The Modulation of Apoptotic Pathways by Gammaherpesviruses

Apoptosis or programmed cell death is a tightly regulated process fundamental for cellular development and elimination of damaged or infected cells during the maintenance of cellular homeostasis. It is also an important cellular defense mechanism against viral invasion. In many instances, abnormal regulation of apoptosis has been associated with a number of diseases, including cancer development. Following infection of host cells, persistent and oncogenic viruses such as the members of the Gammaherpesvirus family employ a number of different mechanisms to avoid the host cell’s burglar alarm and to alter the extrinsic and intrinsic apoptotic pathways by either deregulating the expressions of cellular signaling genes or by encoding the viral homologues of cellular genes. In this review, we summarize the recent findings on how gammaherpesviruses inhibit cellular apoptosis via virus-encoded proteins by mediating modification of numerous signal transduction pathways. We also list the key viral anti-apoptotic proteins that could be exploited as effective targets for novel antiviral therapies in order to stimulate apoptosis in different types of cancer cells

Epstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis

The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities

Kaposi's Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells

The early period of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection involves the dynamic expression of viral genes, which are temporally and epigenetically regulated. KSHV can effectively infect and persist in endothelial as well as human B cells with different gene expression patterns. To understand the temporal epigenetic changes which occur when KSHV infects the lymphocytic compartment, we infected human peripheral blood mononuclear cells (PBMCs) and comprehensively analyzed the changes which occurred at the binding sites of virally encoded lytic as well as latent proteins along with epigenetic modifications across the KSHV genome during early primary infection. Using chromatin immunoprecipitation (ChIP) assays, we showed that the KSHV genome acquires a uniquely distinct histone modification pattern of methylation (H3K4me3, H3K9me3, and H3K27me3) and acetylation (H3Ac) during de novo infection of human PBMCs. This pattern showed that the epigenetic changes were temporally controlled. The binding profiles of KSHV latent protein LANA and the immediate early proteins RTA and K8 showed specific patterns at different times postinfection, which reflects the gene expression program. Further analysis demonstrated that KSHV can concurrently express lytic and latent genes which were associated with histone modifications at these specific regions on the viral genome. We identified three KSHV genes, K3, ORF49, and ORF64, which exhibited different profiles of histone modifications during the early stages of PBMC infection. These studies established a distinct pattern of epigenetic modification which correlates with viral gene expression temporally regulated during the first 7 days of PBMC infection and provides clues to the regulatory program required for successful infection by KSHV of human PBMCs

Tumor-Shed PGE2 Impairs IL2Rγc-Signaling to Inhibit CD4+ T Cell Survival: Regulation by Theaflavins

BACKGROUND:Many tumors are associated with decreased cellular immunity and elevated levels of prostaglandin E2 (PGE2), a known inhibitor of CD4+ T cell activation and inducer of type-2 cytokine bias. However, the role of this immunomodulator in the survival of T helper cells remained unclear. Since CD4+ T cells play critical roles in cell-mediated immunity, detail knowledge of the effect tumor-derived PGE2 might have on CD4+ T cell survival and the underlying mechanism may, therefore, help to overcome the overall immune deviation in cancer. METHODOLOGY/PRINCIPAL FINDINGS:By culturing purified human peripheral CD4+ T cells or Jurkat cells with spent media of theaflavin- or celecoxib-pre-treated MCF-7 cells, we show that tumor-shed PGE2 severely impairs interleukin 2 receptor gammac (IL2Rgammac)-mediated survival signaling in CD4+ T cells. Indeed, tumor-shed PGE2 down-regulates IL2Rgammac expression, reduces phosphorylation as well as activation of Janus kinase 3 (Jak-3)/signal transducer and activator of transcription 5 (Stat-5) and decreases Bcl-2/Bax ratio thereby leading to activation of intrinsic apoptotic pathway. Constitutively active Stat-5A (Stat-5A1 6) over-expression efficiently elevates Bcl-2 levels in CD4+ T cells and protects them from tumor-induced death while dominant-negative Stat-5A over-expression fails to do so, indicating the importance of Stat-5A-signaling in CD4+ T cell survival. Further support towards the involvement of PGE2 comes from the results that (a) purified synthetic PGE2 induces CD4+ T cell apoptosis, and (b) when knocked out by small interfering RNA, cyclooxygenase-2 (Cox-2)-defective tumor cells fail to initiate death. Interestingly, the entire phenomena could be reverted back by theaflavins that restore cytokine-dependent IL2Rgammac/Jak-3/Stat-5A signaling in CD4+ T cells thereby protecting them from tumor-shed PGE2-induced apoptosis. CONCLUSIONS/SIGNIFICANCE:These data strongly suggest that tumor-shed PGE2 is an important factor leading to CD4+ T cell apoptosis during cancer and raise the possibility that theaflavins may have the potential as an effective immunorestorer in cancer-bearer

The Role of Gammaherpesviruses in Cancer Pathogenesis

Worldwide, one fifth of cancers in the population are associated with viral infections. Among them, gammaherpesvirus, specifically HHV4 (EBV) and HHV8 (KSHV), are two oncogenic viral agents associated with a large number of human malignancies. In this review, we summarize the current understanding of the molecular mechanisms related to EBV and KSHV infection and their ability to induce cellular transformation. We describe their strategies for manipulating major cellular systems through the utilization of cell cycle, apoptosis, immune modulation, epigenetic modification, and altered signal transduction pathways, including NF-kB, Notch, Wnt, MAPK, TLR, etc. We also discuss the important EBV latent antigens, namely EBNA1, EBNA2, EBNA3’s and LMP’s, which are important for targeting these major cellular pathways. KSHV infection progresses through the engagement of the activities of the major latent proteins LANA, v-FLIP and v-Cyclin, and the lytic replication and transcription activator (RTA). This review is a current, comprehensive approach that describes an in-depth understanding of gammaherpes viral encoded gene manipulation of the host system through targeting important biological processes in viral-associated cancers

STAT3 in Tumor-Associated Myeloid Cells: Multitasking to Disrupt Immunity

Myeloid immune cells, such as dendritic cells, monocytes, and macrophages, play a central role in the generation of immune responses and thus are often either disabled or even hijacked by tumors. These new tolerogenic activities of tumor-associated myeloid cells are controlled by an oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). STAT3 multitasks to ensure tumors escape immune detection by impairing antigen presentation and reducing production of immunostimulatory molecules while augmenting the release of tolerogenic mediators, thereby reducing innate and adaptive antitumor immunity. Tumor-associated myeloid cells and STAT3 signaling in this compartment are now commonly recognized as an attractive cellular target for improving efficacy of standard therapies and immunotherapies. Hereby, we review the importance and functional complexity of STAT3 signaling in this immune cell compartment as well as potential strategies for cancer therapy

An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation

<div><p>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.</p></div

The EBV Latent Antigen 3C Inhibits Apoptosis through Targeted Regulation of Interferon Regulatory Factors 4 and 8.

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

KSHV encoded LANA contributes to viral latent replication by activating phosphorylation of Survivin

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus casually linked to Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). Previously, we showed that LANA encoded by KSHV upregulates expression of survivin, a member of the inhibitor of apoptosis (IAP) family. This leads to an increase in the rate of cell proliferation of KSHV-infected B cells. LANA is required for tethering of the KSHV episome to the host chromosomes and efficiently segregates the viral genomes into dividing tumor cells. Here we show that LANA interacts with Aurora kinase B (AK-B) and induces phosphorylation of survivin at residue T34. Phosphorylation of survivin specifically on residue T34 enhances the activity of p300 and inhibits the activity of histone deacetylase 1 (HDAC-1), which then leads to an increase in acetylation of histone H3 on the viral genome. Phosphorylation of survivin specifically on residue T34 upregulates the activities of histone acetyltransferases and deacetylases, which then leads to an increase in viral copy number in KSHV-infected B cells. This results in a boost of KSHV replication in latently infected B-lymphoma cells. The studies showed that LANA can also function to regulate viral replication prior to mitosis of the latently infected cells, suggesting that LANA possesses a novel role in regulating KSHV replication in infected B cells