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BACKGROUND/AIMS: The hepatocellular transport pathways and cellular proteins utilized during the packaging and secretion of hepatitis B virus are poorly understood. In this study, we tested if the large GTPase dynamin, a protein involved in vesicle formation and secretion at the trans-Golgi network in hepatocytes, is also used by hepatitis B virus (HBV) in secreting viral proteins. METHODS: Using HepG2.2.15 cells expressing the full-length HBV genome, we tested the effects of wild-type and mutant dynamin on the localization and secretion of two hepatitis B antigens, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Distribution of these two antigens was analyzed morphologically in cells transiently transfected with wild-type or mutant dynamin constructs, whereas secretion of the antigens was measured by testing for antigen levels in the media of transfected cells. RESULTS: Mutant dynamin was found to induce a striking redistribution of HBsAg and HBeAg to a perinuclear compartment, as well as a decrease in the levels of HBsAg and HBeAg present in cell culture media indicating a reduction in viral protein secretion. At the electron microscopy level, cells expressing the mutant dynamin showed a marked accumulation of viral particles in dilated cisternae of an uncharacterized cellular compartment. CONCLUSIONS: Intact dynamin function is required for secretion of HBV proteins from hepatocytes through an uncharacterized cellular compartment. 相似文献
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Jingmin Zhang Yafeng Wang Youmei Peng Chongzhen Qin Yixian Liu Jingjing Li Jinhua Jiang Yubing Zhou Junbiao Chang Qingduan Wang 《The Brazilian journal of infectious diseases》2018,22(6):477-486
Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it “HepG2.RL1”. Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection. 相似文献
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Gong ZJ De Meyer S Clarysse C Verslype C Neyts J De Clercq E Yap SH 《Journal of viral hepatitis》1999,6(3):229-236
Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA), is currently used as an immunosuppressive agent in kidney transplant recipients. After oral administration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP-DH). Inhibition of this enzyme results in a depletion of the intracellular GTP and dGTP pools. MPA has been shown to inhibit the replication of a number of viruses, including arena viruses (Junin and Tacaribe), yellow fever virus, reovirus-1, parainfluenza-3 virus, Coxsackie B4 virus, Epstein-Barr virus and human immunodeficiency virus. To examine whether MPA also has an inhibitory effect on HBV replication, experiments were performed using cultures of primary human hepatocytes and HBV-transfected, HepG2 2.2.15 cells. After in vitro infection with HBV in human hepatocytes, HBV covalently-closed-circular (ccc) DNA and HBV mRNAs were detectable in the cells during the 10 days following infection. HBV DNA and hepatitis B surface antigen (HBsAg) were also secreted into the culture medium. In the presence of 10 microg ml-1 MPA (the therapeutic serum level of MPA as an immunosuppressive agent) in culture medium, HBV ccc DNA and HBV mRNAs became undetectable 5 days after treatment was started. The secretion of HBV DNA and HBsAg into the medium was also markedly reduced. No cytotoxic effect of the drug was noted during the experiments. The effect of MPA on HBV replication was abolished by the presence of guanosine (50 microg ml-1). In HepG2 2.2.15 cells (which contain an integrated tandem dimer of the HBV genome), MPA treatment had no significant inhibitory effect on the secretion of HBV DNA and HBsAg into the culture medium. HBV ccc DNA and HBV mRNAs in HepG2 2.2.15 cells were also not affected. The observed effect of MPA on HBV replication in primary human hepatocyte cultures may involve only episomal replication and may have clinical implications, especially before integration of HBV DNA into the host genome. 相似文献
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目的 基于低密度cDNA Macoarray技术筛选出差异表达的干扰素(IFN)α抗病毒基因,以探讨IFN α抗病毒蛋白的表达与HBV复制的关系. 方法 以一定浓度的IFN α处理肝胚瘤细胞株HepG2和HepG2.2.15细胞6h,用cDNA Macroarray分析比较两细胞株IFN α抗病毒基因表达谱,并筛选出差异表达的IFNα抗病毒基因.将表达HBV核心蛋白(HBc)的质粒pHBc-EGFP转染HepG2细胞,RT-PCR法分析HBc对IFN α抗病毒基因表达的影响.将表达抗黏病毒A蛋白(MxA)的表达质粒pcDNA3.1-Flag-MxA转染HepG2.2.15,以酶联免疫吸附试验、Dot blot、Southern blot等方法分别检测HepG2.2.15细胞表达释放的HBsAg与HBeAg、细胞外HBV DNA和细胞内HBV DNA复制中间体(松弛环状DNA、双股线性DNA),以判断HBV复制情况.两组间数据比较采用t检验,组间不同时间点数据比较采用单因素方差分析.结果 cDNA Macroarray分析显示HepG2和HepG2.2.15细胞的抗病毒基因表达谱具有差异性:IFNa抗病毒基因中干扰素诱导跨膜蛋白(IFITM)1、IFITM2、IFITM3、RING4等在HepG2.2.15细胞的表达被部分抑制,而重要的抗病毒蛋白MxA表达被完全抑制.HBc转染组细胞中MxA mRNA表达的相对水平为0.31±0.05,低于空白对照组的0.74±0.04,差异有统计学意义,P<0.05.MxA蛋白转染HepG2.2.15细胞48、72 h后,MxA转染组细胞上清液中HBsAg的S/CO值分别为1.42+0.21和1.58±0.18,HBeAg的S/CO值为1.44±0.14和2.28±0.24,而空白对照组细胞上清液中HBsAg的S/CO值为1.92±0.19和2.79±0.25,HBeAg的S/CO值为2.31±0.46和3.37±0.29,两组细胞上清液中HBV抗原的S/CO值差异均有统计学意义,P值均<0.05.细胞外HBV DNA、胞内HBV复制中间体DNA均无明显变化.结论 HBV及其抗原成分的复制和表达影响着IFNα抗病毒蛋白的表达;HBV通过抑制IFN α抗病毒蛋白的表达而发挥拮抗IFNα的抗病毒活性. 相似文献
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Zhao ZF Yang H Han DW Zhao LF Zhang GY Zhang Y Liu MS 《World journal of gastroenterology : WJG》2006,12(37):6046-6049
INTRODUCTION Hepatitis B is a severe infectious disease threatening peoples’ health all over the world. There is still no efficient therapy to control HBV persistent replication, which may lead to the development of liver cirrhosis and hepatocellualar ca… 相似文献
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目的探讨维甲酸诱导基因-I(RIG-I)在诱导HBV感染IFN产生中的影响,以期对阐明乙型肝炎慢性化机制,寻找新的治疗靶点提供新的思路。方法以RIG-I表达载体flagRIG-l-ful1转染培养6孔板中的HepG2、HepG2.2.15,24h后用水疱性口炎病毒(VSV)感染细胞,然后在0、8、16和24h收集细胞及培养上清液,采用quantitive RT-PCR、Western印迹检测RIG-I、MDA5、IPS-1、ISG54等基因的表达,ELISA检测培养上清液中分泌的IFN-β水平。结果 HepG2.2.15细胞在VSV感染后IFN-β分泌水平(11.18±1.34)pg/mL明显低于HepG2细胞(275.50±22.97)pg/mL(P<0.01);通过质粒flagRIG-I-ful1转染高表达RIG-I后,HepG2.2.15分泌IFN-β的能力恢复至(548.78±57.99)pg/mL与HepG2细胞(532.10±39.34)pg/mL接近的水平(P=0.7013)。结论 HepG2.2.15细胞感染VSV后IFN产生障碍,高表达RIG-I后IFN表达水平得到恢复,提示RIG-I功能缺陷导致抗病毒免疫应答降低,RIG-I可能在HBV感染清除中起重要作用。 相似文献
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目的 研究转染HBV X基因的人肝癌细胞株中蛋白质表达谱的改变,为筛查在HBV相关性肝细胞癌发生中发挥重要作用的关键蛋白质分子奠定基础.方法 利用分子生物学方法建立稳定表达HBV X蛋白(HBx)的肝癌细胞株HepG2+HBx,同时设空载体peDNA3转染细胞HepG2-pcDNA3及HBV全基因转染的人肝癌细胞株HepG2.2.15为对照.PCR法扩增Neo基因检测质粒DNA片段的插入,免疫印迹法检测HBx蛋白的表达.利用固相pH梯度双向凝胶电泳分离3种肝癌细胞株HepG2-pcDNA3、HepG2-HBx和HepG2.2.15的总蛋白,用图像分析软件比较、分析、识别细胞间的差异表达蛋白质.统计学分析采用t检验.结果 获得分辨率高、重复性好的3种细胞的双向电泳(2-DE)图谱.软件分析表明,HepG2-pcDNA3、HepG2-HBx和HepG2.2.15细胞的2-DE凝胶可识别蛋白点分别为(2 095±137)、(2 188±105)和(2 109±20)个.比较HepG2-pcDNA3与HepG2-HBx细胞的2-DE图谱发现37个差异显著的蛋白点,其中21个在HepG2-HBx表达上调,16个下调.6个表达量差异在5倍以上(t=0.027,P<0.05);HepG2.2.15与HepG2-HBx细胞相比,有38个差异显著的蛋白点,其中35个在HepG2-HBx细胞中上调,3个下调,14个表达量差异在5倍以上(t=0.031,P<0.05).结论 HBx基因转染引起人肝癌细胞株蛋白质表达谱的变化,可能与感染的肝细胞发生恶性转化的分子生物学机制有关. 相似文献
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Liu MC Yu M Zhang NL Gong WB Wang Y Piao WH Wang QH Wang GQ 《Journal of viral hepatitis》2004,11(2):124-129
The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents. 相似文献
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目的研究QSG-7701及HepG2细胞支持HBV复制模式的差异及其内在机制。方法质粒PUC18-HBV1.2转染QSG-7701与HepG2细胞后定量检测细胞上清液中HBV DNA和HBsAg;采用基因芯片技术比较分析二者基因表达差异并用实时定量PCR验证。结果HepG2细胞在转染后6 d内培养上清液可检出HBV DNA及HBsAg,QSG-7701细胞转染后2周内均可检出HBV DNA及HBsAg,且HBV DNA在10 d内保持相对稳定的高水平复制(1×107~3×107拷贝/ml);基因芯片检测结果示QSG-7701细胞中与HBV生活周期相关的因予如HLF、RXRα、IL-6高表达,而HBxIP、SPIK1为低表达,MMP3不表达。结论QSG-7701支持高水平的HBV复制,并可维持cccDNA池的相对稳定,基因差异表达可能为二者支持不同HBV复制模式提供解释。 相似文献
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目的观察HepG2.2.15细胞内HBV核心蛋白的亚细胞定位及转移,了解核心蛋白的入核机制。方法2%二甲亚砜或(和)1μmol/LBay414109处理HepG2.2.15细胞4d;荧光共聚焦显微镜观察HBsAg和HBcAg在细胞内的定位;Westernblot检测胞质和胞核中的HBcAg水平;选择性PCR检测胞核内HBV共价闭合环状DNA(cccDNA)水平。结果二甲亚砜处理提高了胞质和胞核内的HBcAg表达及核内的cccDNA水平;Bay414109处理后胞质中HBcAg水平下降但胞核中HBcAg水平上升,cccDNA水平下降;联合应用二甲亚砜和Bay41—4109处理后HBsAg在胞质内呈条索状分布,胞质中HBcAg水平下降,但胞核内HBcAg明显上升,cccDNA水平下降。结论HepG2.2.15细胞中存在HBV核心颗粒入核障碍,游离核心蛋白易于通过核孔,二甲亚砜可促进核心蛋白进入细胞核,并有助于cccDNA的形成。 相似文献
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目的了解载脂蛋白质BmRNA编辑酶催化多肽3G(APOBEC3G)对乙型肝炎病毒(HBV)和鸭乙型肝炎炎病毒(DHBV)复制的抑制作用。方法从健康人外周血单个核细胞提取RNA,逆转录聚合酶链反应扩增APOBEC3G,将产物克隆到pXF3H载体的EcoRⅠ和Hind Ⅲ酶切位点以构建真核表达质粒;以ayw亚型HBV全长质粒构建具有复制能力的1.3倍HBV质粒(pHBV1.3)。不同剂量的APOBEC3G真核表达质粒与pHBV1.3共转染HepG2细胞;酶联免疫吸附法检测细胞培养上清液的乙型肝炎表面抗原和e抗原水平,Southernblot和Northernblot分析HBV核衣壳相关DNA和RNA的水平变化。不同剂量APOBEC3G真核表达质粒与头尾相接的2倍DHBV质粒共转染LMH鸡肝癌细胞,Southernblot分析DHBV核衣壳相关DNA水平变化。结果成功构建APOBEC3G真核表达质粒和具有复制能力的1.3倍HBV质粒。APOBEC3G抑制乙型肝炎表面抗原和e抗原的分泌,转染细胞内HBV核衣壳相关RNA表达水平下降,而对核心蛋白质的表达没有影响;APOBEC3G对转染细胞内HBV和DHBV核衣壳相关DNA水平具有剂量依赖的抑制效应。结论APOBEC3G对HBV和DHBV复制具有抑制作用。 相似文献
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目的 了解HBV及其抗原成分对干扰素(IFN)αJanus激酶-信号传导和转录激活子(JAK-STAT)信号传导途径分子和抗病毒蛋白表达可能存在的影响.方法以人肝胚瘤细胞株HepG2细胞为研究对象,分别经质粒转染(以能够表达完整HBV病毒颗粒或HBsAg、HBcAg的质粒pSM2、pHBS2-S和pHBc-EGFP)、病毒感染(以HepG2.2.15细胞培养上清液感染HepG2细胞,其含有完整HBV病毒颗粒和HBV抗原)以及与HBV抗原直接接触刺激等方式处理不同组HepG2细胞,以Northern blot和RT-PCR等方法分析各处理组HepG2细胞的IFN α应答情况,如检测抗病毒蛋白[如粘病毒抵抗蛋白A(MxA)、2'-5'寡腺苷酸合成酶(2'-5'OAS)、9-27等]和JAKSTAT信号传导途径分子(如STAT1)的表达.对数据进行t检验.结果 转染pSM2、pHBS2-S和pHBc-EGFP质粒后,HepG2细胞能够表达完整的HBV颗粒或HBV抗原,且随着转染时间的延长,HBV颗粒或抗原表达量逐步增多,转染48、96h的细胞培养上清液中HBsAg的S/CO值为0.81±0.11和2.35±0.33(t=10.84,P<0.05),HBeAg的S/CO值为0.69±0.06和1.79±0.13(t=18.82,P<0.05).Northern blot分析提示HepG2细胞能够表达IFN α抗病毒蛋白MxA、2',5'OAS、9-27等,但质粒转染、病毒感染和HBV抗原直接接触刺激的HepG2细胞,IFN α抗病毒蛋白MxA、2',5'OAS、9-27等的表达量明显减低,且随着转染时间的延长而进一步减低;此外,STAT1的表达也随着HBV颗粒或HBV抗原的表达而受到抑制.结论在体外细胞模型中,HBV及其抗原成分影响IFN αJAK-STAT信号传导途径分子和抗病毒蛋白的表达;HBV具有拮抗或反作用于IFN α抗病毒活性的机制.Abstract: Objective To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN α.Methods The HepG2 cells were transfected with pSM2.pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen,Pre-S antigen and core antigens.The infectious supernatant from HepG2.2.15cells and the pured HBV proteins which contained tIle S.Pre-S antigens were used to treat the HepG2 cells.Northern blot and RT-PCR were applied to analyse the expresssions of the antiviral proteins MxA,2'-5'OAS.9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN αtreatment.Results The HepG2 cells transfected with pSM2,pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg,Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN α antiviral proteins MxA,2' -5' OAS and 9-27.When transfected with pHBV-dimer,pHBS2-S,pHBc-EGFP plasmids,the IFN:A antiviral proteins MxA,2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells,and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore,the expression of IFN α JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells. Conclusions The HBV and its antigens influence the expressions of IFN α JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is idicated that HBV might possess the activity to antagonise or counteract the IFN α antiviral action. 相似文献
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目的 评价骨髓间充质干细胞(BMSC)向肝细胞诱导过程中对HBV的易感性及无涎糖蛋白受体(ASGPR)对BMSC感染HBV的作用.方法 体外使用肝细胞生长因子、成纤维细胞生长因子-4和表皮生长因子,将BMSC诱导分化为肝细胞.检测乙型肝炎患者BMSC的HBV感染情况,并对原代及诱导培养后的BMSC进行体外HBV感染实验,检测BMSC感染后的HBsAg、HBcAg表达情况,并检测BMSC诱导前后ASGPR的表达.每个实验采用来自不同的5个标本,分别重复3次,数据统计采用非参数检验.结果 诱导培养第6天开始,BMSC开始表达甲胎蛋白(AFP)、细胞角蛋白18(CK18)和Alb,并随着诱导时间延长,CK18及Alb表达逐渐增多,而AFP则逐渐减少,并具有糖原合成、尿素分泌及Alb合成的肝细胞功能.BMSC在体内及体外都不能被HBV感染,经过向肝细胞诱导之后,仍然不能被感染,ASGPR在BMSC向肝细胞诱导后表达增多,但是与对照组HepG2细胞相比,仍然呈低水平表达.结论 BMSC在体内外能抵抗HBV感染.ASGPR可能是导致HBV不能感染BMSC的重要原因之一. 相似文献
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Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells 总被引:10,自引:0,他引:10
P Roingeard S L Lu C Sureau M Freschlin B Arbeille M Essex J L Romet-Lemonne 《Hepatology (Baltimore, Md.)》1990,11(2):277-285
The relationship between the presence of hepatitis B virus antigens, their localization and hepatitis B virus replication was studied in different clones of cultured HepG2 hepatoblastoma cells transfected with cloned hepatitis B virus DNA. Intracellular hepatitis B virus antigens were detected by immunofluorescence. The production of these antigens was evaluated in the culture media by enzyme-linked immunoassay. Hepatitis B virus DNA was detected using dot-blot hybridization. Three types of HBcAg staining were observed in transfected HepG2 cells: (a) cells with nuclear HBcAg, (b) cells with cytoplasmic HBcAg and (c) cells with both nuclear and cytoplasmic HBcAg. Cell types b and c also expressed hepatitis B virus DNA in their culture media. Our results suggest that cytoplasmic HBcAg may be more involved than nuclear HBcAg in hepatitis B virus replication. The site of hepatitis B virus formation in hepatocytes was studied by electron microscopic examination of a specific hepatitis B virus producer clone, thereby allowing detection of intracellular Dane particles more easily than liver biopsy samples from infected patients. Dane particles and HBsAg filaments were found in large, dilated structures probably related to the endoplasmic reticulum. Budding of core particles into cisternae of endoplasmic reticulum-related structures appears to be a possible mechanism for hepatitis B virus formation; our results suggest that the exocytosis of cisternae to extracellular spaces may be a mechanism for release of hepatitis B virus particles. 相似文献
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Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus 总被引:4,自引:0,他引:4
BACKGROUND/AIMS: Hepatitis B virus (HBV) cannot be propagated in cultured cells but two human hepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations. METHODS: Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals. RESULTS: HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals. CONCLUSIONS: The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background. 相似文献
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