmed that the demonstrated drop in protein levels of BCL6 was greater than fivefold as seen in the EBV-positive latency III cells Mutu III, Sav III, and Kem III as compared to that of the EBV-positive latency I cells or EBV-negative cell lines (Fig. 1A).These results suggested that EBV infection was responsible for the reduced expression of BCL6 in human Burkitt's and lymphoblastoid cell lines.Additionally, the type I latency cells Mutu I, Sav I, and Kem I did not show a reduction in BCL6, strongly suggesting that this may be due to one or more of the latent antigens expressed during the latency III program.To determine which latent gene contributes to the reduction in BCL6 expression levels, western blot analyses were performed with eight latent genes (EBNAs 1, 2, 3A, 3B, 3C, EBNA-LP, LMP1, and LMP2A) stably expressed in BJAB cell lines.The results of the western blot clearly showed that EBNA3C was able to significantly reduce the expression levels of BCL6 by greater than 60% as evidenced by western blot signals and the induced expression of FBXO11 (Fig. 1B, see lane 7 and bottom graph).
To further validate this finding, we exogenously expressed EBNA3C and FBXO11 in Saos-2 cells.We observed that enhanced expression of FBXO11 from a heterologous system in the presence of increasing concentrations of EBNA3C led to a significant downregulation of BCL6 expression levels (Fig. 2A).To further validate these findings in BJAB cells and EBNA3C expressing BJAB7 cells, the cells were transfected with FLAG-FBXO11 and HA-BCL6.We observed significantly lower levels of BCL6 in cells that were expressing EBNA3C and FBXO11 as compared to cells that were expressing either FBXO11 or EBNA3C alone (Fig. S1A).We additionally knocked down the expression of FBXO11 in Saos-2 cells expressing EBNA3C using shRNA and noticed a significant reduction of BCL6 expression (Fig. S1B), which further confirms the involvement of FBXO11 in the regulation of BCL6 protein levels.
To explore the role of FBXO11, we constructed a carboxy-terminal-deleted Flag-tag ged FBXO11 mutant and co-transfected it with EBNA3C.The Flag-FBXO11MT is devoid of the carboxy-terminal substrate recognition domain (Δ1,753-2,238 bp).The expres sion of exogenous EBNA3C was confirmed by western blot using antibody against the c-Myc epitope, while the expression of FBXO11 and FBXO11MT was confirmed by using antibodies against the Flag epitope.We observed that the expression of full-length FBXO11 and EBNA3C led to a dramatic reduction in levels of BCL6 expression, while the exogenous expression of mutant FBXO11 did not show any reduction in levels of BCL6

Full-Length Text
Journal of Virology as a result of its degradation (Fig. 2B, bottom graph).This underlines the importance of FBXO11 and EBNA3C in regulating BCL6 expression levels.

The amino-terminal domain of EBNA3C binds FBXO11 in human cells
Next, we examined whether EBNA3C could interact directly with FBXO11.Saos-2 cells were transfected with epitope-tagged Myc-EBNA3C and Flag-FBXO11.Immunoprecipi tation with anti-Flag antibody followed by western blot using Myc-specific antibody revealed that Myc-tagged EBNA3C was co-immunoprecipitated with Flag-FBXO11 (Fig. 3A).To further determine the interaction in B-cell lines, BJAB cells, BJAB7 cells that express EBNA3C, and EBV-transformed cells (LCL1) were tested for the interaction of EBNA3C with FBXO11.Western blot analysis demonstrated that endogenous EBNA3C can physically associate with FBXO11 in the background of B cells and more importantly in EBV-transformed lymphoblastoid cell lines (Fig. 3B).
To determine the functional binding domain of EBNA3C that specifically interacts with FBXO11, Myc-tagged full-length and truncated regions of EBNA3C (1-365 aa, 366-620 aa, and 621-992 aa) (Fig. 3C) were co-transfected with Flag-tagged FBXO11 into Saos-2 cells.The targeted protein was immunoprecipitated with anti-Flag-specific antibody.The results demonstrated that FBXO11 was associated with EBNA3C (1-365 aa) along with the full-length EBNA3C protein (1-992 aa) (Fig. 3D).No detectable coimmunoprecipitation was observed with the control vector supporting the associated specificity of the complex between EBNA3C and FBXO11 in these cells.These results showed that EBNA3C amino acid residues 1-365 aa, which include the acidic domain, were responsible for the interaction of EBNA3C with the FBXO11 protein.We further used EBNA3C smaller truncations (1-200 aa, 100-200 aa, and 50-365 aa) to more precisely determine the region of EBNA3C that interacts with FBXO11.We observed Flag-tagged FBXO11 co-immunoprecipitated with 1-200 aa and 50-365 aa fragments of EBNA3C (Fig.

3E
).This result clearly demonstrated that the region containing residues 50-100 aa of EBNA3C was critical for binding with FBXO11.

EBNA3C co-localizes to nuclear compartments with FBXO11 in human cells
To determine the localization of EBNA3C and FBXO11, Saos-2 cells were transfected with constructs expressing Myc-tagged EBNA3C and Flag-tagged FBXO11, and the cellular localization of the expressed proteins was examined using immunofluorescence assays and visualized by fluorescence microscopy.In cells transfected independently with Myc-EBNA3C or Flag-FBXO11 alone, both were found to be primarily expressed in the nucleus (Fig. 4A).In cells co-transfected with Myc-EBNA3C and Flag-FBXO11, the merged yellow fluorescence demonstrated that EBNA3C co-localized with FBXO11 in nuclear compartments in cells expressing these constructs (Fig. 4A).
To further determine the localization of EBNA3C and FBXO11 proteins in more physiologically relevant B cells, immunofluorescence assays were performed using antibodies specific to EBNA3C and FBXO11 proteins.The results further confirmed that EBNA3C co-localized with FBXO11 in nuclear compartments of EBV-transformed cells (Fig. 4B).This was consistent with the results of the above studies, which demonstrated that EBNA3C co-immunoprecipitated with FBXO11 and showed the association of these complexes in the same cellular compartments.

EBNA3C promotes FBXO11-mediated cell proliferation
Previously, we reported that EBNA3C-mediated regulation of BCL6 promoted cell proliferation by targeting BCL2 and CCND1 (8).Although BCL6 is reported to be an oncoprotein, BCL6 also binds and represses BCL2 and BCL-XL expression (18).To examine the involvement of FBXO11, Saos-2 cells were transfected with expression constructs of EBNA3C and FBXO11 and selected with G418 for 2 weeks to monitor colony formation.We observed a significant increase in colony numbers when EBNA3C and FBXO11 were co-transfected in comparison to those transfected with only EBNA3C or FBXO11 (Fig. 5A  and B).We further extended these studies by performing cell proliferation assays as determined by cell counting for 6 days in Saos-2 cells (Fig. 5C).These results demonstra ted that expression of EBNA3C and FBXO11 results in a strong induction in cell prolifera tion as determined by the increase in cell numbers when compared to either EBNA3C or FBXO11 alone and suggests that the complex is important for driving an increase in cell proliferation.

BCL6 expression is regulated by EBNA3C via SCF FBXO11 complex-mediated ubiquitylation
Previous data demonstrated that EBNA3C recruited E3 ligases for targeted degradation of cellular substrates (19,20).Our previous research has shown that EBNA3C promoted the degradation of the BCL6 protein (8).FBXO11 is a member of the F-box protein family, which is the substrate-binding subunit of the Skp1-Cul1-F-box ubiquitin ligase complex (12).To further assess whether this F-box protein is the E3 Ligase interacting with EBNA3C and BCL6, ubiquitination assays were performed with different expression plasmids for Myc-E3C, Flag-FBXO11, Flag-FBXO11mut, HA-Ub, and HA-BCL6 and incubated for 24 hours followed by MG132 treatment for another 12 hours.This was followed by immunoprecipitation and western blot analysis.The results of this assay demonstrated enhanced ubiquitination of BCL6 when both EBNA3C and FBXO11 were co-expressed as compared to FBXO11 alone or co-expression of FBXO11mut and EBNA3C (Fig. 6).This strongly indicated that BCL6 is likely degraded in the presence of EBNA3C through SCF FBXO11 complex-mediated ubiquitylation.

Knockdown of FBXO11 suppresses the transformation of LCLs
To evaluate the effect of FBXO11 on EBNA3C-mediated transformation of B cells, FBXO11 was stably knocked down in BJAB and LCL1 cells by lentivirus transduction using shRNA targeting FBXO11 and selected using puromycin for 3 weeks (Fig. 7A through C).The ability of these selected cell lines to form colonies was determined by soft agar assays.
The results showed that a smaller number of colonies were observed in FBXO11 knocked down BJAB cells as compared to the shControl cells (Fig. 7).However, in FBXO11 knocked down EBV-transformed LCL1 cells, the relative colony number was dramatically less (Fig. 7D and E), when compared to the shControl cells.This result clearly demonstrated the significance of EBNA3C and FBXO11 on cell growth and proliferation of EBV-transformed B cells.
To dissect the role of FBXO11 and EBNA3C on the cell cycle, propidium iodide assays were performed on FBXO11 knocked down BJAB and LCL1 cells, and the percentages of cells in different phases of the cell cycle were determined (Fig. 7F and G).The results showed that the knockdown of FBXO11 in BJAB cells increased the percentage of cells in the S phase.However, when FBXO11 was knocked down in LCL1 cells, the percentage of cells in the S phase dramatically decreased.These results indicated that in EBV-infected LCL cells, knockdown of FBXO11 suppressed G1 to S transition, while in EBV-negative BJAB cells, the cell cycle progressed as expected with time.

DISCUSSION
BCL6 belongs to the BTB/POZ/Zinc Finger protein family and is a nuclear phosphopro tein.It functions as a transcription repressor, suppressing target genes by binding to specific DNA sequences and recruiting corepressors such as SMRT, MTA3, NCoR, and HDAC (21)(22)(23)(24)(25)(26).BCL6 plays a crucial role in germinal center formation and somatic hypermutation during B-cell development.Disruptions in the regulatory region of BCL6 caused by chromosomal translocations and mutations result in dysregulation of BCL6 expression, observed in approximately 40% of DLBCL and 5%-10% of FL cases (27).While BCL6 expression is associated with the EBV latent antigens EBNA2 and LMP1, conflicting results have not provided a comprehensive explanation or detailed mechanism for EBVmediated BCL6 degradation in B-cell lymphoma (28)(29)(30).A recent study suggested that EBNA3C has no effect on BCL6 expression (31).However, a previous report indicated that BCL6 expression can increase more than 10-fold in EBNA3C-deleted EBV-infected cells (31,32).Our previous study conclusively demonstrated that EBNA3C specifically downregulates BCL6 expression at both the transcriptional and post-transcriptional levels (8).
FBXO11 is categorized as a member of the F-box protein family, known for its characteristic F-box domain, which consists of approximately 40 amino acids.This protein family serves as the substrate-binding component within the SCF ubiquitin ligase complex (33).FBXO11 exerts crucial regulatory functions in cell cycle regulation, tumorigenesis, and metastasis of tumor cells through its ability to bind to substrate proteins and facilitate their degradation (34).The SCF FBXO11 complex mediates ubiquityla tion and degradation of BCL6, a transcription repressor that is required for normal germinal center development (9).FBXO11 interacts with CDT2 (a DCAF protein that controls cell-cycle progression) and recruits CDT2 to the SCF FBXO11 complex to promote its proteasomal degradation (35,36).FBXO11 was shown to mediate the ubiquitylation and degradation of SNAIL, a transcription factor that promotes epithelial-mesenchymal transition (EMT) (37).The recognition of SNAIL by FBXO11 appears to be dependent on Ser-11 phosphorylation of SNAIL by protein kinase D1 (PDK1).FBXO11 blocks SNAILinduced EMT, tumor initiation, and metastasis in multiple breast cancer models (37).
In the present study, we demonstrated that EBNA3C specifically interacts with FBXO11 and mediates the degradation of BCL6 through the ubiquitin-proteasome dependent pathway (Fig. 8).A previous study showed that the BCL6 protein can be targeted for degradation by cellular factor FBXO11 in DLBCL (9).In our study, we have successfully demonstrated that the recruitment of FBXO11 and degradation of BCL6 by EBNA3C require this E3 ligase.Earlier research suggests that acetylation of BCL6 within its PEST domain leads to the inactivation of its co-repressor recruitment function (38).Moreover, the activation of the MAPK signaling pathway induces the phosphorylation of BCL6, subsequently resulting in its degradation through the ubiquitin-proteasome pathway (39).Additional investigations are needed to ascertain the potential connec tions between EBNA3C-mediated BCL6 degradation, BCL6 acetylation, and phosphoryla tion.
The significance of BCL6's role in GC B cells is evident from its ability to regulate multiple functional pathways within the cell.BCL6 has been found to target over 1,000 genes by binding to their promoters, thereby influencing downstream signaling pathways crucial for GC development.These pathways are involved in various cellular processes such as apoptosis, cell cycle regulation, and cell differentiation (40,41).One of the important proteins targeted by BCL6 is BCL2, which plays a critical role in antiapoptosis.BCL6 directly interacts with Miz1 and binds to the BCL2 promoter, resulting in the inhibition of Miz1-induced transcriptional activity of BCL2 in B cells (42).Dysregula tion of BCL6-mediated BCL2 expression is a common occurrence in DLBCL and FL (43).Our previous findings demonstrated that EBNA3C induces cell proliferation by the degradation of BCL6, thus inhibiting its expression, thereby alleviating the suppression of BCL2 and activating the anti-apoptosis pathway for tumorigenesis (8).Additionally, CCND1, a direct BCL6 target in human B cells, is de-repressed, promoting G1-S transition in EBV-transformed LCLs.The control of other cyclin proteins by BCL6 remains unclear.Notably, studies suggest that CCND2 is another BCL6 target, but its expression is negatively correlated (44)(45)(46)(47)(48). EBNA3C upregulates the expression of activation-induced cytidine deaminase (AID), responsible for somatic hypermutation and class-switch recombination, in EBV-infected cells (31,49).BCL6 can also enhance AID expression by inhibiting miR-155 and miR-361 (50).The regulation of AID expression by EBNA3C without BCL6 involvement warrants further exploration (51).Recent research identified BCL6-targeted genes in T follicular helper cells, underscoring BCL6's genome-wide occupancy and role in transcriptional regulatory networks (52).Although BCL6 smallmolecular inhibitors show promise as therapeutic targets for human lymphomas (53), the BCL6-mediated regulatory networks in EBV-transformed LCLs remain elusive.Xenografts of LCLs in BCL6 knockout mice may elucidate BCL6's biological function in EBV-related lymphomagenesis.However, a more efficient in vivo model is crucial for uncovering the roles of EBNA3C and other latent antigens in the GC reaction.
In summary, the inhibition of BCL6 expression by the essential EBV antigen EBNA3C provides a novel perspective on EBV's role in lymphomagenesis by impeding the GC reaction.While various EBV latent proteins are expressed in infected cells, understanding how these proteins collaborate to regulate B-cell development or induce B-cell lym phoma requires further investigation.Nevertheless, our findings have implications for emerging strategies targeting EBV-associated cancers.

Cell culture and plasmid constructs
EBV-negative Burkitt's lymphoma BJAB cells were kindly provided by Elliot Kieff (Harvard Medical School, Boston, MA, USA).Mutu I and Mutu III cells were kindly provided by Yan Yuan (School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA).Sav I and Sav III cell lines, Kem I and Kem III cell lines were kindly provided by Dr. Paul M. Lieberman (The Wistar Institute, Philadelphia, PA, USA).BJAB stably expressing EBNA3C cells (BJAB7 and BJAB10) were prepared by transfecting pZipneo EBNA3C into BJAB cells, followed by neomycin selection (54).LCL1 cells were EBV-transformed immortalized LCLs generated in our laboratory (55).
Plasmids expressing full-length EBNA3C or its truncations such as 1-365, 366-620, and 621-992 with C-terminal Myc-tag have been previously described (56).HA-tagged full-length BCL6 were kindly provided by Dr. Riccardo Dalla-Favera (Columbia University, New York, USA).Plasmid expressing the full-length FBXO11 was cloned by using Kpn1 and BamH1 restriction enzyme in the pA3F vector as previously described (56).

Immunoblotting
Cells were lysed with RIPA buffer (10 mM Tris, pH 7.5, 1% Nonidet-P40, 2 mM EDTA, and 150 mM NaCl plus protease inhibitors), and protein concentration was determined using Bradford assays.The lysates were analyzed by Western blots using the appropri ate primary antibodies and conjugated fluorescent secondary antibodies.The results were scanned with an Odyssey Infrared scanner.Densitometry analysis was performed with the Odyssey V3.0 software.The western blots were repeated three times, and the quantification is a representation of one of the assays based on densitometry.

Confocal microscopy
Saos-2 cells were cultivated in 12-well plates on coverslips and then transfected with the specified plasmids.After 24 hours of transfection, the cells were washed three times with ice-cold PBS and fixed using 4% paraformaldehyde.B cells, totaling 2 million, were harvested, washed with PBS, and air-dried.These cells were also fixed with 4% paraformaldehyde at room temperature for 20 minutes.Subsequently, the fixed cells were washed with PBS and permeabilized with 0.2% Triton X-100 for 20 minutes at room temperature.To block nonspecific binding, the cells were treated with 3% bovine serum albumin for 0.5 hour at room temperature.Next, the cells were incubated overnight at 4°C with either rabbit anti-EBNA3C antibody, mouse anti-FBXO11 antibody, or mouse anti-Flag antibody.Following this incubation, the cells were washed three times with PBS and then incubated with secondary antibodies: Alexa Fluor 594 goat anti-rabbit (ThermoFisher; 1:250) or Alexa Fluor 488 goat anti-mouse (ThermoFisher 1:250) for 1 hour at room temperature.To visualize the nuclei, the cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for 10 minutes.After another three washes with PBS, the coverslips were flipped over and placed on a glass slide with a drop of mounting media.Confocal images were captured using a Fluoview FV300 confocal microscope.The images were edited in ImageJ, and the co-localization was calculated using Pearson's coefficient (r) and was presented in the figure."r" value ranges from 0 to 1, where 0 represents no co-localization, while 1 represents perfect co-localization (17).

Colony formation assay
Saos-2 cells and HEK293 in 6-well plates were transfected with GFP, control vector, Flag-FBXO11, Flag-FBXO11mut alone, or together with Myc-EBNA3C.The transfected cells were selected in DMEM with 2 mg/mL G418 (Sigma-Aldrich, St. Louis, MO, USA).Two weeks later, 4% paraformaldehyde was used to fix the cell colonies at room temperature for 30 minutes and stained with 0.1% crystal violet for 0.5 hour.The colonies were scanned by BioRAD ChemiDOC MP Imaging system, and the relative colony number was measured by Image J software.All assays were repeated three times for reproducibility.

Generation of lentiviral particles
ShControl and shFBXO11 lentiviruses were produced through the transfection of HEK293T cells with transfer plasmids, along with third-generation packaging and envelop plasmids, following previously described protocols (57,58).Briefly, HEK293T cells were cultured in 10 cm cell culture dishes until reaching 40%-60% confluency.Transfec tion involved the use of 10 µg of transfer plasmids along with packaging and envelop plasmids, employing the calcium phosphate method.After discarding the initial culture medium containing the transfection mix, the supernatants were collected at 12-hour intervals between 24 and 96 hours.These supernatants were then filtered through a 0.45 µm syringe filter, and lentiviruses were concentrated via ultracentrifugation at 23,500 rpm for 2 hours (59).The resulting pelleted lentiviruses were resuspended in 1 mL of complete medium and stored frozen until needed for transduction.Transduction was carried out by mixing cells with the resuspended lentiviral stock in the presence of 8 µg/mL polybrene.Cells were subjected to selection with 2 µg/mL puromycin 48 hours post-transduction.

Soft agar assays
The soft agar assays were performed in FBXO11 knocked down BJAB or LCL1 cells.One milliliter of 0.5% agar supplemented with RPMI media was poured into a 6-well plate and set aside to solidify. 1 × 10 5 cells were mixed with 0.5 mL of 0.3% agar/medium and poured on top of the 0.5% agar layer.Two weeks later, colonies were stained with 0.005% crystal violet overnight and scanned using the BioRAD ChemiDOC MP Imaging system.The relative colony numbers were measured by Image J software.All assays were repeated three times for reproducibility.

Flow cytometry
Flow cytometry was performed with FBXO11 knocked down BJAB or LCL1 cells to detect the cell cycle and DNA content.One million stable cells were collected and suspended with 300 µL of PBS containing 2% FBS.Then, the cells were fixed with 1 mL ice-cold 100% ethanol for 24 hours at 4°C.After washing with PBS containing 2% FBS once, the cells were stained with PI staining buffer (0.5 mg/mL propidium iodide in PBS and 50 µg/mL RNase A) for 30 minutes at room temperature and analyzed using a FACS Calibur (BD LSR II Special Order System, USA) and FlowJo software (Treestar, Inc., San Carlos, CA, USA).

Statistical analysis
Each experiment was repeated at least three times.The mean scores were examined by using Student's t-test.All statistical tests were performed using Microsoft Office Excel.A P-value of 0.05 was considered to be a statistically significant difference.A P-value of 0.01 indicated high statistical significance.

FIG 1
FIG 1 BCL6 level is reduced in latency III EBV-positive cells.(A) Fifteen million BJAB, LCL1, Mutu I, Sav I, and Kem I cells and corresponding latency III EBV-positive cells and (B) eight latent genes (EBNAs 1, 2, 3A, 3B, 3C, EBNA-LP, LMP1, and LMP2A) expressing stable BJAB cells were harvested and lysed with RIPA buffer.The expression levels of E3C, FBXO11, and BCL6 were detected by western blot.The relative density (RD) of BCL6 was quantitated for a representive blot and shown.

FIG 2
FIG 2 BCL6 levels are decreased in the presence of EBNA3C.Half a million Saos-2 cells were transfected with HA-BCL6 and (A) an increasing amount of Myc-tagged EBNA3C (10 and 15 µg) alone or together with Flag-FBXO11, (B) Myc-tagged EBNA3C (15 µg), Flag-FBXO11, and Flag-FBXO11mut separately or together.Total amounts of plasmids were kept constant by co-transfecting with the vector.At 48 hours post-transfection, the cells were harvested and lysed with RIPA buffer.The expression levels of E3C, FBXO11, and BCL6 were detected by western blot.The relative density (RD) of BCL6 was quantitated for a representative blot and shown.

FIG 3 (
FIG 3 (Continued) immunoprecipitated samples fractionated, and specific signals were detected by western blot by using antibodies against Flag and Myc.(B) EBNA3C associated with endogenous FBXO11.Sixty million BJAB, BJAB7, and LCL1 were collected and lysed for immunoprecipitation with 2 µg anti-FBXO11 antibody.Western blot was used to detect specific signals in the inputs and immunoprecipitated samples.(C) The schematic diagram summarizes the binding domains between different regions of EBNA3C.Jκ, RBP-Jκ; LZ, leucine zipper domain; AD, acidic domains; P rich, proline-rich; Q rich, glutamine-proline-rich; and NLS, nuclear localization signal.(D and E) The N terminus of EBNA3C is critical for EBNA3C and FBXO11 interaction.Ten million Saos-2 cells were transfected with Flag-tagged FBXO11 alone or together with Myc-tagged full-length EBNA3C or EBNA3C truncated mutants.At 48 hours post-transfection, the cells were harvested and lysed for immunoprecipitation with 2 µg anti-Flag antibody.The input and immunoprecipitated samples were resolved using 8% polyacrylamide (D) and 12%polyacrylamide (E) and blotted using specific antibodies.

FIG 4 Full
FIG 4 Co-localization of EBNA3C and FBXO11 in human cells.(A) EBNA3C co-localized with FBXO11 in 293T and Saos-2 cells.A total of 0.1 million Saos-2 cells were plated on coverslips and transfected with Myc-tagged EBNA3C and Flag-tagged FBXO11.At 24 hours post-transfection, cells were subjected to immunofluorescence assays.(B) EBNA3C co-localized with endogenous FBXO11 in B cells.BJAB, BJAB7, and LCL1 cells were plated on the slide and air-dried.The cells were fixed and subjected to immunofluorescence assays as described in Materials and Methods.The colocalization between the green and red channels was calculated using Pearson's coefficient (r) in ImageJ software (17).

FIG 5
FIG 5 EBNA3C and FBXO11 promote cell proliferation.(A and B) Saos-2 cells were transfected with the indicated plasmids and eGFP.The cells were selected with G418 (neomycin) antibiotics for 2 weeks.The cells were fixed, and the cell colonies were stained with 0.1% crystal violet.The relative colony number was measured by Image J software.(C) 5 × 10 5 selected cells were plated and cultured for 6 days.Viable cells were counted every day using trypan blue staining.

FIG 6
FIG 6 EBNA3C mediates BCL6 degradation through the ubiquitin pathway.EBNA3C enhanced poly-ubiquitination of BCL2.Ten million Saos-2 cells were transfected with the indicated constructs.At 24 hours post-transfection, cells were incubated with 10 µM MG132 for another 16 hours.Then, the cells were harvested and subjected to immunoprecipitation using an antibody against BCL6.The input and immunoprecipitated samples were detected by western blot.

FIG 7 (
FIG 7 (Continued) and FBXO11 mRNA expression was detected using real-time PCR.(C) FBXO11 knocked down BJAB and LCL1 cells were constructed by lentivirus transduction and selected by puromycin for 3 weeks.GFP fluorescence was determined in the selected cells.(D and E) Colony formation was measured in FBXO11 knocked down BJAB and LCL1 cells by soft agar assays.The relative colony number was measured by Image J software.(F and G) FBXO11 knocked down BJAB and LCL1 cells were stained with PI staining buffer and analyzed by flow cytometry.