Epstein-Barr Virus LMP1 Modulates the CD63 Interactome.
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ABSTRACT:
SUBMITTER: Cheerathodi M
PROVIDER: S-EPMC8071190 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC5309960 | biostudies-other
Project description:The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells.IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication.
| S-EPMC5809724 | biostudies-literature
Project description:Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy. The principal oncogene of EBV, latent membrane protein 1 (LMP1), induces the expression of programmed death-ligand 1 (PD-L1), which is an immunosuppressive transmembrane protein and a promising therapeutic target for various malignancies. Recent studies have revealed an association between the level of soluble PD-L1 (sPD-L1) and disease progression. However, the role of sPD-L1 in NPC or its relevance to LMP1 has not been elucidated. This study aimed to examine whether LMP1 induces sPD-L1 in vitro and analyze the clinical relevance of LMP1, PD-L1, and sPD-L1 in NPC patients. Analysis of nasopharyngeal cell lines revealed that LMP1 induces both cellular PD-L1 and sPD-L1. Analysis of biopsy specimens from 32 NPC patients revealed that LMP1 expression was significantly correlated with PD-L1 expression. Finally, the serum sPD-L1 level in NPC patients was higher than that in the controls. Moreover, the sPD-L1 level in the advanced stage was higher than that in the early stage. However, LMP1 expression, PD-L1 expression, and sPD-L1 levels were not associated with prognosis. These results suggest that LMP1 induces both sPD-L1 and PD-L1, which are associated with NPC progression.
| S-EPMC7998736 | biostudies-literature
Project description:Epstein-Barr virus (EBV) infection transforms B cells in vitro and is associated with human B cell lymphomas. The major EBV oncoprotein, latent membrane protein 1 (LMP1), mimics constitutively active CD40 and is essential for outgrowth of EBV-transformed B cells in vitro; however, EBV-positive diffuse large B cell lymphomas and Burkitt lymphomas often express little or no LMP1. Thus, EBV may contribute to the development and maintenance of human lymphomas even in the absence of LMP1. Here, we found that i.p. injection of human cord blood mononuclear cells infected with a LMP1-deficient EBV into immunodeficient mice induces B cell lymphomas. In this model, lymphoma development required the presence of CD4+ T cells in cord blood and was inhibited by CD40-blocking Abs. In contrast, LMP1-deficient EBV established persistent latency but did not induce lymphomas when directly injected into mice engrafted with human fetal CD34+ cells and human thymus. WT EBV induced lymphomas in both mouse models and did not require coinjected T cells in the cord blood model. Together, these results demonstrate that LMP1 is not essential for EBV-induced lymphomas in vivo and suggest that T cells supply signals that substitute for LMP1 in EBV-positive B cell lymphomagenesis.
| S-EPMC4382240 | biostudies-other
Project description:Nasal NK/T cell lymphomas (NKTCL) are a subset of aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin's lymphomas. The role of EBV in pathogenesis of NKTCL is not clear. Intriguingly, EBV encodes more than 40 microRNAs (miRNA) that are differentially expressed and largely conserved in lymphocryptoviruses. While miRNAs play a critical role in the pathogenesis of cancer, especially lymphomas, the expression and function of EBV transcribed miRNAs in NKTCL are not known. To examine the role of EBV miRNAs in NKTCL, we used microarray profiling and qRT-PCR to identify and validate expression of viral miRNAs in SNK6 and SNT16 cells, which are two independently derived NKTCL cell lines that maintain the type II EBV latency program. All EBV BART miRNAs except BHRF-derived miRNAs were expressed and some of these miRNAs are expressed at higher levels than in nasopharyngeal carcinomas. Modulating the expression of BART9 with antisense RNAs consistently reduced SNK6 and SNT16 proliferation, while antisense RNAs to BARTs-7 and -17-5p affected proliferation only in SNK6 cells. Furthermore, the EBV LMP-1 oncoprotein and transcript levels were repressed when an inhibitor of BART9 miRNA was transfected into SNK6 cells, and overexpression of BART9 miRNA increased LMP-1 protein and mRNA expression. Our data indicate that BART9 is involved in NKTCL proliferation, and one of its mechanisms of action appears to be regulating LMP-1 levels. Our findings may have direct application for improving NKTCL diagnosis and for developing possible novel treatment approaches for this tumor, for which current chemotherapeutic drugs have limited effectiveness.
| S-EPMC3213120 | biostudies-literature
Project description:The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation.
| S-EPMC2826061 | biostudies-literature
Project description:Approximately 20% of global cancer incidence is causally linked to an infectious agent. Epstein-Barr virus (EBV) accounts for around 1% of all virus-associated cancers and is associated with nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP1), the major oncoprotein encoded by EBV, behaves as a constitutively active tumour necrosis factor (TNF) receptor activating a variety of signalling pathways, including the three classic MAPKs (ERK-MAPK, p38 MAPK and JNK/SAPK). The present study identifies novel signalling properties for this integral membrane protein via the induction and secretion of activin A and TGFβ1, which are both required for LMP1's ability to induce the expression of the extracellular matrix protein, fibronectin. However, it is evident that LMP1 is unable to activate the classic Smad-dependent TGFβ signalling pathway, but rather elicits its effects through the non-Smad arm of TGFβ signalling. In addition, there is a requirement for JNK/SAPK signalling in LMP1-mediated fibronectin induction. LMP1 also induces the expression and activation of the major fibronectin receptor, α5β1 integrin, an effect that is accompanied by increased focal adhesion formation and turnover. Taken together, these findings support the putative role for LMP1 in the pathogenesis of NPC by contributing to the metastatic potential of epithelial cells.
| S-EPMC4726061 | biostudies-other
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.
| S-EPMC5021435 | biostudies-literature
Project description:Background:Epstein-Barr virus-encoded LMP1 plays a critical role in the carcinogenesis of nasopharyngeal carcinoma (NPC), but the mechanism remains elusive. We aimed to analyze the expression and clinical pathological significance of provirus integration site for Moloney murine leukemia virus 1 (Pim1) in clinical NPC, and to elucidate the effect of LMP1 on Pim1 expression and its mechanism. Methods:Immunohistochemical staining was used to detect the expression of Pim1 in clinical NPC tissues and control nasopharyngeal chronic inflammation (NPI) tissues, and the correlation between Pim1 and clinical parameters of NPC patients was analyzed. The LMP1 stable expression cell line CNE1-LMP1-OV was constructed through infecting the well-differentiated nasopharyngeal carcinoma cells CNE1 with LMP1 overexpressing lentivirus. Then the in vivo experiments were conducted. Results:Among 89 NPC patients, 48 cases (53.93%) were positive for Pim1, while only one case was Pim1 positive in 15 NPI controls (6.67%). Pim1 expression was not correlated with gender, age, smoking status and clinical classification of NPC patients, but positively correlated with T, N and M classification. CNE1-LMP1-OV cell line was successfully established, which displayed a higher cell proliferation ability and Pim1 expression. NF-?B inhibitor PDTC, PKC inhibitor GF109203X and STAT3 inhibitor Stattic significantly attenuated LMP1-induced Pim1 expression, and while AP-1 inhibitor SR11302 showed no inhibitory effect. Interestingly, Pim1 inhibitor quercetagetin significantly inhibited the proliferation of CNE1-LMP1-OV cells. Conclusion:LMP1 mediates Pim1 expression through NF-?B, PKC and STAT3 signaling, which promotes the proliferation of NPC cells and participate in the clinical progression of NPC.
| S-EPMC6376889 | biostudies-literature
Project description:Our goal in this work was to illustrate the Epstein-Barr virus (EBV)-modulated global biochemical profile and provide a novel metabolism-related target to improve the therapeutic regimen of nasopharyngeal carcinoma (NPC). We used a metabolomics approach to investigate EBV-modulated metabolic changes, and found that the exogenous overexpression of the EBV-encoded latent membrane protein 1 (LMP1) significantly increased glycolysis. The deregulation of several glycolytic genes, including hexokinase 2 (HK2), was determined to be responsible for the reprogramming of LMP1-mediated glucose metabolism in NPC cells. The upregulation of HK2 elevated aerobic glycolysis and facilitated proliferation by blocking apoptosis. More importantly, HK2 was positively correlated with LMP1 in NPC biopsies, and high HK2 levels were significantly associated with poor overall survival of NPC patients following radiation therapy. Knockdown of HK2 effectively enhanced the sensitivity of LMP1-overexpressing NPC cells to irradiation. Finally, c-Myc was demonstrated to be required for LMP1-induced upregulation of HK2. The LMP1-mediated attenuation of the PI3-K/Akt-GSK3beta-FBW7 signaling axis resulted in the stabilization of c-Myc. These findings indicate a close relationship between EBV and glycolysis in NPC. Notably, LMP1 is the key regulator of the reprogramming of EBV-mediated glycolysis in NPC cells. Given the importance of EBV-mediated deregulation of glycolysis, anti-glycolytic therapy might represent a worthwhile avenue of exploration in the treatment of EBV-related cancers.
| S-EPMC4162460 | biostudies-literature