Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.     

乙型肝炎病毒X蛋白对细胞凋亡的影响存在剂量依赖性

乙型肝炎病毒(HBV)X蛋白(HBx)与HBV相关肝细胞肝癌(HCC)的发生和发展密切相关.深入研究HBx在HCC形成中的作用将为探索HBV致癌机制提供重要依据.HBx是多功能蛋白,其对细胞凋亡的影响至今仍存在分歧.许多研究表明,HBx既有促进细胞凋亡又有抑制细胞凋亡的功能,但原因不清楚.本研究中,将表达HBx的质粒短...     

A comprehensive molecular interaction map for Hepatitis B virus and drug designing of a novel inhibitor for Hepatitis B X protein

Hepatitis B virus (HBV) infection is a leading source of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. In this study, we use computation methods in order to improve our understanding of the complex interactions that occur between molecules related to Hepatitis B virus (HBV). Due to the complexity of the disease and the numerous molecular players involved, we devised a method to construct a systemic network of interactions of the processes ongoing in patients affected by HBV. The network is based on high-throughput data, refined semi-automatically with carefully curated literature-based information. We find that some nodes in the network that prove to be topologically important, in particular HBx is also known to be important target protein used for the treatment of HBV. Therefore, HBx protein is the preferential choice for inhibition to stop the proteolytic processing. Hence, the 3D structure of HBx protein was downloaded from PDB. Ligands for the active site were designed using LIGBUILDER. The HBx protein's active site was explored to find out the critical interactions pattern for inhibitor binding using molecular docking methodology using AUTODOCK Vina. It should be noted that these predicted data should be validated using suitable assays for further consideration.     

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.     

The hepatitis B virus protein MHBs(t) sensitizes hepatoma cells to TRAIL-induced apoptosis through ERK2

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Infectious agents, such as hepatitis B virus (HBV), may play important roles in regulating the sensitivity of hepatocytes to TRAIL. Our previous studies showed that HBx, a protein encoded by the HBV genome, enhanced TRAIL-induced apoptosis through upregulating Bax. We report here that another HBV protein called MHBs(t) (C-terminally truncated middle hepatitis B surface protein) is also a potent regulator of TRAIL-induced apoptosis. Overexpressing MHBs(t) in hepatoma cells enhanced TRAIL-induced apoptosis. Mechanistic studies reveal that MHBs(t) had no effect on Bax or TRAIL receptor expression or procaspase-8 activation, but selectively enhanced the activation of ERK2 (extracellular signal-regulated kinase 2) and the degradation of procaspases-3 and 9. ERK2 activation is required for the MHBs(t) effect because ERK2 inhibition by its inhibitor PD98059 significantly reversed TRAIL-induced apoptosis of MHBs(t)-transfected cells. These results establish that unlike HBx, MHBs(t) enhances TRAIL-induced hepatocyte apoptosis through a novel mechanism that involves ERK2. Therefore, manipulating the ERK2 signaling pathway may provide new therapeutic opportunities to contain hepatic cell death during HBV infection. Xiaohong Liang and Juan Du contributed equally to this work.     

Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

Death receptors of TNFSF10/TRAIL (tumor necrosis factor superfamily member 10) contribute to immune surveillance against virus-infected or transformed cells by promoting apoptosis. Many viruses evade antiviral immunity by modulating TNFSF10 receptor signaling, leading to persistent infection. Here, we report that hepatitis B virus (HBV) X protein (HBx) restricts TNFSF10 receptor signaling via macroautophagy/autophagy-mediated degradation of TNFRSF10B/DR5, a TNFSF10 death receptor, and thus permits survival of virus-infected cells. We demonstrate that the expression of the TNFRSF10B protein is dramatically reduced both in liver tissues of chronic hepatitis B patients and in cell lines transfected with HBV or HBx. HBx-mediated downregulation of TNFRSF10B is caused by the lysosomal, but not proteasomal, degradation pathway. Immunoblotting analysis of LC3B and SQSTM1, and microscopy analysis of tandem-fluorescence-tagged LC3B revealed that HBx promotes complete autophagy. Inhibition of autophagy with a pharmacological inhibitor and LC3B knockdown revealed that HBx-induced autophagy is crucial for TNFRSF10B degradation. Immunoprecipitation and GST affinity isolation assays showed that HBx directly interacts with TNFRSF10B and recruits it to phagophores, the precursors to autophagosomes. We confirmed that autophagy activation is related to the downregulation of the TNFRSF10B protein in liver tissues of chronic hepatitis B patients. Inhibition of autophagy enhanced the susceptibility of HBx-infected hepatocytes to TNFSF10. These results identify the dual function of HBx in TNFRSF10B degradation: HBx plays a role as an autophagy receptor–like molecule, which promotes the association of TNFRSF10B with LC3B; HBx is also an autophagy inducer. Our data suggest a molecular mechanism for HBV evasion from TNFSF10-mediated antiviral immunity, which may contribute to chronic HBV infection.     

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.     

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。     

CRL4在增殖的肝细胞系中上调乙型肝炎病毒抗原的分泌表达

乙型肝炎病毒（hepatitis B virus,HBV）编码的X蛋白（hepatitis B virus X protein,HBx）对HBV感染的起始和维持至关重要。HBx可能作为病毒来源的接头分子,介导Cullin-RING E3泛素连接酶4（cullin-RING ubiquitin E3 ligase 4,CRL4）复合物对染色体外DNA限制因子SMC5/6的降解。最近研究发现CRL4接头分子DNA损伤结合蛋白1（DNA damage-binding protein 1,DDB1）可不依赖与HBx的相互作用而直接上调病毒的表达和复制。本研究基于HBx基因删除（X-null）的HBV重组cccDNA（recombinant covalently closed circular DNA,rcccDNA）模型系统,在多种体外培养肝细胞系中证实上述发现。有意思的是,CRL4刺激rcccDNA^X-null转染细胞抗原分泌表达的效应能被血清饥饿实验抵消。应用尾静脉高压注射小鼠模型,同样发现CRL4并不上调rcccDNA^X-null在非增殖小鼠肝脏细胞中的表达。以上结果提示,细胞增殖特征与CRL4不依赖HBx上调病毒抗原表达的效应密切相关,有助于HBx生物学意义的准确分析和理解。     

肝胚瘤细胞株HepG_2中HBx蛋白对SOCS3表达的影响及其机制

目的探讨乙型肝炎病毒X蛋白(Hepatitis B virus X protein,HBx)对肝胚瘤细胞株HepG2表达信号抑制因子3(SOCS3)的影响及其机制。方法将表达HBx蛋白的重组质粒FL1-145HBx转染HepG2细胞,以不同浓度的SRC抑制剂PP2处理转染细胞,运用Western blot和RT-PCR技术分析HBx、SOCS3 mRNA和蛋白的表达情况,并以免疫细胞化学分析转染细胞中p-SRC的表达水平。结果重组质粒FL1-145HBx转染HepG2细胞后,转染细胞HBx能够表达HBx蛋白,并且细胞中SOCS3 mRNA和蛋白表达水平随着HBx蛋白的表达而显著增加(P0.05),同时p-SRC蛋白的表达增强;而SRC抑制剂PP2处理转染细胞后,随着p-SRC蛋白表达的减少SOCS3蛋白表达逐渐下降。结论在肝胚瘤细胞株HepG2中,HBx蛋白很可能通过促进SRC蛋白的磷酸化而诱导SOCS3蛋白的表达。     

Turnover of hepatitis B virus X protein is facilitated by Hdj1, a human Hsp40/DnaJ protein

Hepatitis B virus X (HBX) protein is required for the productive infection of hepatitis B virus (HBV) in vivo and implicated in the development of hepatocellular carcinoma. We have previously shown that hTid-1 and Hdj1, the human Hsp40/DnaJ chaperone proteins, bind the HBV core protein and inhibit viral replication in cell culture system. Here, we report evidences to suggest that HBX is the major target of Hdj1 in the inhibition of HBV replication. Expression of Hdj1 in cultured human hepatoma HepG2 cells facilitated degradation of HBX by the proteasome pathway, and thereby inhibited replication of the wild-type HBV as well as that of the HBX-deficient mutant virus rescued by HBX supplied in trans. Mutational analyses indicated that J domain of Hdj1 is required for the process. These results might provide a molecular basis for the antiviral effect of cellular chaperones.     

HBx-EGFP-TLM融合蛋白表达载体的构建、蛋白表达纯化及活性检测

乙型肝炎病毒X蛋白 (HBx) 具有广泛的转录激活作用,但是由于细胞类型和实验条件的差异,外源启动子介导的HBx瞬时表达水平常表现出不均一性,为其功能的研究带来困难。为了解决HBx研究过程中遇到的这些难题,利用PCR扩增获得HBx及含转导肽TLM的EGFP编码序列,经酶切后插入pGEX-4T-1原核表达载体,成功构建重组质粒pGEX-HBx-EGFP-TLM和pGEX-EGFP-TLM。重组质粒转化大肠杆菌BL21(DE3) 感受态细胞,融合蛋白经IPTG诱导表达,采用?KTATM Purifier 蛋白纯化系统纯化并进行SDS-PAGE和Western blotting分析,确定为目的蛋白。将纯化后的融合蛋白分别与AML12和SMMC-7721细胞共孵育,Western blotting和激光共聚焦显微镜检测证实TLM转导肽能够介导HBx-EGFP和EGFP进入细胞,同时进入细胞的HBx-EGFP-TLM能够发挥转录激活活性,为HBx功能的深入研究奠定了基础。     

Intracellular hepatitis B virus increases hepatic cholesterol deposition in alcoholic fatty liver via hepatitis B core protein

Hepatitis B virus (HBV) infection is a prevalent infectious disease with serious outcomes like chronic and acute hepatitis, cirrhosis, and hepatocellular carcinoma. However, the metabolic alteration by HBV is rarely taken into consideration. With the high prevalence of alcohol consumption and chronic HBV infection, their overlap is assumed to be an increasing latent hazard; although the extent has not been calculated. Moreover, the impact of chronic alcohol consumption combined with HBV on cholesterol metabolism is unknown. Six-week-old male FVB/Ncrl mice were hydrodynamically injected with a pGEM-4Z-1.3HBV vector and then fed an ethanol diet for 6 weeks. Serum biomarkers and liver histology, liver cholesterol levels, and cholesterol metabolism-related molecules were measured. In vitro assays with HBx, hepatitis B surface (HBs), or hepatitis B core (HBc) protein expression in HepG2 cells costimulated with ethanol were conducted to assess the cholesterol metabolism. HBV expression synergistically increased cholesterol deposition in the setting of alcoholic fatty liver. The increase of intrahepatic cholesterol was due to metabolic alteration in cholesterol metabolism, including increased cholesterol synthesis, decreased cholesterol degradation, and impaired cholesterol uptake. Overexpression of HBV component HBc, but not HBs or HBx, selectively promoted the hepatocellular cholesterol level.     

乙肝病毒感染对细胞基本自噬的影响

【目的】慢性乙肝病毒(Hepatitis B virus,HBV)感染在肝硬化和肝癌的发生过程中起着重要的作用,通过研究HBV感染对细胞基本自噬的影响,为HBV感染诱发肝癌以及HBV的免疫逃逸机理研究提供新的思路。【方法】本研究利用乙肝病毒表达质粒瞬时或稳定转染不同肝细胞,通过计数绿色荧光蛋白(greenfluorescent protein,GFP)聚集数目检测自噬小体形成,western blot检测LC3(microtubule-associated proteinlight chain 3,微管相关蛋白质轻链3)脂酰化和p62的降解,通过构建HBV B型和C型X蛋白(HBx)的表达质粒并瞬时转染肝癌细胞和正常肝细胞,对不同基因型X蛋白对细胞自噬的影响进行了分析。【结果】乙肝病毒感染后促进了LC3的脂酰化和p62的降解,增加了自噬小体的形成,增强了细胞的基本自噬。进一步研究发现,HBV感染增强的细胞基本自噬水平由HBx所引发,且C型HBx比B型对细胞基本自噬的增加更加显著。【结论】HBV通过HBx增强细胞的基本自噬,且不同基因型HBx对细胞基本自噬的增强程度不同,为进一步阐明HBV感染机理奠定了基础。