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The Epstein-Barr virus (EBV) episome is known to interact with the three-dimensional structure of the human genome in infected cells. However, the exact locations of these interactions and their potential functional consequences remain unclear. Recently, high-resolution chromatin conformation capture (Hi-C) assays in lymphoblastoid cells have become available, enabling us to precisely map the contacts between the EBV episome(s) and the human host genome. Using available Hi-C data at a 10-kb resolution, we have identified 15,000 reproducible contacts between EBV episome(s) and the human genome. These contacts are highly enriched in chromatin regions denoted by typical or super enhancers and active markers, including histone H3K27ac and H3K4me1. Additionally, these contacts are highly enriched at loci bound by host transcription factors that regulate B cell growth (e.g., IKZF1 and RUNX3), factors that enhance cell proliferation (e.g., HDGF), or factors that promote viral replication (e.g., NBS1 and NFIC). EBV contacts show nearly 2-fold enrichment in host regions bound by EBV nuclear antigen 2 (EBNA2) and EBNA3 transcription factors. Circular chromosome conformation capture followed by sequencing (4C-seq) using the EBV origin of plasmid replication (oriP) as a “bait” in lymphoblastoid cells further confirmed contacts with active chromatin regions. Collectively, our analysis supports interactions between EBV episome(s) and active regions of the human genome in lymphoblastoid cells. IMPORTANCE EBV is associated with ∼200,000 cancers each year. In vitro, EBV can transform primary human B lymphocytes into immortalized cell lines. EBV-encoded proteins, along with noncoding RNAs and microRNAs, hijack cellular proteins and pathways to control cell growth. EBV nuclear proteins usurp normal transcriptional programs to activate the expression of key oncogenes, including MYC, to provide a proliferation signal. EBV nuclear antigens also repress CDKN2A to suppress senescence. EBV membrane protein activates NF-κB to provide survival signals. EBV genomes are maintained by EBNA1, which tethers EBV episomes to the host chromosomes during mitosis. However, little is known about where EBV episomes are located in interphase cells. In interphase cells, EBV promoters drive the expression of latency genes, while oriP functions as an enhancer for these promoters. In this study, integrative analyses of published lymphoblastoid cell line (LCL) Hi-C data and our 4C-seq experiments position EBV episomes to host genomes with active epigenetic marks. These contact points were significantly enriched for super enhancers. The close proximity of EBV episomes and the super enhancers that are enriched for transcription cofactors or mediators in lymphoblasts may benefit EBV gene expression, suggesting a novel mechanism of transcriptional activation.

Epstein-Barr virus (EBV) infection is highly prevalent in humans and is implicated in various diseases, including cancer1,2. Chronic active EBV infection (CAEBV) is an intractable disease classified as a lymphoproliferative disorder in the 2016 World Health Organization lymphoma classification1,2. CAEBV is characterized by EBV-infected T/natural killer (NK) cells and recurrent/persistent infectious mononucleosis-like symptoms3. Here, we show that CAEBV originates from an EBV-infected lymphoid progenitor that acquires DDX3X and other mutations, causing clonal evolution comprising multiple cell lineages. Conspicuously, the EBV genome in CAEBV patients harboured frequent intragenic deletions (27/77) that were also common in various EBV-associated neoplastic disorders (28/61), including extranodal NK/T-cell lymphoma and EBV-positive diffuse large B-cell lymphoma, but were not detected in infectious mononucleosis or post-transplant lymphoproliferative disorders (0/47), which suggests a unique role of these mutations in neoplastic proliferation of EBV-infected cells. These deletions frequently affected BamHI A rightward transcript microRNA clusters (31 cases) and several genes that are essential for producing viral particles (20 cases). The deletions observed in our study are thought to reactivate the lytic cycle by upregulating the expression of two immediate early genes, BZLF1 and BRLF14-7, while averting viral production and subsequent cell lysis. In fact, the deletion of one of the essential genes, BALF5, resulted in upregulation of the lytic cycle and the promotion of lymphomagenesis in a xenograft model. Our findings highlight a pathogenic link between intragenic EBV deletions and EBV-associated neoplastic proliferations.

Epstein-Barr virus (EBV) was discovered as the first human tumor virus in endemic Burkitt lymphoma, the most common childhood cancer in sub-Saharan Africa. In Burkitt lymphoma and in 200,000 EBV-associated cancers per year, epigenetic mechanisms maintain viral latency, during which lytic cycle factors are silenced. This property complicated EBV’s discovery and facilitates tumor immunoevasion. DNA methylation and chromatin-based mechanisms contribute to lytic gene silencing. Here, we identified histone chaperones CAF1 and HIRA, which have key roles in host DNA replication-dependent and replication-independent pathways, respectively, as important for EBV latency. EBV strongly upregulates CAF1 in newly infected B-cells, where viral genomes acquire histone 3.1 and 3.3 variants prior to the first mitosis. Since histone chaperones ATRX and DAXX also function in maintenance of EBV latency, our results suggest that EBV coopts multiple histone pathways to reprogram viral genomes and highlight targets for lytic induction therapeutic strategies., Epstein-Barr virus (EBV) infects 95% of adults worldwide and causes infectious mononucleosis. EBV is associated with endemic Burkitt lymphoma, Hodgkin lymphoma, posttransplant lymphomas, nasopharyngeal and gastric carcinomas. In these cancers and in most infected B-cells, EBV maintains a state of latency, where nearly 80 lytic cycle antigens are epigenetically suppressed. To gain insights into host epigenetic factors necessary for EBV latency, we recently performed a human genome-wide CRISPR screen that identified the chromatin assembly factor CAF1 as a putative Burkitt latency maintenance factor. CAF1 loads histones H3 and H4 onto newly synthesized host DNA, though its roles in EBV genome chromatin assembly are uncharacterized. Here, we found that CAF1 depletion triggered lytic reactivation and virion secretion from Burkitt cells, despite also strongly inducing interferon-stimulated genes. CAF1 perturbation diminished occupancy of histones 3.1 and 3.3 and of repressive histone 3 lysine 9 and 27 trimethyl (H3K9me3 and H3K27me3) marks at multiple viral genome lytic cycle regulatory elements. Suggestive of an early role in establishment of latency, EBV strongly upregulated CAF1 expression in newly infected primary human B-cells prior to the first mitosis, and histone 3.1 and 3.3 were loaded on the EBV genome by this time point. Knockout of CAF1 subunit CHAF1B impaired establishment of latency in newly EBV-infected Burkitt cells. A nonredundant latency maintenance role was also identified for the DNA synthesis-independent histone 3.3 loader histone regulatory homologue A (HIRA). Since EBV latency also requires histone chaperones alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX) and death domain-associated protein (DAXX), EBV coopts multiple host histone pathways to maintain latency, and these are potential targets for lytic induction therapeutic approaches.

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.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that causes infectious mononucleosis and several malignancies, such as endemic Burkitt lymphoma and nasopharyngeal carcinoma. Herpesviruses carry genes that can modify cell functions, including transcription and ubiquitination, thereby facilitating viral growth and survival in infected cells. Using a reporter screening system, we revealed the involvement of several EBV gene products in such processes. Of these, BGLF2 activated the AP-1 signaling pathway through phosphorylation of p38 and c-Jun N-terminal kinase (JNK). Knockout of the BGLF2 gene did not affect viral gene expression and viral genome DNA replication, but resulted in marked reduction of progeny titer. We also found that the BGLF2 disruption resulted in significant loss of infectivity upon de novo infection. Interestingly, expression of a binding partner, BKRF4, repressed the activation of AP-1 by BGLF2. These results shed light on the physiological role of the tegument protein BGLF2. IMPORTANCE Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, carries ~80 genes. While several genes have been investigated extensively, most lytic genes remain largely unexplored. Therefore, we cloned 71 EBV lytic genes into an expression vector and used reporter assays to screen for factors that activate signal transduction pathways, viral and cellular promoters. BGLF2 activated the AP-1 signaling pathway, likely by interacting with p38 and c-Jun N-terminal kinase (JNK), and increased infectivity of the virus. We also revealed that BKRF4 can negatively regulate AP-1 activity. Therefore, it is suggested that EBV exploits and modifies the AP-1 signaling pathway for its replication and survival.

The Epstein–Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, and occasionally switches from the latent state to the lytic cycle. In this report, we identified and examined the role of a lytic gene, BRRF2. We first prepared an antibody against BRRF2 and identified the gene product as a viral lytic protein expressed in B95-8 cells with late kinetics. Immunofluorescence revealed that BRRF2 localized in the cytoplasm of cells during the lytic phase. We also found that BRRF2 protein was phosphorylated in lytic cells, but the only viral protein kinase, BGLF4, was not involved in the phosphorylation. Knockout EBV and a repaired strain were then prepared, and we found that BRRF2 disruption did not affect viral gene expression and DNA replication, but decreased virus production. These results demonstrated that BRRF2 is involved in production of infectious progeny, although it is not essential for lytic replication.

Latent membrane protein 1 (LMP1) is a major oncogene encoded by Epstein-Barr virus (EBV) and is essential for immortalization of B cells by the virus. Previous studies suggested that several transcription factors, such as PU.1, RBP-Jκ, NFκB, EBF1, AP-2 and STAT, are involved in LMP1 induction; however, the means by which the oncogene is negatively regulated remains unclear. Here, we introduced short mutations into the proximal LMP1 promoter that includes recognition sites for the E-box and Ikaros transcription factors in the context of EBV-bacterial artificial chromosome. Upon infection, the mutant exhibited increased LMP1 expression and EBV-mediated immortalization of B cells. However, single mutations of either the E-box or Ikaros sites had limited effects on LMP1 expression and transformation. Our results suggest that this region contains a suppressive cis-regulatory element, but other transcriptional repressors (apart from the E-box and Ikaros transcription factors) may remain to be discovered.

Epstein-Barr virus (EBV) transforms B cells to continuously proliferating lymphoblastoid cell lines (LCLs), which represent an experimental model for EBV-associated cancers. EBV nuclear antigens (EBNAs) and LMP1 are EBV transcriptional regulators that are essential for LCL establishment, proliferation, and survival. Starting with the 3D genome organization map of LCL, we constructed a comprehensive EBV regulome encompassing 1,992 viral/cellular genes and enhancers. Approximately 30% of genes essential for LCL growth were linked to EBV enhancers. Deleting EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets included MCL1, IRF4, and EBF. MYC ESE looping to the transcriptional stat site of MYC was dependent on EBNAs. Deleting MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study provides a comprehensive view of the spatial organization of chromatin during EBV-driven cellular transformation.

Reactivation of Epstein-Barr virus (EBV) from latency is dependent on expression of the viral transactivator BZLF1 protein, whose promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers. Using a reporter assay system, we screened for factors that can activate Zp and isolated genes, including those encoding MEF2B, KLF4, and some cellular b-Zip family transcription factors. After confirming their importance and functional binding sites in reporter assays, we prepared recombinant EBV-BAC, in which the binding sites were mutated. Interestingly, the MEF2 mutant virus produced very low levels of BRLF1, another transactivator of EBV, in addition to BZLF1 in HEK293 cells. The virus failed to induce a subset of early genes, such as that encoding BALF5, upon lytic induction, and accordingly, could not replicate to produce progeny viruses in HEK293 cells, but this restriction could be completely lifted by exogenous supply of BRLF1, together with BZLF1. In B cells, induction of BZLF1 by chemical inducers was inhibited by point mutations in the ZII or the three SP1/KLF binding sites of EBV-BAC Zp, while leaky BZLF1 expression was less affected. Mutation of MEF2 sites severely impaired both spontaneous and induced expression of not only BZLF1, but also BRLF1 in comparison to wild-type or revertant virus cases. We also observed that MEF2 mutant EBV featured relatively high repressive histone methylation, such as H3K27me3, but CpG DNA methylation levels were comparable around Zp and the BRLF1 promoter (Rp). These findings shed light on BZLF1 expression and EBV reactivation from latency.

Summary Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication., Graphical Abstract, Highlights • Unbiased global analysis of host and EBV proteome remodeling in lytic replication • Temporal profiles of >8,000 host and 69 viral proteins, using type I and II EBV • Both EBV types target the B cell receptor complex for degradation • Conserved EBV and HCMV lytic cycle host targets are identified, Ersing et al. present a temporal proteomic map of EBV B cell lytic replication. Tandem-mass-tag-based proteomics uncover extensive remodeling of the human proteome by EBV, conserved across the two major EBV strains. Cell-cycle, innate, and adaptive immune pathways are modulated, complement is upregulated, and the B cell receptor is degraded by infection.

The Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with several malignancies. It establishes a latent infection in B lymphocytes and is occasionally reactivated to enter the lytic cycle. Here we examined the role of the EBV gene BRRF1, which is expressed in the lytic state. We first confirmed, using a DNA polymerase inhibitor, that the BRRF1 gene is expressed with early kinetics. A BRRF1-deficient recombinant virus was constructed using a bacterial artificial chromosome system. No obvious differences were observed between the wild-type, BRRF1-deficient mutant and the revertant virus in HEK293 cells in terms of viral lytic protein expression, viral DNA synthesis, progeny production, pre-latent abortive lytic gene expression and transformation of primary B cells. However, reporter assays indicated that BRRF1 may activate transcription in promoter- and cell type-dependent manners. Taken together, BRRF1 is dispensable for viral replication in HEK293 cells and transformation of B cells, but it may have effects on transcription.

In the version of this Letter originally published, in the sentence beginning "The major driver role of DDX3X mutations...", the citation "Fig. 2a-f" should have been "Fig. 2". In addition, in the sentence beginning "Another finding of interest was the presence of identical driver mutations...", the citation "Fig. 3a,b and Fig. 4" should have been "Fig. 3". This has now been amended in all versions of the Letter.

Introduction Epstein-Barr virus (EBV) is a double-stranded DNA virus that infects >95% of the human population and is associated with a substantial risk of cancer development. Most infections in children and adolescents are asymptomatic or result in infectious mononucleosis; however, in some patients, EBV is associated with various hematological malignancies including Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), and extranodal NK/T-cell lymphoma. EBV infection is also present in a portion of epithelial cell neoplasms such as gastric cancer and nasopharyngeal carcinoma. Despite the large population risk of cancer associated with EBV, it is poorly understood why only a small subset of EBV-infected individuals develop neoplasms, while others do not. Patients and Methods We designed a target enrichment system to capture several EBV strains including the Akata strain, which is responsible for the majority of EBV infections in Japan. We analyzed the genomes of EBV strains in 139 patients with various EBV-associated diseases and 17 EBV-positive cell lines. Next-generation sequencing reads were aligned to the Akata reference genome to analyze nucleotide variations, copy number alterations, and structural variations including sequence insertions in the human genome. The institutional review board of Nagoya University Graduate School of Medicine approved this study. Results We identified a median of 645 single nucleotide variants (SNVs) in the EBV genomes, 78% of which affected coding sequences. SNVs in coding sequences were significantly biased toward synonymous variants, suggesting negative selection pressure. The SNVs detected in noncoding sequences were enriched in two evolutionarily conserved viral noncoding RNAs (EBER1 and EBER2), particularly in the PAX5-binding domain of EBER2. However, most SNVs identified in the EBV genome do not seem to affect the development of neoplasms, as hierarchical clustering of EBV genomes from neoplastic and non-neoplastic diseases based on SNVs revealed no significant association between the EBV strain and disease type. In addition to SNVs, we identified frequent intragenic deletions in the EBV genomes of patients with EBV-positive DLBCL (10/14, 71%), extranodal NK/T-cell lymphoma (10/23, 43%), chronic active EBV infection (27/77, 35%), and other EBV-associated neoplasms (2/7). Such deletions were also identified in several EBV-associated cell lines (6/17), but not in non-neoplastic diseases such as infectious mononucleosis (0/4) and post-transplant lymphoproliferative disorders (0/14), suggesting a unique role of these mutations in the neoplastic proliferation of EBV-infected cells. Frequent deletions were detected in BamHI A rightward transcripts microRNA clusters (31/156), which suppress viral transcription factors (BZLF1 and BRLF1) required for the lytic reactivation of EBV. Deletions also were associated with several genes essential for virus production (20/156). These observed deletions are thought to upregulate lytic cycle-associated genes, some of which benefit neoplasms by inducing genomic instability and immune escape and mitigate cell damage caused by the production of viral particles. In fact, deletion of one essential gene, BALF5, resulted in upregulation of the lytic cycle and promotion of lymphomagenesis in a xenograft model. Discussion Although the essential roles of several latency-associated genes, such as LMP-1 and EBNA-2, in EBV-mediated immortalization and transformation of human lymphocytes have long been discussed, our finding raises the possibility that lytic cycle-associated genes also contribute to lymphomagenesis. This agrees with reports that lytic cycle-associated genes are expressed in Burkitt lymphoma, DLBCL, and chronic active EBV infection, and that BZLF1-deficient lymphoblastoid cells exhibit significantly impaired tumorigenicity in mice. In addition, essential gene deletions lead to the protection of EBV-infected cells from lysis. Further studies are warranted to exploit these findings for the design of novel therapeutics for EBV-associated neoplasms. Disclosures Kiyoi: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria. Nakamura:Roche/Chugai,: Research Funding; Kyowa-Kirin: Research Funding.

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, but a small percentage of infected cells switch from the latent state to the lytic cycle, leading to potent viral DNA replication and progeny viruses production. We here focused on a lytic gene BGLF3.5, and first established BGLF3.5 mutants by marker cassette insertion. Unexpectedly, this insertion mutant failed to produce BGLF4 protein and thus progeny production was severely inhibited. Then we carefully made two point mutant viruses (stop codon insertion or frame-shift mutation) and found that BGLF3.5 is not essential for EBV lytic replication processes, such as viral gene expression, DNA replication, or progeny production in the HEK293 cells although its homolog in murine gammaherpesvirus 68 (MHV-68) was reported to be essential. In addition, we examined the roles of two short, upstream open reading frames within the 5′UTR of BGLF3.5 gene in translation of BGLF4.

Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication.