The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation

The Epstein-Barr Virus LF2 Protein Inhibits Viral Replication▿

The switch from Epstein-Barr virus (EBV) latent infection to lytic replication is governed by two transcriptional regulators, Zta and Rta. We previously reported that the EBV protein encoded by the LF2 gene binds to Rta and can inhibit Rta activity in reporter gene assays. We now report that LF2 associates with Rta in the context of EBV-infected cells induced for lytic replication. LF2 inhibition of Rta occurs in both epithelial and B cells, and this downregulation is promoter specific: LF2 decreases Rta activation of the BALF2, BMLF1, and BMRF1 promoters by 60 to 90% but does not significantly decrease Rta activation of its own promoter (Rp). LF2 decreases Rta activation by at least two mechanisms: decreased DNA binding and interference with transcriptional activation by the Rta acidic activation domain. Coexpression of LF2 also specifically induces modification of Rta by the small ubiquitin-like modifiers SUMO2 and SUMO3. We further demonstrate that LF2 overexpression blocks lytic activation in EBV-infected cells induced with Rta or Zta. Our results demonstrate that LF2, a gene deleted from the EBV reference strain B95-8, encodes a potent inhibitor of EBV replication, and they suggest that future studies of EBV replication need to account for the potential effects of LF2 on Rta activity

EBNA3C associates with the WDR48/USP46 complex in EBNA3C-F-HA LCLs.

<p>Immunoprecipitation assay using Flag agarose to retrieve protein complexes from EBNA3C-F-HA LCLs (E3C-F-HA) is compared to flag immunoprecipiates from untagged wildtype (WT) LCLs. One percent of total cell lysate (Input) or immunoprepicitated specimens using Flag agarose (Flag IP) were separated by SDS PAGE and probed using antibodies to EBNA3C, RBPJ, CtBP1, WDR48, WDR20, USP46, or NF-kB p65.</p

Epstein-Barr Virus Nuclear Protein 3C Domains Necessary for Lymphoblastoid Cell Growth: Interaction with RBP-Jκ Regulates TCL1▿

B lymphocytes converted into lymphoblastoid cell lines (LCLs) by an Epstein-Barr virus that expresses a conditional EBNA3C require complementation with EBNA3C for growth under nonpermissive conditions. Complementation with relatively large EBNA3C deletion mutants identified amino acids (aa) 1 to 506 (which includes the RBP-Jκ/CSL [RBP-Jκ] binding domain) and 733 to 909 to be essential for LCL growth, aa 728 to 732 and 910 to 992 to be important for full wild-type (wt) growth, and only aa 507 to 727 to be unimportant (S. Maruo, Y. Wu, T. Ito, T. Kanda, E. D. Kieff, and K. Takada, Proc. Natl. Acad. Sci. USA 106:4419-4424, 2009). When mutants with smaller deletions were used, only aa 51 to 400 and 851 to 900 were essential for LCL growth; aa 447 to 544, 701 to 750, 801 to 850, and 901 to 992 were important for full wt growth; and aa 4 to 50, 401 to 450, 550 to 707, and 751 to 800 were unimportant. These data reduce the EBNA3C essential residues from 68% to 40% of the open reading frame. Point mutations confirmed RBP-Jκ binding to be essential for wt growth and indicated that SUMO and CtBP binding interactions were important only for full wt growth. EBNA3C aa 51 to 150, 249 to 311, and 851 to 900 were necessary for maintaining LCL growth, but not RBP-Jκ interaction, and likely mediate interactions with other key cell proteins. Moreover, all mutants null for LCL growth had fewer S+G2/M-phase cells at 14 days, consistent with EBNA3C interaction with RBP-Jκ as well as aa 51 to 150, 249 to 311, and 851 to 900 being required to suppress p16INK4A (S. Maruo, Y. Wu, S. Ishikawa, T. Kanda, D. Iwakiri, and K. Takada, Proc. Natl. Acad. Sci. USA 103:19500-19505, 2006). We have confirmed that EBNA3C upregulates TCL1 and discovered that EBNA3C upregulates TCL1 through RBP-Jκ, indicating a central role for EBNA3C interaction with RBP-Jκ in mediating cell gene transcription

Deletion of EBNA3A residues 920–944 disrupts WDR48 binding without affecting CtBP1 association.

<p>Co-immunoprecipitation assay to assess binding of EBNA3A mutants to WDR48 (A) and CtBP1 (B). For these assays, flag tagged full length EBNA3A (1–944), an EBNA3A CtBP1 binding mutant (mCtBP), an EBNA3A mutant lacking the C-terminal 25 residues (1–919), or vector control (pSG5) was co-transfected with Xpress-WDR48 or HA-CtBP1. Lysates were immunoprecipitated with Flag agarose (A) or HA agarose (B), separated by SDS PAGE, and probed with WDR48, RBPJ, flag, EBNA3A and HA antibodies.</p

Summary of peptides identified by mass spec of tandem affinity purifications (TAP).

<p>Total number of peptides detected for each EBNA3 protein and the host proteins RBPJ, CtBP1, WDR48, WDR20, USP46 and USP12 in purified complexes is indicated. No peptides corresponding to any of these proteins were detected in the control TAP from wildtype LCLs.</p><p>Summary of peptides identified by mass spec of tandem affinity purifications (TAP).</p

WDR48 coimmunoprecipitates with RBPJ in EBV infected cells.

<p>Co-immunoprecipitation assays comparing the association of RBPJ with WDR48 in LCLs with that observed in EBV negative BL41 cells. Cell lysates were immunoprecipitated with polyclonal RBPJ sera, separated by SDS PAGE, and probed for EBNA3A, EBNA3C, WDR48, and RBPJ (as indicated).</p

ChIP assay for WDR48 at the p14ARF promoter.

<p>Chromatin immunoprecipitation (ChIP) assays were performed using antibodies for WDR48 (A) from EBNA3C-HT LCLs that were grown in the presence of 4HT (dark gray) or after 14 days of growth in the absence of 4HT (light gray). Amount of genomic DNA was measured by real time PCR using primers specific to the EBNA3C binding site in the p14^ARF promoters or sites near the EIF2AK3 and PPIA genes which bind cell transcription factors but not EBNA3C. The bar graph represents the amount of DNA precipitated relative to the amount of DNA in the corresponding input sample. The experiment shown is representative of four independent experiments and error bars indicating standard error of the mean within this experiment. Asterisk denotes that the difference in ChIP signal seen at the p14^ARF promoter is statistically significant (p = 0.01). (B) Western blot for USP46, WDR48, and tubulin levels in whole cell lysates from EBNA3CHT LCLs grown in the presence of 4HT or after 14 days of growth in the absence of 4HT.</p

Purified EBNA3 complexes exhibit DUB activity.

<p>(A) Tandem affinity purification was performed on wild type or EBNA3-F-HA expressing LCLs. Purified complexes from wild type (X), E3A-F-HA (closed circle), E3B-F-HA (closed triangle), or E3C-F-HA (closed square) LCLs were assayed for DUB activity by fluorometric assay using Ub-AMC as a substrate. (B) Western blot of purified EBNA3 complexes used in (A) demonstrating selective precipitation of tagged EBNA3 protein complexes and co-purification of RBPJ and USP46 proteins. Lysates derived from the equivalent of approximately 1.5x10⁶ cells (input), 2.4x10⁶ cells (Flag elution), or 10x10⁶ cells (HA elution) were loaded on each lane, separated by SDS PAGE, and probed with EBNA3s, Flag, EBNA3A, and HA antibodies.</p

EBNA3A1-919, which associates with CtBP1 but not WDR48, is impaired for LCL growth maintenance.

<p>(A) Transcomplementation assay comparing growth of EBNA3A-HT cells transfected with EBNA3A WT (closed diamond), EBNA3A 1–826 (open square), EBNA3A mCtBP1 (open diamond), EBNA3A mRBPJ (open triangle), or EBNA3A 1–919 (X) in the absence of 4HT. EBNA3A-HT cells were also transfected with a control GFP expression plasmid, split, and maintained in either the presence (closed square) or absence (open circle) of 4HT. Cells were counted every 3 to 4 days, and diluted in fresh media to maintain a concentration of 200,000 cells/mL. Based on dilution factors, total cell number was calculated and is plotted on the Y-axis versus time. (B) Wild type EBNA3A, not mutant EBNA3A, suppresses p16 expression level in trans-complemented cells. After 15 days of EBNA3A WT or EBNA3A mutant (1–826, mCtBP1, mRBPJ, or 1–919) transfection, cells were harvested and protein expression was detected by immunoblotting with p16 antibody and actin as an internal control. As a control experiment, GFP expression plasmid was transfected into the cells and cultured with or without 4HT for 15 days. The ration of the p16 and actin bands was quantified and is indicated in the bottom panel.</p