乙型肝炎病毒C蛋白和X蛋白的修饰及其意义

乙型肝炎病毒(HBV)作为一个外来入侵的病毒可以激发人体的免疫反应,导致人体一系列与HBV感染有关的病理变化与临床表现.HBC和HBX是由HBV基因编码的两种重要蛋白.HBC对病毒基因组的复制和成熟是必不可少的,其蛋白修饰后的产物对病毒在宿主细胞内的组装、成熟和对外分泌中发挥着作用.HBX能与多种蛋白发生相互作用,在HBV的复制中起着不可或缺的作用,也影响着肝细胞癌的形成.现有的对HBC和HBX的磷酸化和泛素化过程的研究,为进一步揭示HBV的致病机制提供了依据,希望最终为乙型肝炎的临床治疗寻找一条新的途径.     

HBXIP基因对乙肝病毒X蛋白诱导细胞凋亡的影响

探讨乙型肝炎病毒X蛋白结合蛋白(hepatitisBXinteractingprotein ,HBXIP)基因在乙型肝炎病毒X蛋白(HBX)诱导肝癌细胞凋亡时对细胞周期的影响.构建HBXIP基因真核表达载体pcDNA3 hbxip ,进行瞬时基因转染,将克隆有HBx基因的pCMV X (分别为1μg、2 μg和3μg)和pcDNA3 hbxip质粒分别和共转染至人H74 0 2肝癌细胞中(总体积分别为5 0 μl) .发现瞬时转染3μgpCMV X质粒后,肝癌细胞凋亡发生率为34 4 % ,肝癌细胞的细胞周期相关蛋白p2 7表达水平发生明显上调;与对照组相比,瞬时转染1μg、2 μg和3μg时,细胞周期蛋白D和细胞周期蛋白E的表达水平均发生明显上调,但随着HBX水平的增加细胞周期蛋白D和细胞周期蛋白E的表达水平发生明显下降;在稳定转染pCMV X质粒的H74 0 2 X肝癌细胞中无明显的细胞凋亡发生,研究发现p2 7的表达水平发生了明显下调,而细胞周期蛋白D和细胞周期蛋白E的表达水平发生了明显上调;当pcDNA3 hbxip质粒与pCMV X质粒进行共瞬时转染时,细胞凋亡发生率由pcDNA3质粒与pCMV X质粒共转染时的2 9 2 %下降为13 3% ,p2 7的表达水平发生了下调,但细胞周期蛋白D和细胞周期蛋白E的表达水平无明显变化.研究结果表明,瞬时转染一定剂量的x基因可导致肝癌细胞发生凋亡,细胞周期相关蛋白p2 7、细胞周期蛋白D和     

乙肝病毒X蛋白结合蛋白(HBXIP)增强肝癌细胞NF-κB转录活性的实验研究

前期研究结果表明,乙型肝炎病毒X蛋白结合蛋白(hepatitis B virus X-interacting protein,HBXIP)具有促进细胞增殖的作用.为了进一步阐明其分子机制,观察了HBXIP对核因子κB(NF-κB)转录活性的影响.实验中通过基因共转染将NF-κB报告基因质粒pNF-κB-Luc和HBXIP真核表达载体pcDNA3-hbxip导入人肝癌H7402细胞系中,进行荧光素酶活性分析.结果显示:H7402细胞过表达HBXIP后NF-κB的转录活性明显增强;此外,基因转染后经免疫印迹检测显示,与NF-κB二聚体结合的抑制亚基IκBα的磷酸化水平明显增加;同时,提取H7402细胞的核蛋白,然后应用免疫印迹检测细胞核中p65/NF-κB的水平.结果显示,H7402细胞中HBXIP过表达后细胞核中p65/NF-κB的水平明显增加.当应用RNA干扰技术抑制了细胞内源性的HBXIP基因表达后,则出现与上述结果相反的效果.上述结果提示,HBXIP可增加核内p65/NF-κB蛋白水平,进而发挥NF-κB促转录调控的作用.因此,HBXIP可通过调控NF-κB信号途径而促进细胞增殖.     

乙型肝炎病毒X蛋白的产生和作用机理

乙型肝炎病毒（ＨＢＶ）基因组的４个开放读框（ＯＲＦ）,分别编码包括Ｘ蛋白在内的７种病毒蛋白;其中Ｘ蛋白具有广泛的反式激活作用,并且可能和与ＨＢＶ感染相关的肝细胞癌的发生有直接关系〔１〕。ＨＢＶＸ蛋白已证明不与双链ＤＮＡ相结合,其反式激活作用的发挥有赖...     

乙型肝炎病毒X蛋白(HBx):一种多功能的病毒调节因子

乙型肝炎病毒(HBV)的慢性感染是导致肝细胞癌(HCC)的主要危险因子。X蛋白(HBx)被认为在肝细胞癌的发生发展中起重要作用。X基因是HBV基因组最小的开放读码框,它编码的X蛋白含154个氨基酸,分子量约为16．5kD。HBx是一种多功能的病毒调节因子,通过调节细胞和病毒的转录活性、信号传导途径、基因毒性压力反应、蛋白质降解等,直接或间接地影响HBV的复制和增殖。HBx亦可影响细胞周期调控、细胞凋亡,从而可能在慢性活动性肝炎和肝硬化的病程中起到起始肿瘤形成的作用。     

S100A9参与乙型肝炎病毒X蛋白介导的HepG2细胞增殖与迁移

目的:探讨S100A9在乙型肝炎病毒X(HBx)介导的HepG2细胞增殖及迁移中的作用。方法:用表达HBx蛋白的重组腺病毒AdHBx感染HepG2细胞后,用CCK-8实验检测细胞增殖能力及划痕愈合实验检测细胞迁移能力;在HepG2/AdHBx细胞中转染S100A9-siRNA及其对照siRNA后,检测HepG2细胞增殖及迁移能力;在HepG2/Ad HBx和对照组HepG2/AdGFP细胞中,采用Real-time PCR及Western Blot检测S100A9基因及蛋白的表达情况;在HepG2/AdHBx细胞中,加入不同剂量的NF-κB抑制剂BAY11-7082后,检测各组中S100A9的基因及蛋白表达情况。结果:HBx促进HepG2细胞的增殖与迁移; S100A9-siRNA抑制S100A9的表达后,HBx促进HepG2细胞的增殖与迁移的作用降低,HBx介导的HepG2细胞的增殖与迁移部分依赖于S100A9; S100A9基因及蛋白表达在HepG2/AdHBx中较对照组HepG2/Ad GFP显著升高,HBx可致S100A9表达增加;抑制NF-κB转录活性后,AdHBx+BAY11-7082组S100A9基因及蛋白表达较对照组显著降低,阻断NF-κB转录活性可部分抑制HBx调控的S100A9表达。结论:HBx可调控S100A9的表达且与NF-κB活化有关,S100A9参与HBx介导的HepG2细胞的增殖与迁移。     

乙型肝炎病毒X基因及HBx蛋白的研究进展

我国是乙型肝炎高发区,乙型肝炎病毒(HBV)X基因及其编码的多功能蛋白HBx是乙型肝炎病毒基因转录所必需的作用因子,在乙肝的致病过程中起到重要作用。HBx可直接或间接改变肝脏结构细胞的结构和功能,引发肝脏细胞的凋亡;具有广泛的非特异性反式激活作用,反式激活细胞内的某些癌基因或病毒基因,与乙型肝炎和肝细胞癌的发生关系十分密切。本文从多方面综述了X基因及HBx蛋白目前的研究进展,展现了X基因功能的多样性。     

乙型肝炎病毒X蛋白与端粒酶的关系

端粒（telomere）是真核细胞染色体末端的重复序列,对维持染色体的稳定性具有重要作用 .端粒酶（telomerase）是维持端粒长度必需的一种逆转录酶,在细胞增殖、衰老、永生化 和癌变等方面具有重要的作用.在肿瘤细胞中端粒酶发生明显变化,有较高水平的表达和很 强的活性,成为肿瘤细胞的重要特征之一.乙型肝炎病毒（hepatitis B virus）X蛋白（HBx ）与肝癌的发生密切相关,HBx可以通过多种途径发挥致癌作用,其中一个重要的分子机制 ,即激活人端粒酶反转录酶hTERT（为端粒酶的催化亚单位）使细胞发生永生化而癌变.因此 ,详细阐明HBx与hTERT的关系对于揭示乙肝病毒致癌机制具有重要的意义.本文对HBV的分子 结构和HBx的分子生物学特性进行了详细的阐述,对HBx与hTERT及其调控因子的关系进行了 归纳和总结,有助于进一步阐明HBx的致癌机制.     

乙肝病毒X蛋白诱导肝癌细胞凋亡的信号转导途径

乙型肝炎病毒(hepatitisBvirus,HBV)X蛋白(HBX)与肝癌(hepatocellularcarcinoma,HCC)的发生具有密切的关系.HBX不但具有拮抗细胞凋亡的作用,还具有促进细胞凋亡的作用.为了进一步探讨HBX促细胞凋亡作用的分子机制,通过脂质体转染的方法将携带X基因的真核表达载体pCMVX导入H7402肝癌细胞,使乙肝病毒x基因(HBx)瞬时过量表达.流式细胞仪检测结果显示,在瞬时转染3μgpCMVX质粒后,肝癌细胞发生凋亡.为阐明HBX诱导细胞凋亡的信号转导途径,对HBX与线粒体释放细胞色素c的关系做了初步探讨.通过罗丹明123染色,经流式细胞仪分析,显示在转染HBx基因后细胞线粒体膜电位明显下降,表明HBX可促进细胞色素c从线粒体释放增加.Western印迹检测结果显示,肝癌细胞在导入HBx基因后,细胞凋亡线粒体转导途径中细胞色素c、Apaf1、procaspase3和procaspase9等的表达水平均上调.研究结果说明,HBX可通过影响线粒体凋亡途径促进肝癌细胞凋亡.     

乙肝病毒X蛋白通过激活NF-κB信号通路上调ABCG2的表达

目的：研究乙肝病毒X蛋白（HBx）通过核因子-κB（NF-κB）信号通路对半转运蛋白（ABCG2）的调节作用。方法：用特异性的NF-κB信号通路阻断剂PDTC阻断NF-κB信号通路,荧光双标激光扫描共聚焦显微镜观察L02细胞系转染HBx基因前后及PDTC加入前后NF-κB信号通路的激活、失活情况,同时用Real-time PCR和Western Blot技术检测转染前后及PDTC加入前后ABCG2在mRNA及蛋白水平的表达变化。结果：以L02细胞为参照,转染HBx基因后的L02-HBx细胞NF-κB信号通路被激活,ABCG2 mRNA和蛋白水平分别增加3.62±0.15和4.61±0.73倍,差异有统计学意义（P〈0.05）;PDTC作用24h后L02/HBx细胞NF-κB信号通路阻断,ABCG2 mRNA和蛋白表达分别为2.15±0.32倍和2.37±0.55倍,与未加入PDTC作用的L02-HBx细胞相比均有统计学意义（P〈0.05）。结论：NF-κB信号通路是HBx上调ABCG2表达的途径之一。     

COX-2 mediates hepatitis B virus X protein abrogation of p53-induced apoptosis

The oncogenic hepatitis B virus X protein (HBx) and cyclooxygenase (COX)-2 are highly co-expressed in chronic hepatitis, cirrhosis and well-differentiated hepatocellular carcinoma (HCC). Although HBx is shown to activate COX-2, the functional consequences of this interaction in hepatocarcinogenesis remain unknown. Using an engineered hepatoma cell system in which the expression of wild-type p53 can be chemically modulated, we show here that COX-2 mediates HBx actions in opposing p53. Enforced expression of HBx sequestrates p53 in the cytoplasm and significantly abolishes p53-induced apoptosis. The anti-apoptotic Mcl-1 protein is suppressed by p53 but reactivated by HBx. The abrogation of apoptosis is completely reversed by specific COX-2 inhibition, suggesting that HBx blocks p53-induced apoptosis via activation of COX-2/PGE₂ pathway. We further show that COX-2 inhibition blocks HBx reactivation of Mcl-1, linking this protein to the anti-apoptotic function of COX-2. These results demonstrate that COX-2 is an important survival factor mediating the oncogenic actions of HBx. Over-expression of HBx and COX-2 may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to the initiation and progression of HCC.     

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.     

Putative tumor suppressor YueF affects the functions of hepatitis B virus X protein in hepatoma cell apoptosis and p53 expression

Previously, we identified YueF as a novel Hepatitis B virus X protein (HBx)-interacting protein. Herein, we studied the functions of YueF and HBx in hepatocarcinogenesis. YueF was expressed at high levels in normal human hepatic cells and tissues, but scarcely found in hepatoma cells or other tumor tissues. Over-expression of YueF, or YueF and HBx could induce cell apoptosis and enhance p53 expression in hepatoma cells, whereas over-expression of HBx alone behaved contrarily. These results indicate that YueF has tumor suppressor activity and affects the functions of HBx in cell apoptosis and p53 expression in hepatoma cells.     

乙型肝炎病毒X蛋白的优势氨基酸序列与热点突变位点的生物信息学分析

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)全长154个氨基酸,与肝癌发生密切相关.为确定HBx的优势氨基酸序列和热点突变位点,在GenBank中下载所有HBx的氨基酸序列13950条,剔除插入突变、缺失突变和起始密码子非甲硫氨酸的序列,最后保留7126条.通过分析这7126条序列,计算出HBx每个位点的氨基酸分布情况,出现频率最高的氨基酸为该位点的优势氨基酸,其他氨基酸为突变氨基酸.154个位点的优势氨基酸组成HBx优势氨基酸序列.突变率>10.0%的热点突变位点有32个.其中第36、42、44、87、88和127位氨基酸有4种(突变率>1.0%)以上突变形式,具有较高的多态性.与肝癌密切相关的K130M/V131I双突变率为34.7%.通过7126条HBx序列与优势序列的同源性比较,随机选出其中50条序列(2条与优势序列同源性<75%,48条同源性为76%~99%),与23条参考序列及优势序列共同构建系统发生树.结果显示,HBx优势氨基酸序列属于基因型C,这与基因型C为全球主要流行型一致.本研究首次系统性分析了GenBank中HBx的优势序列,确定了32个HBx热点突变位点和6个多态性较高的位点,为基于HBx突变的基础和应用研究奠定了基础.     

Interaction of hepatitis B virus X protein with damaged DNA-binding protein p127: Structural analysis and identification of antagonists

The hepatitis B virus X protein is a multifunctional protein that is essential for natural infection and has also been implicated in liver cancer development. Previous studies have identified the DDB1 subunit of the damaged-DNA binding complex as a critical partner of X protein in the infection process, X-mediated cytotoxicity and stability of the viral protein. Here, we investigated the structural and functional constraints of X-DDB1 interaction using various mutational analyses. Our data show that the interaction interface of X with DDB1 is confined to a 15-residue epitope. All substitutions responsible for loss of binding mapped to this core-binding domain. In contrast, a marked increase in affinity for DDB1 resulted from substitutions at clustered positions lying close to the DDB1-binding epitope and correlated with loss of apoptotic potential. Selection of mutations in DDB1 that partially rescue the binding defect of an X mutant gave further insight into the contacts established between the two proteins. Importantly, both the core-binding domain of X and the gain-of-affinity X mutants inhibited DDB1-mediated stabilization of wild-type X protein. These X protein derivatives thus provide the basis for the development of therapeutic agents that antagonize X function through competitive inhibition of X-DDB1 interaction.     

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a modelin vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL-induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.     

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is one member of TNF superfamily[1]. It is unique, for it could specifically induce the apoptosis of tumor cells or virus-infected cells but have no cytotoxic effects onnormal cells[1,2]. Owing to this characteristic, it has become a promising candidate molecule for biological therapy for tumor. Many factors could affect the sensitivity towardsTRAIL-induced apoptosis, including cytokines, virus infection, drugs, radials, etc. Studies show tha…