BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2

Abstract

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection.

Fig 1. Structural analysis of the BS69_CC-MYND domains in complex with the EBNA2_381–389 peptide.

(A) Domain architecture of BS69 and EBNA2. Regions responsible for EBNA-LP activation or RBP-Jk binding, transactivation domain (TAD) and nuclear localization sequence (NLS) of EBNA2 are labeled. Note that CR5 contributes to RBP-Jk binding only indirectly [16]. (B) Ribbon representation of the BS69_CC-MYND domains (coiled-coil: red; MYND: light blue) in complex with the EBNA2_381–389 peptide (yellow sticks). The zinc ions are shown in orange spheres. Selected intermolecular interactions from the coiled-coil domain are shown in the expanded view. (C) Structure of the BS69_CC-MYND-EBNA2_381–389 complex in a view different from (B). (D) Close-up view of the BS69_CC-MYND-EBNA2_381–389 interactions. The hydrogen bonds are shown in dashed line. The water molecules are shown in magenta spheres. Residue W536 from the neighboring MYND domain is labeled with an apostrophe mark.

Fig 2. (A-H) ITC analysis of BS69 wild type and mutants binding to the EBNA2 peptides.

In (A-G) EBNA2 peptides were titrated against BS69. In (H) BS69_CC-MYND was titrated against EBNA2_381–445. For the R560A mutant, the K _d value was estimated by fixing the parameter of the stoichiometric ratio (N) to 1.

Fig 3. Comparison of the BS69_MYND-EBNA2_381–389 interaction with those of other MYND domains.

(A) Structure-based sequence alignment of BS69_MYND with the MYND domains from other proteins. Residues Q546 and W550 of BS69_MYND, marked with red asterisk, were mutated for in vivo functional assay. The other EBNA2-interacting residues of BS69_MYND are marked with blue asterisks. (B-D) Surface view of the BS69_MYND with the EBNA2_381–389 peptide (B), the ETO_MYND domain in contact with SMRT_1101–1113 (C), and the DEAF1_MYND domain (D), with the protein interaction sites highlighted in magenta. EBNA2_381–389 P383, L385 and P387, and the corresponding residues in SMRT_1101–1113 are highlighted in blue in (B) and (C), respectively. Note that the BS69_MYND-EBNA2_381–389 interaction is enhanced by dimerization of the BS69 coiled-coil domain.

Fig 4. Expression and interaction analysis of BS69 and EBNA2 in EBV-infected cells.

(A) The relative mRNA expression of endogenous BS69 over GAPDH in different EBV-infected cell lines or B cells with EBV-infection at various days of post-infection (dpi) was identified by qRT-PCR. (B) Expression of endogenous BS69, EBNA2 and EBNA1 in B cells with EBV infection at 0, 1, 4, and 7 dpi, LCLs, and B lymphoma cells was analyzed by Western blotting using antibodies against BS69, EBNA2 and EBNA1, respectively. The images with short exposure (S.E.) versus long exposure (L.E.) were shown. The expression of β-actin was used as internal control. (L.E.) (C) Transfection-mediated Co-IP analysis was performed using BJAB cells co-transfected with the plasmids of flag-BS69_CC-MYND wild type, Q546A, Q546A/W550A and EBNA2 (E2). The anti-flag M2 antibody was used for immunoprecipitation of flag-BS69_CC-MYND wild type or Q546A. The slower migration of the Q546A/W550A band was due to the fact that its linker sequence bridging the flag-tag and BS69_CC-MYND is 13-amino acid longer than that of wild type or Q546A. (D) Resting primary B cells with the indicated dpi of EBV infection were used to evaluate the EBNA2 and BS69 expression and localization by confocal immunofluorescence microscopy. The nuclei were counterstained with DAPI (blue). Both individual and merge images were shown. EBNA2 was shown in green versus BS69 was shown in RED. Cells infected with EBV were indicated as (+) while uninfected cells were indicated as (-). The enlarged view of the marked regions were shown, respectively.

Fig 5. Recruitment of BS69_CC-MYND to EBNA2 target promoter through protein interactions leads to down regulation of EBNA2 dependent transcription.

(A) EBNA2 specific reporter plasmids, LMP1-Luc and Cp-Luc, EBNA1 specific oriP-Luc reporter plasmid, CMV-βGal internal control, and the indicated expression plasmids were subjected to a procedure of transfection-mediated transcription reporter assay. The effects of flag-BS69_CC-MYND wild type, Q546A or Q546A/W550A on EBNA2-mediated transcription were determined by the resulting luciferase activity corrected for β-gal activity. (B) The experimental design of the transfection-mediated ChIP assay was shown. M2-conjugated sepharose was used to precipitate flag-BS69_CC-MYND wild type, Q546A, or Q546A/W550A while H3ac was used to precipitated acetylated-H3. PE2 (EBNA2) ChIP was used to assay EBNA2 enrichment at transfected LMP1 DNA. IgG was used as negative control. The amount of ChIPed DNA was quantified by real time PCR. The enrichment of BS69_CC-MYND wild type, Q546A, or Q546A/W550A at the LMP1 promoter and the enrichment of H3ac at GAPAH promoter were represented as % of input DNA, respectively.

Fig 6. Induced expression of BS69_CC-MYND in LCLs leads to a strong debilitation of cell growth.

(A) The retroviral vector mediated inducible expression vector of DD-BS69_CC-MYND wild type, Q546A, or Q546A/W550A was used to transduce two LCLs, LCL#1 and LCL#2, and BJAB control cell lines. DD-BS69: DD-BS69 wild-type; DD-Q546: DD-BS69 Q546A; DD-BS69 Q/W: DD-BS69 Q546A/W550A (B) 10⁵ per mL of the above retrovirus transduced cell lines were aliquoted into 6-well plates and cell viability assays were conducted with the treatment of inducer (shield 1) or PBS (negative control) every 24 hrs for five consecutive days. The expression of actin was used internal control.