EB病毒编码的潜伏膜蛋白与泛素蛋白酶系统

EB病毒(Epstein-Barr virus,EBV)是人类疱疹病毒,与淋巴系统、上皮细胞肿瘤相关。其编码潜伏性膜蛋白(LMPl、LMP2A和LMP2B)特别是LMP1,由于它是众多EBV编码蛋白质中唯一被明确证明能恶性转化原代B细胞、鼠成纤维细胞和人上皮细胞的蛋白质,所以被列为癌基因。最近对潜伏膜蛋白的研究显示．潜伏膜蛋白与病毒利用泛素蛋白酶系统来达到逃避宿主免疫应答等机制有关,研究这个过程也许可以开发新的策略来防治EBV相关肿瘤。     

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.     

Role of the Immunoreceptor Tyrosine-Based Activation Motif of Latent Membrane Protein 2A (LMP2A) in Epstein-Barr Virus LMP2A-Induced Cell Transformation

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is widely expressed in EBV-associated malignancies. We demonstrate that LMP2A has a transformation ability. This study shows that LMP2A-induced transformation in several human nonhematopoietic cell lines was blocked in those cells expressing an immunoreceptor tyrosine-based activation motif (ITAM) LMP2A mutant. The Syk inhibitor or Syk-specific small interfering RNA (siRNA) inhibited LMP2A-induced transformation. These results indicate that the interaction of the LMP2A ITAM with Syk is a key step for LMP2A-mediated transformation.     

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.     

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.     

EB病毒潜伏膜蛋白1在鼻咽癌细胞中通过ERK介导Ets-1表达

为了探讨EB病毒编码的潜伏膜蛋白1(LMP1)对核转录因子Ets-1表达和活化的影响,并证实细胞外信号调节激酶1/2（ERK1/2）参与了该过程,选用可调控表达LMP1的鼻咽癌细胞系L7,应用蛋白质印迹法检测Ets-1、p-ERK蛋白质表达,免疫共沉淀-蛋白质印迹法检测Ets-1磷酸化状态,使用ERK1/2特异性小分子阻断物PD98059作用后,蛋白质印迹法检测p-ERK、Ets-1表达及磷酸化变化.结果显示：在L7细胞中诱导性LMP1可促进p-ERK、Ets-1蛋白质表达及其苏氨酸残基磷酸化,在一定范围呈时间和剂量效应;通过PD98059对诱导性LMP1作用的干预发现,p-ERK大部分表达被阻断,而Ets-1表达及其苏氨酸磷酸化也被部分阻断,以上结果提示ERK部分介导了LMP1诱导Ets-1表达和活化.     

Establishment of Novel Monoclonal Fabs Specific for Epstein-Barr Virus Encoded Latent Membrane Protein 1

<正>Dear Editor,Epstein-Barr virus (EBV, also termed human herpesvirus-4) was the first identified human tumor virus. Since its discovery in 1964, studies have shown that EBV infects over 90%of all people by the time they are adults(Williams and Crawford 2006). EBV infection can result in     

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 Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.     

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.     

鼻咽癌细胞中EB病毒编码的潜伏膜蛋白1活化cyclinD1的表达

为了探讨EB病毒编码的潜伏膜蛋白1(EBV-LMP1)促进细胞增殖,参与EBV相关疾病致瘤的分子机制,研究了LMP1在鼻咽癌细胞中调节cyclinD1表达,进而影响细胞周期行进及细胞恶性表型改变,并初步确定了LMP1发挥该功能的结构域.利用已建株的Tet-on-LMP1-HNE2鼻咽癌细胞系,蛋白质印迹实验分析LMP1诱导cyclinD1蛋白质表达的表达动力学,包括时间效应及剂量效应;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照,确定LMP1活化cyclinD1表达的结构域.同时结合基因诱导表达及反义寡聚核酸技术阻断基因表达的实验方法,进一步确定LMP1上调的cyclinD1功能,即对细胞周期行进及细胞恶性表型的影响.结果表明LMP1确实可以诱导cyclinD1的表达（2～4倍）,且诱导具有时间依赖性及剂量依赖性;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照,结合报道基因分析法,确定与空白载体细胞系比较,野生型LMP1从转录水平可反式激活cyclinD1报道基因活性约11.2倍,其中CTAR1及CTAR2均可活化cyclinD1表达,但以CTAR2为主,与野生型LMP1诱导cyclinD1反式激活活性比较,CTAR1缺失导致cyclinD1报道基因活性下降23.6%,CTAR2缺失导致cyclinD1活性下降约80.7%,C端均缺失时cyclinD1活性只有野生型的17.7%.流式细胞仪分析显示,强力霉素诱导后cyclinD1高表达的细胞停留于G0/G1期明显减少,较未经诱导的细胞,从66.42%减至56.55%,而进入S期及G2/M期的细胞明显增多.在稳定表达LMP1的细胞中,与导入正义LMP1比较,导入反义LMP1 PS-ODNs及反义cylinD1,可以使细胞软琼脂集落形成率明显降低(从30.48%分别降至15.21%,21.76%).EBV-LMP1可以活化cyclinD1的表达,且发挥这种功能的结构域以CTAR2为主,活化的cyclinD1参与细胞周期行进,抑制LMP1及cyclinD1的表达均可导致细胞软琼脂集落形成率降低.     

EB病毒潜伏膜蛋白1调控细胞凋亡的cDNA阵列分析

为探讨EB病毒潜伏膜蛋白（LMP1）介导细胞凋亡和抑制细胞凋亡双重效应的机制,采用已建立的受四环素调控的LMP1表达的鼻咽癌系统（pTet-on-LMP1 HNE2),定量诱导pTet-on-LMP1 HNE2细胞LMP1动态表达,分别与含有细胞凋亡相关基因为主的Atlas apoptosis cDNA expression array膜杂交,分析LMP1介导 的表达差异基因。结果表明：①LMP1介导细胞凋亡和抑制细胞凋亡的基因的表达,同时上调或下调某些细胞凋亡相关基因的表达;②LMP1不仅介导细胞凋亡和抑制凋亡基因的表达,同时介导细胞分裂分化和增殖基因的表达,LMP1同时介导功能不同甚至功能相反的基因表达;③LMP1介导的基因表达与其表达持续时间和表达水平相关,不同表达水平和不同表达时间介导的基因表达谱不同。因此,LMP1具有介导细胞凋亡和抑制凋亡基因表达的双重生物学效应,同时介导细胞增殖、细胞周期、细胞凋亡等多种生物学效应基因的表达,从而参与细胞的癌变。     

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。