Chih-Wen Peng

Four EBNA2 Domains Are Important for EBNALP Coactivation

ABSTRACT EBNA2 transcriptional activation and regulated EBNALP coactivation are critical for Epstein-Barr virus-infected primary B-lymphocyte growth transformation. EBNALP coactivation requires the EBNA2 acidic activation domain (E2AD); EBNALP can bind to E2AD. EBNALP has now been found to bind less well to EBNA2 amino acids 1 to 58, which has been identified to be a second transcriptional activation domain, E2AD2. E2AD2 was specifically coactivated by EBNALP. Moreover, E2AD, E2AD2, EBNA2 RG domain, and the intermediate domain between RG and E2AD had significant roles in EBNA2-mediated activation and EBNALP coactivation.

Nucleolin is important for Epstein–Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription

Significance Epstein–Barr Virus (EBV) genome persistence and expression in human cells can result in human lymphocyte or epithelial cell malignancies. EBV genome persistence and gene expression depends on the EBV encoded Epstein–Barr virus nuclear antigen 1 (EBNA1) protein binding to cognate sites in EBV episomes. Search for a cell protein necessary for EBNA1 interaction with EBV episomes identified a low-level nucleolin (NCL) association with EBNA1. Despite low-level stable association, NCL shRNA knockdowns revealed wild-type NCL, but not an NCL ATP binding site point mutant to be critical for EBNA1 binding to EBV episomes, transcription, and genome maintenance. These experiments identify NCL and NCL K429 as potential targets for inhibition of EBNA1’s role in EBV oncogenesis. Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1s N-terminal 100 aa and NCLs RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.

EGCG debilitates the persistence of EBV latency by reducing the DNA binding potency of nuclear antigen 1

Because the expression of EBNA1 is prevalent in all EBV-associated tumors, it has become one of the most attractive drug targets for the discovery of anti-EBV compounds. In a cell-based reporter system, EBNA1 consistently upregulated the transcription of an oriP-Luc mini-EBV episome by 6- to 8-fold. The treatment of cells with 50 μM EGCG effectively blocked the binding of EBNA1 to oriP-DNA both in vivo and in vitro, which led to the abrogation of EBNA1-dependent episome maintenance and transcriptional enhancement. Importantly, the anti-EBNA1 effects caused by EGCG ultimately impaired the persistence of EBV latent infection. Our data suggest that the inhibition of EBNA1 activity by EGCG could be a promising starting point for the development of new protocols for anti-EBV therapy.

Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

Epstein-Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine-Glycine repeat (RG) domain at amino acid positions 335-360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2.

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.

Impact of cuticle photoluminescence on the color morphism of a male damselfly Ischnura senegalensis (Rambur, 1842)

In this study the damselfly Ischnura senegalensis (Rambur, 1842) was first found to produce strong photoluminescence (PL) emissions from various colored-body portions, such as the eighth abdominal segment of the tail. The colors of the colored-body portions can be enhanced or modified by the PL emissions for assistance in reducing intrasexual and male harassment, and improving mature mating and conspecific identity. Therefore, the PL emissions that contribute to the color modification and coloration are involved in the cuticle evolution of the damselflies. The micro-PL confocal images verify that the PL emissions can strongly influence the surface colors of the cuticle, and demonstrate why the damselfly Ischnura senegalensis is called a bluetail.

Ribosome Protein L4 is essential for Epstein-Barr Virus Nuclear Antigen 1 function

Significance Epstein–Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) is essential for establishing long-term EBV infection in human cells and is implicated in EBV-related malignancies. The maintenance of the EBV genome and viral gene expression from the episome are specifically driven by EBNA1 through binding to origin of plasmid replication (oriP). This study finds that EBNA1 complexes with Ribosome Protein L4 (RPL4) and Nucleolin to stabilize EBNA1 binding to oriP. Cooperation of RPL4’s N terminus with Nucleolin K429 is necessary for EBNA1 oriP binding and episome maintenance, whereas RPL4s C-terminal K380 and K393 induce H3K4me2, which promotes EBNA1 transactivation of oriP. These discoveries enhance our understanding of the complexity of EBNA1’s interactions with host proteins and EBV episomes and reveal new targets for inhibition of EBV genome persistence. Epstein–Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection.

Epstein-Barr Virus Nuclear Antigen Leader protein co-activates EP300

ABSTRACT Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs. IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.