Human Tumor Virus Research Articles

An insect cell-derived extracellular vesicle-based gB vaccine elicits robust adaptive immune responses against Epstein-Barr virus.

Epstein-Barr virus (EBV), the first identified human tumor virus, is implicated in various human malignancies, infectious mononucleosis, and more recently, multiple sclerosis. Prophylactic vaccines have the potential to effectively prevent EBV infection. Glycoprotein B (gB) serves as the fusogen and plays a pivotal role in the virus entry process, making it a critical target for EBV vaccine development. Surface membrane proteins of enveloped viruses serve as native conformational antigens, making them susceptible to immune recognition. Utilizing lipid membrane-bound viral antigens is a promising strategy for effective vaccine presentation in this context. In this study, we employed a truncated design for gB proteins, observing that these truncated gB proteins prompted a substantial release of extracellular vesicles (EVs) in insect cells. We verified that EVs exhibited abundant gB proteins, displaying the typical virus particle morphology and extracellular vesicle characteristics. gB EVs demonstrated a more efficient humoral and cellular immune response compared with the gB ectodomain trimer vaccine in mice. Moreover, the antisera induced by the gB EVs vaccine exhibited robust antibody-dependent cytotoxicity. Consequently, gB EVs-based vaccines hold significant potential for preventing EBV infection and offer valuable insights for vaccine design.

Metabolic regulation of the tumour and its microenvironment: The role of Epstein-Barr virus.

The Epstein-Barr virus (EBV), the first identified human tumour virus, infects over 95% of the individuals globally and has the potential to induce different types of cancers. It is increasingly recognised that EBV infection not only alters cellular metabolism, contributing to neoplastic transformation, but also utilises several non-cell autonomous mechanisms to shape the metabolic milieu in the tumour microenvironment (TME) and its constituent stromal and immune cells. In this review, we explore how EBV modulates metabolism to shape the interactions between cancer cells, stromal cells, and immune cells within a hypoxic and acidic TME. We highlight how metabolites resulting from EBV infection act as paracrine factors to regulate the TME, and how targeting them can disrupt barriers to immunotherapy.

Human Viral Oncoproteins and Ubiquitin-Proteasome System.

Some human cancers worldwide may be related to human tumor viruses. Knowing, controlling, and managing the viruses that cause cancers remain a problem. Also, tumor viruses use ubiquitin-proteasome system (UPS) that can alter host cellular processes through UPS. Human tumor viruses cause persistent infections, due to their ability to infect their host cells without killing them. Tumor viruses such as Epstein-Barr virus, hepatitis C virus, hepatitis B virus, human papillomaviruses, human T cell leukemia virus, Kaposi's sarcoma-associated herpesvirus, and Merkel cell polyomavirus are associated with human malignancies. They interfere with the regulation of cell cycle and control of apoptosis, which are important for cellular functions. These viral oncoproteins bind directly or indirectly to the components of UPS, modifying cellular pathways and suppressor proteins like p53 and pRb. They can also cause progression of malignancy. In this review, we focused on how viral oncoproteins bind to the components of the UPS and how these interactions induce the degradation of cellular proteins for their survival.

Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.

Epigenetic Mechanisms in Latent Epstein-Barr Virus Infection and Associated Cancers.

The Epstein-Barr Virus (EBV) is a double-stranded DNA-based human tumor virus that was first isolated in 1964 from lymphoma biopsies. Since its initial discovery, EBV has been identified as a major contributor to numerous cancers and chronic autoimmune disorders. The virus is particularly efficient at infecting B-cells but can also infect epithelial cells, utilizing an array of epigenetic strategies to establish long-term latent infection. The association with histone modifications, alteration of DNA methylation patterns in host and viral genomes, and microRNA targeting of host cell factors are core epigenetic strategies that drive interactions between host and virus, which are necessary for viral persistence and progression of EBV-associated diseases. Therefore, understanding epigenetic regulation and its role in post-entry viral dynamics is an elusive area of EBV research. Here, we present current outlooks of EBV epigenetic regulation as it pertains to viral interactions with its host during latent infection and its propensity to induce tumorigenesis. We review the important epigenetic regulators of EBV latency and explore how the strategies involved during latent infection drive differential epigenetic profiles and host-virus interactions in EBV-associated cancers.

Human Mammary Tumor Virus (HMTV) Reshapes Risk of Women Developing Breast Cancer: Re-visiting an Under considered Variable in Tumorigenesis of the Breast.

Human Mammary Tumor Virus (HMTV) Reshapes Risk of Women Developing Breast Cancer: Re-visiting an Under considered Variable in Tumorigenesis of the Breast.

A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Human tumor viruses: induction of three-dimensional alterations in the host genome structure.

Certain viruses called tumor viruses or oncoviruses are capable to change the gene expression pattern of distinct human or animal cell types in tissue culture, resulting in uncontrolled proliferation as well as a change in the social behavior of the infected cells: the oncovirus-transformed, immortalized cells are capable to form malignant neoplasms in suitable animal models. At present, seven human viruses are categorized as causative agents of distinct human malignancies. The genomes of human tumor viruses, typically encode viral oncoproteins and non- translated viral RNAs that affect the gene expression pattern of their target cells or induce genetic and epigenetic alterations contributing to oncogenesis. Recently, the application of chromatin conformation capture technologies and three-dimensional (3D) molecular imaging techniques revealed how the gene products or genomes of certain human tumor viruses interact with and induce alterations in the 3D host genome structure. This Mini Review aims to cover selected aspects of these developments. The papers, discussed briefly, describe how insertion of a novel viral binding site for the 3D genome organizer cellular protein CCCTC-binding factor (CTCF) into the DNA of T cells infected by human T-cell lymphotropic virus type 1 (HTLV-1) may contribute to lymphomagenesis, as well as how integration of high risk human papillomavirus genome into the host cell DNA may facilitate cervical carcinogenesis. Recent results regarding the interactions of cellular genomes with the episomal, chromatinized DNA genomes of oncogenic human herpesvirus, Epstein-Barr virus (EBV) will also be summarized, similarly to available data regarding contacts formed by episomal or integrated hepatitis B virus (HBV) DNA with host chromatin. Finally, a putative mechanism of hepatitis C virus (HCV) induced chromatin alterations will be presented, which may solve the riddle, how a cytoplasmic RNA virus without a viral oncogene could induce malingnant transfrormation of hepatocytes.

Anoikis resistance and immune escape mediated by Epstein-Barr virus-encoded latent membrane protein 1-induced stabilization of PGC-1α promotes invasion and metastasis of nasopharyngeal carcinoma

BackgroundEpstein-Barr virus (EBV) is the first discovered human tumor virus that is associated with a variety of malignancies of both lymphoid and epithelial origin including nasopharyngeal carcinoma (NPC). The EBV-encoded latent membrane protein 1 (LMP1) has been well-defined as a potent oncogenic protein, which is intimately correlated with NPC pathogenesis. Anoikis is considered to be a physiological barrier to metastasis, and avoiding anoikis is a major hallmark of metastasis. However, the role of LMP1 in anoikis-resistance and metastasis of NPC has not been fully identified.MethodsTrypan blue staining, colony formation assay, flow cytometry, and TUNEL staining, as well as the detection of apoptosis and anoikis resistance‐related markers was applied to evaluate the anoikis-resistant capability of NPC cells cultured in ultra-low adhesion condition. Co-immunoprecipitation (Co-IP) experiment was performed to determine the interaction among LMP1, PRMT1 and PGC-1α. Ex vivo ubiquitination assay was used to detect the ubiquitination level of PGC-1α. Anoikis- resistant LMP1-positive NPC cell lines were established and applied for the xenograft and metastatic animal experiments.ResultsOur current findings reveal the role of LMP1-stabilized peroxisome proliferator activated receptor coactivator-1a (PGC-1α) in anoikis resistance and immune escape to support the invasion and metastasis of NPC. Mechanistically, LMP1 enhances PGC-1α protein stability by promoting the interaction between arginine methyltransferase 1 (PRMT1) and PGC-1α to elevate the methylation modification of PGC-1α, thus endowing NPC cells with anoikis-resistance. Meanwhile, PGC-1α mediates the immune escape induced by LMP1 by coactivating with STAT3 to transcriptionally up-regulate PD-L1 expression.ConclusionOur work provides insights into how virus-encoded proteins recruit and interact with host regulatory elements to facilitate the malignant progression of NPC. Therefore, targeting PGC-1α or PRMT1-PGC-1α interaction might be exploited for therapeutic gain for EBV-associated malignancies.

EBV-induced T-cell responses in EBV-specific and nonspecific cancers.

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus associated with various malignancies, including B-lymphoma, NK and T-lymphoma, and epithelial carcinoma. It infects B lymphocytes and epithelial cells within the oropharynx and establishes persistent infection in memory B cells. With a balanced virus-host interaction, most individuals carry EBV asymptomatically because of the lifelong surveillance by T cell immunity against EBV. A stable anti-EBV T cell repertoire is maintained in memory at high frequency in the blood throughout persistent EBV infection. Patients with impaired T cell immunity are more likely to develop life-threatening lymphoproliferative disorders, highlighting the critical role of T cells in achieving the EBV-host balance. Recent studies reveal that the EBV protein, LMP1, triggers robust T-cell responses against multiple tumor-associated antigens (TAAs) in B cells. Additionally, EBV-specific T cells have been identified in EBV-unrelated cancers, raising questions about their role in antitumor immunity. Herein, we summarize T-cell responses in EBV-related cancers, considering latency patterns, host immune status, and factors like human leukocyte antigen (HLA) susceptibility, which may affect immune outcomes. We discuss EBV-induced TAA-specific T cell responses and explore the potential roles of EBV-specific T cell subsets in tumor microenvironments. We also describe T-cell immunotherapy strategies that harness EBV antigens, ranging from EBV-specific T cells to T cell receptor-engineered T cells. Lastly, we discuss the involvement of γδ T-cells in EBV infection and associated diseases, aiming to elucidate the comprehensive interplay between EBV and T-cell immunity.

Immunohistochemical Detection of the Expressed BRCA1 and BRCA2 Proteins in Microenvironment of Malignant Breast Cancerous Tissues Infected with Human Mammary Tumor Virus.

The objective of the study was to examine the prevalence of HMTV infection, its associations with breast malignant tissues, and the expression of BRCA1 and BRCA2 proteins. One hundred archival breast tissues, 40 biopsies from female patients with breast cancer (BC), and 20 healthy breast tissues from the control group were used in the study. Immunohistochemical analysis was conducted to detect the expressed BRCA1 and BRCA2 proteins. Digoxigenin-labeled HMTV probes were used in chromogenic in situ hybridization for the identification of HMTV in breast tumor tissues. The complementary sequence sites of the HMTV probe sequences were stained by NBT/BCIP as blue signals. There were 12 out of 40 (30%) benign breast tumorous tissues and 14 out of 40 (35%) BC tissues, while healthy control breast tissues were 10% (2 out of 20 tissues). Positive immunohistochemical (IHC) reactions for BRCA2 protein were observed in 12 out of 40 BC tissues (30.0%), 25% of benign breast tumorous tissues, and 5% of the control group. A significant (p < 0.05) statistical difference in the percentages of HMTV in the studied groups was found. HMTV might contribute to the development of subsets of benign and malignant breast tumors. The observed rates of defective or mutated BRCA1 and BRCA2 genes in healthy tissues indicate a role in the development of breast tumors.

Methyltransferase-independent function of enhancer of zeste homologue 2 maintains tumorigenicity induced by human oncogenic papillomavirus and polyomavirus

Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients.

The Impact of Human Mammary Tumor Virus (HMTV) on the Expression of Epidermal Growth Factor Receptor (EGFR) and Death-Domain Associated Protein (DAXX) in Breast Carcinoma Tissues.

The presence and characteristics of HMTV in Iraqi breast cancer women are still unknown. Furthermore, the identification of HMTV in human breast carcinoma tissue of patients differs by country, and the factors that influence it are still unknown. In many epithelial tumor types, the EGFR and its signaling pathways outcomes are necessary for the behavior of cells and regulating their proliferation, and it has been discovered that DAXX has strong carcinogenic characteristics and could be a new treatment target. This case-control retrospective study investigated the presence of HMTV in paraffin-embedded blocks (FFPT) of tumor samples from 60 Iraqi women patients diagnosed with primary breast cancer and 20 cases of benign tumors as a control group. HMTV env sequences were identified by Real-time PCR. EGFR and DAXX expression were immun0-detected by the immuno-histochemistry technique. HMTV sequences were detected in 15 (25%) samples of malignant breast tumors and 8 (40%) samples of benign breast tumors. There was no statistically significant association between the detection of env sequences of HMTV and age, grade, hormone receptors, EGFR, or DAXX expression compared to clinicopathological characteristics. However, statistically, the data showed a highly significant difference in the Expression of EGFR between study groups, age, and histological types (P=0.0001), and a significant negative association was observed between EGFR and both Her2 and TNBC. There was a statistically significant difference between DAXX (+) and DAXX (-) in study groups (P=0.002), and it was significantly associated with age and histological types of breast cancer (P=0.031 and 0.007, respectively). No significant association was found between DAXX and EGFR, grade, Her2. TNBC of breast cancer. The current study found HMTV env sequences in breast tumors of Iraqi women, suggesting that a larger sample size is needed to illustrate the potential causative role of HMTV in the development of human breast malignancy. Moreover, a negative association was found between HMTV and DAXX and EGFR Expression.

Human DNA tumor viruses evade uracil-mediated antiviral immunity.

Human DNA tumor viruses evade uracil-mediated antiviral immunity.

Exploration and analysis of differentially expressed genes in Epstein-Barr virus negative and positive plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) often associated with Epstein-Barr virus (EBV) infection. Despite recent advances in treatment, PBL still has a poor prognosis. EBV is listed as one of the human tumor viruses that may cause cancer, and is closely related to the occurrence of some nasopharyngeal carcinoma (NPC), lymphoma and 10% of gastric cancer (GC). It is very important to explore the differentially expressed genes (DEGs) between EBV-positive and EBV-negative PBL. Through bioinformatics analysis of DEGs between EBV-positive PBL and EBV-negative PBL, we gain a deeper understanding of the pathogenesis of EBV-positive PBL. We selected the GSE102203 data set, and screened the DEGs between EBV-positive PBL and EBV-negative PBL. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied. The protein-protein interaction (PPI) network was constructed, and screened for the hub genes. Finally, Gene Set Enrichment Analysis (GSEA) was performed. In EBV-positive PBL, the immune-related pathway is upregulated and Cluster of differentiation 27 (CD27) and programmed cell death-ligand 1 (PD-L1) are hub genes. In EBV-positive PBL, EBV may affect tumorigenesis through activation of immune-related pathways and upregulation of CD27, PD-L1. Immune checkpoint blockers of CD70/CD27 and programmed cell death 1 (PD-1)/PD-L1 pathways may be one of the effective strategies for the treatment of EBV-positive PBL.

Epstein-Barr Virus B Cell Growth Transformation: The Nuclear Events.

Epstein-Barr virus (EBV) is the first human DNA tumor virus identified from African Burkitt's lymphoma cells. EBV causes ~200,000 various cancers world-wide each year. EBV-associated cancers express latent EBV proteins, EBV nuclear antigens (EBNAs), and latent membrane proteins (LMPs). EBNA1 tethers EBV episomes to the chromosome during mitosis to ensure episomes are divided evenly between daughter cells. EBNA2 is the major EBV latency transcription activator. It activates the expression of other EBNAs and LMPs. It also activates MYC through enhancers 400-500 kb upstream to provide proliferation signals. EBNALP co-activates with EBNA2. EBNA3A/C represses CDKN2A to prevent senescence. LMP1 activates NF-κB to prevent apoptosis. The coordinated activity of EBV proteins in the nucleus allows efficient transformation of primary resting B lymphocytes into immortalized lymphoblastoid cell lines in vitro.

A DNA tumor virus globally reprograms host 3D genome architecture to achieve immortal growth

Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) to lymphoblastoid cell lines (LCLs) models human DNA tumor virus oncogenesis. RBL and LCL chromatin interaction maps are compared to identify the spatial and temporal genome architectural changes during EBV B cell transformation. EBV induces global genome reorganization where contact domains frequently merge or subdivide during transformation. Repressed B compartments in RBLs frequently switch to active A compartments in LCLs. LCLs gain 40% new contact domain boundaries. Newly gained LCL boundaries have strong CTCF binding at their borders while in RBLs, the same sites have much less CTCF binding. Some LCL CTCF sites also have EBV nuclear antigen (EBNA) leader protein EBNALP binding. LCLs have more local interactions than RBLs at LCL dependency factors and super-enhancer targets. RNA Pol II HiChIP and FISH of RBL and LCL further validate the Hi-C results. EBNA3A inactivation globally alters LCL genome interactions. EBNA3A inactivation reduces CTCF and RAD21 DNA binding. EBNA3C inactivation rewires the looping at the CDKN2A/B and AICDA loci. Disruption of a CTCF site at AICDA locus increases AICDA expression. These data suggest that EBV controls lymphocyte growth by globally reorganizing host genome architecture to facilitate the expression of key oncogenes.

Co-Infection of the Epstein-Barr Virus and the Kaposi Sarcoma-Associated Herpesvirus.

The two human tumor viruses, Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV), have been mostly studied in isolation. Recent studies suggest that co-infection with both viruses as observed in one of their associated malignancies, namely primary effusion lymphoma (PEL), might also be required for KSHV persistence. In this review, we discuss how EBV and KSHV might support each other for persistence and lymphomagenesis. Moreover, we summarize what is known about their innate and adaptive immune control which both seem to be required to ensure asymptomatic persistent co-infection with these two human tumor viruses. A better understanding of this immune control might allow us to prepare for vaccination against EBV and KSHV in the future.

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line.

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world's adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles. Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 °C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.