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The Epstein-Barr Virus Oncoprotein, LMP1, Regulates the Function of SENP2, a SUMO-protease.

ABSTRACT: Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) activates numerous signal transduction pathways using its C-terminal activating regions. We reported that LMP1 increased global levels of sumoylated proteins, which aided the oncogenic nature of LMP1. Because increased protein sumoylation is detected in numerous cancers, we wanted to elucidate additional mechanisms by which LMP1 modulates the sumoylation machinery. Results indicated that SUMO-protease activity decreased in a LMP1-dependent manner, so we hypothesized that LMP1 inhibits SUMO-protease activity, resulting in reduced de-sumoylation of cellular proteins, which contributes to the detected accumulation of sumoylated proteins in EBV-positive lymphomas. Focusing on SENP2, findings revealed that LMP1 expression corresponded with increased sumoylation of SENP2 at K48 and K447 in a CTAR-dependent manner. Interestingly, independent of LMP1-induced sumoylation of SENP2, LMP1 also decreased SENP2 activity, decreased SENP2 turnover, and altered the localization of SENP2, which led us to investigate if LMP1 regulated the biology of SENP2 by a different post-translational modification, specifically ubiquitination. Data showed that expression of LMP1 inhibited the ubiquitination of SENP2, and inhibition of ubiquitination was sufficient to mimic LMP1-induced changes in SENP2 activity and trafficking. Together, these findings suggest that LMP1 modulates different post-translational modifications of SENP2 in order to modulate its biology and identify a third member of the sumoylation machinery that is manipulated by LMP1 during latent EBV infections, which can affect oncogenesis.

SUBMITTER: Selby TL

PROVIDER: S-EPMC6606635 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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The Epstein-Barr Virus Oncoprotein, LMP1, Regulates the Function of SENP2, a SUMO-protease.

Selby Thomas L TL Biel Natalie N Varn Matthew M Patel Sheetal S Patel Akash A Hilding Leslie L Ray Ashley A Ross Tabithia T Cramblet Wyatt T WT Moss C Randall CR Lowrey Angela J AJ Bentz Gretchen L GL

Scientific reports 20190702 1

Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) activates numerous signal transduction pathways using its C-terminal activating regions. We reported that LMP1 increased global levels of sumoylated proteins, which aided the oncogenic nature of LMP1. Because increased protein sumoylation is detected in numerous cancers, we wanted to elucidate additional mechanisms by which LMP1 modulates the sumoylation machinery. Results indicated that SUMO-protease activity decreased in a LMP1-dependen ...[more]

PMID: 31266997

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Project description:The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation.EBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

Project description:Generating oxidative stress is a critical mechanism by which host cells defend against infection by pathogenic microorganisms. Radiation resistance is a critical problem in radiotherapy against cancer. Epstein-Barr virus (EBV) is a cancer-causing virus and its reactivation plays an important role in the development of EBV-related tumors. This study aimed to explore the inner relationship and regulatory mechanism among oxidative stress, EBV reactivation, and radioresistance and to identify new molecular subtyping models and treatment strategies to improve the therapeutic effects of radiotherapy. Methods: ROS, NADP+/NADPH, and GSSG/GSH were detected to evaluate the oxidative stress of cells. 8-OHdG is a reliable oxidative stress marker to evaluate the oxidative stress in patients. Its concentration in serum was detected using an ELISA method and in biopsies was detected using IHC. qPCR array was performed to evaluate the expression of essential oxidative stress genes. qPCR, Western blot, and IHC were used to measure the level of EBV reactivation in vitro and in vivo. A Rta-IgG ELISA kit and EBV DNA detection kit were used to analyze the reactivation of EBV in serum from NPC patients. NPC tumor tissue microarrays was used to investigate the prognostic role of oxidative stress and EBV reactivation. Radiation resistance was evaluated by a colony formation assay. Xenografts were treated with NAC, radiation, or a combination of NAC and radiation. EBV DNA load of tumor tissue was evaluated using an EBV DNA detection kit. Oxidative stress, EBV reactivation, and the apoptosis rate in tumor tissues were detected by using 8-OHdG, EAD, and TUNEL assays, respectively. Results: We found that EBV can induce high oxidative stress, which promotes its reactivation and thus leads to radioresistance. Basically, EBV caused NPC cells to undergo a process of 'Redox Resetting' to acquire a new redox status with higher levels of ROS accumulation and stronger antioxidant systems by increasing the expression of the ROS-producing enzyme, NOX2, and the cellular master antioxidant regulator, Nrf2. Also, EBV encoded driving protein LMP1 promotes EBV reactivation through production of ROS. Furthermore, high oxidative stress and EBV reactivation were positively associated with poor overall survival of patients following radiation therapy and were significant related to NPC patients' recurrence and clinical stage. By decreasing oxidative stress using an FDA approved antioxidant drug, NAC, sensitivity of tumors to radiation was increased. Additionally, 8-OHdG and EBV DNA could be dual prognostic markers for NPC patients. Conclusions: Oxidative stress mediates EBV reactivation and leads to radioresistance. Targeting oxidative stress can provide therapeutic benefits to cancer patients with radiation resistance. Clinically, we, for the first time, generated a molecular subtyping model for NPC relying on 8-OHdG and EBV DNA level. These dual markers could identify patients who are at a high risk of poor outcomes but who might benefit from the sequential therapy of reactive oxygen blockade followed by radiation therapy, which provides novel perspectives for the precise treatment of NPC.

Project description:Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.

Dataset Information

The Epstein-Barr Virus Oncoprotein, LMP1, Regulates the Function of SENP2, a SUMO-protease.

Publications

The Epstein-Barr Virus Oncoprotein, LMP1, Regulates the Function of SENP2, a SUMO-protease.

Similar Datasets

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