EB病毒及其癌基因LMP1对上皮细胞的转化作用

EB病毒(EBV)是一种人群感染率较高的致病性疱疹病毒,与伯基特氏淋巴瘤、鼻咽癌的关系最为明确。近年来EBV在上皮源性肿瘤发生、发展中的作用受到众多学的关注,而其癌基因LMP1在上皮源性肿瘤发病的病因学方面的作用更成为研究的热点。本综述就EBV进入上皮细胞、永生化上皮细胞的机制、LMP1的基本结构及其对上皮细胞的生物学功能作一较为详细的阐述。     

Epstein-Barr Virus Latent Membrane Protein 2 Induces Autophagy To Promote Abnormal Acinus Formation

Epstein-Barr virus latent membrane protein 2A (LMP2A) induces many characteristics of carcinoma, including transformation, migration, invasion, and impaired differentiation. The MCF10A cell line differentiates to form hollow acini when grown in Matrigel, and expression of LMP2A inhibited differentiation and anoikis induced by loss of matrix attachment. LMP2A-infected cells formed large, lobular structures rather than hollow acini. Autophagy inhibitors impaired this abnormal growth and induced caspase 3 activation and acinus formation. LMP2A also increased autophagosome formation and expression of proteins in the autophagosome pathway. These findings suggest that LMP2A may inhibit anoikis and luminal clearance in acini through induction of autophagy.     

EB病毒LMP1-CTAR3对NP69细胞增殖和蛋白质表达的影响

为了探讨EB病毒潜伏性膜蛋白1(LMP1)第三个功能活性区域(CTAR3)在鼻咽上皮细胞NP69中的转化作用机制,采用逆病毒感染的方法,将浓缩的逆病毒RV-LMP1和RV-LMP1△232～351分别感染鼻咽上皮细胞NP69,建立NP69-LMP1与NP69-LMP1△232～351稳定表达细胞系.通过绘制生长曲线、平皿克隆形成试验和软琼脂集落形成试验比较野生型和突变型LMP1对NP69细胞增殖的影响,运用蛋白质组学方法鉴定NP69-LMP1与NP69-LMP1△232～351细胞间的差异表达蛋白,选用实时荧光定量RT-PCR与Western blot对其中部分蛋白质点差异表达进行验证.结果发现:a.突变型LMP1△232～351促NP69细胞增殖的能力较野生型LMP1明显降低(n=3,P<0.05);b.鉴定了LMP1-CTAR3在NP69细胞中参与调节的16个蛋白质(表达上调的蛋白质8个,下调的8个).c.实时荧光定量RT-PCR和Western blot证实了部分上述蛋白质的差异表达.以上结果说明,LMP1-CTAR3是其发挥促细胞增殖的重要活性部位,可能通过参与调节G蛋白和异柠檬酸脱氢酶等蛋白质的表达而起作用.     

EB病毒潜伏膜蛋白1多态性与鼻咽癌的关系

为了分析广东地区鼻咽癌病人和非鼻咽癌病人携带的Epstein-Barr病毒（EBV）潜伏膜蛋白1（LMP1）基因多态性,探讨LMP1基因羧基端缺失30个碱基的EBV是否与华南地区鼻咽癌高发有关。为此收集了初始的107例鼻咽癌病人和106例非鼻咽癌肿病人的漱口液,用巢式PCR扩增LMP1羧基端,观察缺失型LMP1的构成比。同时,测序分析缺失型LMP1编码区序列,了解缺失型LMP1多态性与鼻咽癌的关联度。结果：在50％的样品中可检出LMP1基因,缺失型LMP1在鼻咽癌病人和非鼻咽病人的构成比相似,约占70？％,原型和缺失型LMP1混合感染少见（0－6％）。另外,广州地区的缺失型LMP序列与上海、台湾、香港地区的缺失型LMP1基因高度同源,鼻咽癌病人是和非鼻咽癌病人携带的缺失型LMP1基因序列基本相同。此结果表明,广州地区流行的LMP1羧基端缺失30个碱基的EBV株,漱口液中检测出的缺失型与原型LMP1构成比约为7：3,在鼻咽癌病人和非鼻咽癌病人此分布情况相似。     

Epstein-Barr Virus Latent Membrane Protein 2A Contributes to Anoikis Resistance through ERK Activation

Epstein-Barr virus (EBV) is associated with various malignancies, including epithelial cancers. In this study, we analyzed the effect of EBV infection on epithelial cells by using EBV-converted epithelial cells. In EBV-positive cells, the extracellular signal-regulated kinase (ERK) pathway is constitutively activated. Inhibition of ERK activity leads to reduced anoikis resistance; therefore, EBV-positive cells are more resistant to anoikis, a type of apoptosis induced by cell detachment, than are EBV-negative cells. Among the viral genes expressed in EBV-positive cells, the latent membrane protein 2A (LMP2A) is responsible for induction of ERK-mediated anoikis resistance, although the expression level of LMP2A is much lower in EBV-positive cells than in EBV-transformed B cells. Further analysis demonstrated that LMP2A downregulation of the proanoikis mediator Bim through proteasomal degradation is dependent on the immunoreceptor tyrosine-based activation motif (ITAM). These findings suggest that LMP2A-mediated ERK activation is involved in the generation of EBV-associated epithelial malignancies.     

EB病毒LMP1在鼻咽癌细胞中调控核转录因子κB活性研究

为了探讨ＥＢ病毒潜伏膜蛋白１（ＬＭＰ１）的致瘤机制,对鼻咽癌细胞中ＬＭＰ１通过核转录因子ｋＢ（ＮＦｋＢ）介导的信号传导途径在鼻咽癌变中的意义进行了研究。利用ＬＭＰ１受四环素衍生物强力霉素诱导表达的鼻咽癌细胞Ｔｅｔ－ｏｎ－ＬＭＰ１ＨＮＥ２,通过ＮＦｋＢ报道基因分析法、凝胶迁移率分析（ＥＭＳＡ）及细胞集落形成率等方法,结合硫代磷酸反义寡核苷酸阻断技术,证实ＬＭＰ１增强鼻咽癌细胞ＮＦｋＢ的ＤＮＡ结合活性     

Role of the TRAF Binding Site and NF-κB Activation in Epstein-Barr Virus Latent Membrane Protein 1-Induced Cell Gene Expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-κB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-κB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IκBα blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-κB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.     

Tet调控的EB病毒LMP1基因导入鼻咽癌细胞系表达的研究

本文利用Ｔｅｔ－ｏｎ基因表达系统建立了一株可诱导ＥＢ病毒ＬＭＰ１基因表达的鼻咽癌细胞株。首先将Ｔｅｔ－ｏｎ基因调控系统的调节质粒ｐＴｅｔ－ｏｎ导入鼻咽癌细胞系ＨＮＥ２中,用ｒｔＴＡ反应性芝光素酶报道基因ｐＴＲＥ－ｌｕｃ挑选高诱导、低背景的克隆,第二轮转染将构建的反应质粒ｐＴＲＥ－ＬＭＰ１导入得到稳定转染的阳性克隆,对各克隆用Ｗｅｓｔｅｒｎ印迹方法进行强力霉素诱导效应检测,其中Ｌ７对Ｄｏｘ具有良好的     

Tyrosine 112 of Latent Membrane Protein 2A Is Essential for Protein Tyrosine Kinase Loading and Regulation of Epstein-Barr Virus Latency

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.     

Epstein-Barr Virus Latent Membrane Protein 2 Effects on Epithelial Acinus Development Reveal Distinct Requirements for the PY and YEEA motifs

Epstein-Barr virus (EBV) is a gammaherpesvirus associated with numerous cancers, including the epithelial cancers nasopharyngeal carcinoma (NPC) and gastric carcinoma. The latent membrane protein 2 (LMP2) encoded by EBV is consistently detected in NPC tumors and promotes a malignant phenotype when expressed in epithelial cells by inducing transformation and migration and inhibiting differentiation. Grown in three dimensions (3D) on Matrigel, the nontumorigenic mammary epithelial cell line MCF10A forms hollow, spherical acinar structures that maintain normal glandular features. Expression of oncogenes in these cells allows for the study of multiple aspects of tumor development in a 3D culture system. This study sought to examine the effects of LMP2 on the generation of MCF10A acini. LMP2 expression induced abnormal acini that were large, misshapen, and filled, indicating that LMP2 induced proliferation, impaired cellular polarization, and induced resistance to cell death, leading to luminal filling. Induction of cell death resistance required the PY, immunoreceptor tyrosine activation motif (ITAM), and YEEA signaling domains of LMP2 and activation of the Src and Akt signaling pathways. The PY domain was required for the inhibition of anoikis and also the delayed proliferative arrest of the LMP2-expressing cells. In addition to directly altering acinus formation, expression of LMP2 also induced morphological and protein expression changes consistent with epithelial-mesenchymal transition (EMT) in a manner that required only the YEEA signaling motif of LMP2. These findings indicate that LMP2 has considerable transforming properties that are not evident in standard tissue culture and requires the ability of LMP2A to bind ubiquitin ligases and Src family kinases.     

Tyrosylprotein Sulfotransferase-1 and Tyrosine Sulfation of Chemokine Receptor 4 Are Induced by Epstein-Barr Virus Encoded Latent Membrane Protein 1 and Associated with the Metastatic Potential of Human Nasopharyngeal Carcinoma

The latent membrane protein 1 (LMP1), which is encoded by the Epstein-Barr virus (EBV), is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC), a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1), an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR) pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.