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Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins–EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed., Author summary Epstein-Barr virus (EBV) establishes latent infection in B-lymphocytes and is associated with a number of human malignancies, both of epithelial and lymphoid origin. EBV encoded EBNA3 family of nuclear latent antigens comprising of EBNA3A, EBNA3B, and EBNA3C are unique to immunoblastic lymphomas. While EBNA3A and EBNA3C are involved in blocking many important tumor suppressive mechanisms, EBNA3B exhibits tumor suppressive functions. Although EBNA3 proteins, in particular EBNA3C, interact with and employ different protein degradation machineries to induce B-cell lymphomagenesis, these viral proteins are extremely stable in growing B-lymphocytes. To this end, we now demonstrate that proteasomal inhibition leads to specifically degradation of oncogenic EBNA3A and EBNA3C proteins, whereas EBNA3B remains unaffected. Upon proteasomal inhibition, EBNA3C degradation occurs via autophagy-lysosomal pathway, through labeling with K63-linked polyubiquitination and participating in p62-LC3B complex involved in ubiquitin-mediated autophagy substrate selection and degradation through autolysosomal process. We also demonstrate that the N-terminal domain is responsible for autophgy-lysosomal mediated degradation, while the C-terminal domain plays a crucial role in cytoplasmic localization. Fascinatingly, while proteasomal inhibition reduces EBNA3C’s oncogenic property, it induces both latent and lytic gene expressions and promotes viral reactivation from EBV transformed B-lymphocytes. This is the first report which demonstrates a viral oncoprotein degrades through autophagy-lysosomal pathway upon proteasomal inhibition. In sum, the results promise development of novel strategies specifically targeting proteolytic pathway for the treatment of EBV associated B-cell lymphomas, particularly those are generated in immunocompromised individuals.

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., Author summary 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.

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., Author Summary 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.

Studies have suggested that Epithelial–Mesenchymal Transition (EMT) and transformation is an important step in progression to cancer. Par3 (partitioning-defective protein) is a crucial factor in regulating epithelial cell polarity. However, the mechanism by which the latency associated nuclear antigen (LANA) encoded by Kaposi's Sarcoma associated herpesvirus (KSHV) regulates Par3 and EMTs markers (Epithelial-Mesenchymal Transition) during viral-mediated B-cell oncogenesis has not been fully explored. Moreover, several studies have demonstrated a crucial role for EMT markers during B-cell malignancies. In this study, we demonstrate that Par3 is significantly up-regulated in KSHV-infected primary B-cells. Further, Par3 interacted with LANA in KSHV positive and LANA expressing cells which led to translocation of Par3 from the cell periphery to a predominantly nuclear signal. Par3 knockdown led to reduced cell proliferation and increased apoptotic induction. Levels of SNAIL was elevated, and E-cadherin was reduced in the presence of LANA or Par3. Interestingly, KSHV infection in primary B-cells led to enhancement of SNAIL and down-regulation of E-cadherin in a temporal manner. Importantly, knockdown of SNAIL, a major EMT regulator, in KSHV cells resulted in reduced expression of LANA, Par3, and enhanced E-cadherin. Also, SNAIL bound to the promoter region of p21 and can regulate its activity. Further a SNAIL inhibitor diminished NF-kB signaling through upregulation of Caspase3 in KSHV positive cells in vitro. This was also supported by upregulation of SNAIL and Par3 in BC-3 transplanted NOD-SCID mice which has potential as a therapeutic target for KSHV-associated B-cell lymphomas., Author Summary Par3 (partitioning-defective protein) is characterized as a cell polarity protein with a crucial role in regulating EMT progression in KSHV-infected cells. Notably, SNAIL, a major EMT regulator is targeted through expression of Par3 and Latency associated nuclear antigen (LANA) during KSHV infection. In this study we show that LANA positively regulated Par3 and SNAIL while E-cadherin was inversely regulated during KSHV infection of B-cells. Furthermore, SNAIL binding with the potent kinase inhibitor p21 in KSHV positive cells enhanced NF-kB signaling. These results demonstrate the importance of Par3 and SNAIL in development of KSHV-induced B-cells cancers through regulation of EMT related antigens, and may be critical for driving KSHV-associated primary effusion lymphoma.

Kaposi's sarcoma associated herpesvirus is tightly linked to multiple human malignancies including Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). KSHV like other herpesviruses establishes life-long latency in the infected host by persisting as chromatin and tethering to host chromatin through the virally encoded protein Latency Associated Nuclear Antigen (LANA). LANA, a multifunctional protein, is capable of binding to a large number of cellular proteins responsible for transcriptional regulation of various cellular and viral pathways involved in blocking cell death and promoting cell proliferation. This leads to enhanced cell division and replication of the viral genome, which segregates faithfully in the dividing tumor cells. The mechanism of genome segregation is well known and the binding of LANA to nucleosomal proteins, throughout the cell cycle, suggests that these interactions play an important role in efficient segregation. Various biochemical methods have identified a large number of LANA binding proteins, including histone H2A/H2B, histone H1, MeCP2, DEK, CENP-F, NuMA, Bub1, HP-1, and Brd4. These nucleosomal proteins may have various functions in tethering of the viral genome during specific phases of the viral life cycle. Therefore, we performed a comprehensive analysis of their interaction with LANA using a number of different assays. We show that LANA binds to core nucleosomal histones and also associates with other host chromatin proteins including histone H1 and high mobility group proteins (HMGs). We used various biochemical assays including co-immunoprecipitation and in-vivo localization by split GFP and fluorescence resonance energy transfer (FRET) to demonstrate their association.

Epstein-Barr virus (EBV) is linked to a broad spectrum of B-cell malignancies. EBV nuclear antigen 3C (EBNA3C) is an encoded latent antigen required for growth transformation of primary human B-lymphocytes. Interferon regulatory factor 4 (IRF4) and 8 (IRF8) are transcription factors of the IRF family that regulate diverse functions in B cell development. IRF4 is an oncoprotein with anti-apoptotic properties and IRF8 functions as a regulator of apoptosis and tumor suppressor in many hematopoietic malignancies. We now demonstrate that EBNA3C can contribute to B-cell transformation by modulating the molecular interplay between cellular IRF4 and IRF8. We show that EBNA3C physically interacts with IRF4 and IRF8 with its N-terminal domain in vitro and forms a molecular complex in cells. We identified the Spi-1/B motif of IRF4 as critical for EBNA3C interaction. We also demonstrated that EBNA3C can stabilize IRF4, which leads to downregulation of IRF8 by enhancing its proteasome-mediated degradation. Further, si-RNA mediated knock-down of endogenous IRF4 results in a substantial reduction in proliferation of EBV-transformed lymphoblastoid cell lines (LCLs), as well as augmentation of DNA damage-induced apoptosis. IRF4 knockdown also showed reduced expression of its targeted downstream signalling proteins which include CDK6, Cyclin B1 and c-Myc all critical for cell proliferation. These studies provide novel insights into the contribution of EBNA3C to EBV-mediated B-cell transformation through regulation of IRF4 and IRF8 and add another molecular link to the mechanisms by which EBV dysregulates cellular activities, increasing the potential for therapeutic intervention against EBV-associated cancers., Author Summary Interferon regulatory factor (IRF) family members have different roles in context of pathogen response, signal transduction, cell proliferation and hematopoietic development. IRF4 and IRF8 are members of the IRF family and are critical mediators of B-cell development. Enhanced expression of IRF4 is often associated with multiple myeloma and adult T-cell lymphomas. Furthermore, IRF8 can function as a tumor suppressor in myeloid cancers. Epstein-Barr virus (EBV), one of the first characterized human tumor viruses is associated with several lymphoid malignancies. One of the essential antigens, EBV encoded nuclear antigen 3C (EBNA3C), plays a critical role in EBV-induced B-cell transformation. In our study, we now demonstrate that EBNA3C forms a molecular complex with IRF4 and IRF8 specifically through its N-terminal domain. We show that IRF4 is stabilized by EBNA3C, which resulted in downregulation of IRF8 through proteasome-mediated degradation and subsequent inhibition of its tumor suppressive activity. Moreover, si-RNA-mediated inhibition of IRF4 showed a substantial reduction in EBV transformed B-cell proliferation, and also enhanced their sensitivity to DNA-damage induced apoptosis. Therefore, our findings demonstrated that targeted disruption of EBNA3C-mediated differential regulation of IRF4 and IRF8 may have potential therapeutic value for treating EBV induced B-cell malignancies.

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.