Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers. Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that contributes to approximately 2% of all cancers by modulating a myriad of host cell activities. M6A is the most abundant RNA modification which is important for infection with HIV-1, HCV, Zika virus, KSHV and SV40 virus. The role of m6A modification and the associated enzymes in EBV infection and related oncogenic activities has not been explored. We investigated the role of m6A modification on EBV-infection and examined its effects on the proliferative activities of the virus which contributes to cancer. We showed that the m6A methyltransferase METTL14 is an important factor in EBV-induced oncogenesis. METTL14 was dramatically increased in EBV latently infected cells. Knock-down of METTL14 led to decreased latent viral gene expression. The essential EBV latent antigen EBNA3C was up-regulated by METTL14-mediated m6A modification, and its expression led to a feedback loop in up-regulation of METTL14 transcription, and the stability of its protein. We now provide a more comprehensive understanding of EBV and the host cell RNA processing machinery critical for regulation of EBV activities to maintain latent infection and its oncogenic properties.

Kaposi’s sarcoma associated herpesvirus (KSHV) infection stabilizes hypoxia inducible factors (HIFs). The interaction between KSHV encoded factors and HIFs plays a critical role in KSHV latency, reactivation and associated disease phenotypes. Besides modulation of large-scale signaling, KSHV infection also reprograms the metabolic activity of infected cells. However, the mechanism and cellular pathways modulated during these changes are poorly understood. We performed comparative RNA sequencing analysis on cells with stabilized hypoxia inducible factor 1 alpha (HIF1α) of KSHV negative or positive background to identify changes in global and metabolic gene expression. Our results show that hypoxia induces glucose dependency of KSHV positive cells with high glucose uptake and high lactate release. We identified the KSHV-encoded vGPCR, as a novel target of HIF1α and one of the main viral antigens of this metabolic reprogramming. Bioinformatics analysis of vGPCR promoter identified 9 distinct hypoxia responsive elements which were activated by HIF1α in-vitro. Expression of vGPCR alone was sufficient for induction of changes in the metabolic phenotype similar to those induced by KSHV under hypoxic conditions. Silencing of HIF1α rescued the hypoxia associated phenotype of KSHV positive cells. Analysis of the host transcriptome identified several common targets of hypoxia as well as KSHV encoded factors and other synergistically activated genes belonging to cellular pathways. These include those involved in carbohydrate, lipid and amino acids metabolism. Further DNA methyltranferases, DNMT3A and DNMT3B were found to be regulated by either KSHV, hypoxia, or both synergistically at the transcript and protein levels. This study showed distinct and common, as well as synergistic effects of HIF1α and KSHV-encoded proteins on metabolic reprogramming of KSHV-infected cells in the hypoxia. Hypoxia inducible factors (HIFs) play a critical role in survival and growth of cancerous cells, in addition to modulating cellular metabolism. Kaposi’s sarcoma associated herpesvirus (KSHV) infection stabilizes HIFs. Several factors encoded by KSHV are known to interact with up or downstream targets of HIFs. However, the process by which KSHV infection leads to stabilized HIF1α and modulation of the cellular metabolism is not understood. Comparative RNA sequencing analysis on cells with stabilized hypoxia inducible factor 1 alpha (HIF1α), of KSHV negative or positive cells led to identification of changes in global and metabolic gene expression. Our results show that hypoxia induces glucose dependency of KSHV positive cells with high glucose uptake and high lactate release. KSHV-encoded vGPCR was identified as a novel target of HIF1α regulation and a major viral antigen involved in metabolic reprogramming. Silencing of HIF1α rescued the hypoxia associated phenotype of KSHV positive cells. Analysis of the host transcriptome identified several common targets of hypoxia and KSHV-encoded factors, as well as other synergistically activated genes belonging to cellular metabolic pathways. This study showed unique, common and the synergistic effects of both HIF1α and KSHV-encoded proteins on metabolic reprogramming of KSHV-infected cells in hypoxia.

Among all of the known biological carcinogens, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are two of the classical oncogenic herpesviruses known to induce the oncogenic phenotype. Many studies have revealed important functions related to epigenetic alterations of the EBV and KSHV genomes that mediate oncogenesis, but the detailed mechanisms are not fully understood. It is also challenging to fully describe the critical cellular events that drive oncogenesis as well as a comprehensive map of the molecular contributors. This review introduces the roles of epigenetic modifications of these viral genomes, including DNA methylation, histone modification, chromatin remodeling, and noncoding RNA expression, and elucidates potential strategies utilized for inducing oncogenesis by these human gammaherpesviruses.

The latent EBV nuclear antigen 3C (EBNA3C) is required for transformation of primary human B lymphocytes. Most mature B-cell malignancies originate from malignant transformation of germinal center (GC) B-cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is Bcl6, a key regulator of this process. We now demonstrate that EBNA3C contributes to B-cell transformation by targeted degradation of Bcl6. We show that EBNA3C can physically associate with Bcl6. Notably, EBNA3C expression leads to reduced Bcl6 protein levels in a ubiquitin-proteasome dependent manner. Further, EBNA3C inhibits the transcriptional activity of the Bcl6 promoter through interaction with the cellular protein IRF4. Bcl6 degradation induced by EBNA3C rescued the functions of the Bcl6-targeted downstream regulatory proteins Bcl2 and CCND1, which resulted in increased proliferation and G1-S transition. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction, and raises the possibility of developing new targeted therapies against EBV-associated cancers. Epstein-Barr virus (EBV) is the first characterized human tumor virus, which is associated with a broad range of human cancers. One of the latent proteins, EBV encoded nuclear antigen 3C (EBNA3C) plays a critical role in EBV-mediated B-cell transformation. Bcl6 is a master regulator required in mature B-cells during germinal center (GC) reaction. As a transcriptional repressor, Bcl6 can be targeted during malignant transformation and therefore contributes to its function as an oncoprotein during lymphomagenesis. In this study, we demonstrated that EBNA3C interacts with Bcl6 and facilitates its degradation through the ubiquitin-proteasome dependent pathway, and suppresses Bcl6 mRNA expression by inhibiting the transcriptional activity of its promoter. Furthermore, EBNA3C-mediated Bcl6 regulation significantly promotes cell proliferation and cell cycle by targeting Bcl2 and CCND1. Therefore, our findings offer new insights into the functions of EBNA3C during B-cell transformation in GC reaction and B-cell lymphoma development. This increases the possibility of developing new therapies for treating EBV-associated cancers.

The Epstein-Barr virus (EBV) is the first human tumor virus identified that can transform quiescent B lymphocytes into lymphoblastoid cell lines (LCLs) in vitro. EBV can establish asymptomatic life-long persistence and is associated with multiple human malignancies, including non-Hodgkin lymphoma and Hodgkin lymphoma, as well as infectious mononucleosis. Although EBV-associated lymphomagenesis has been investigated for over 50 years, viral-mediated transformation is not completely understood, and the development of EBV-specific therapeutic strategies to treat the associated cancers is still a major challenge. However, the rapid development of several novel therapies offers exciting possibilities to target EBV-induced lymphomas. This review highlights targeted therapies with potential for treating EBV-associated lymphomas, including small molecule inhibitors, immunotherapy, cell therapy, preventative and therapeutic vaccines, and other potent approaches, which are novel strategies for controlling, preventing, and treating these viral-induced malignances.

Epstein–Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. EBV is associated with a broad range of human cancers. EBV-encoded nuclear antigen 3C (EBNA3C) is one of the essential latent antigens important for deregulating the functions of numerous host transcription factors which play vital roles in B-cell immortalization. The family of E2F transcription factors are involved in diverse cellular functions. More specifically, E2F6 is one of the E2F family members with a unique property of transcriptional repression. Our current study now demonstrates that EBNA3C can enhance E2F6 repressive functions, and is also responsible for increased E2F6 protein expression in EBV-transformed LCLs. EBNA3C directly interacts with E2F6 at its amino and carboxy terminal domains. Additionally, E2F6 was stabilized by EBNA3C and co-localized in nuclear compartments. Our study also demonstrated that EBNA3C and E2F6 expression resulted in decreased transcriptional activity of E2F1, and that EBNA3C, E2F6 and E2F1 can form a protein complex, and EBNA3C competes with E2F1 for E2F6 binding. The recruitment of E2F6 by EBNA3C was also shown to be important for its related cell proliferation. These results showed a crucial role for EBNA3C-mediated deregulation of E2F6 and its impact on the activities of other E2F family members. Our findings also provide new insights for targeting these E2F transcription factors as potential therapeutic intervention strategies in EBV-associated cancers.