【作者】 赵冬久；

【导师】 章明；

【作者基本信息】 浙江大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 乙型肝炎病毒(Hepatitis B virus, HBV)是一种DNA病毒,能引起急性及慢性肝脏病变,每年有大约100万人死于HBV感染所致的肝功能衰竭、肝硬化和原发性肝细胞癌。肝炎的转归是宿主免疫与HBV体内复制之间进行复杂的相互作用的最终结果,固有免疫是机体抵御病毒感染的第一道防线,发挥重要的抗病毒作用。人载脂蛋白B mRNA编辑酶催化多肽样蛋白3G(Apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 protein G,APOBEC3G)是被新发现的固有免疫分子中的一员,有广泛的抗病毒作用。APOBEC3G通过与Gag结合进入艾滋病病毒-1(HIV-1)的病毒颗粒中并在逆转录时使脱氧胞嘧啶脱氨变为脱氧尿嘧啶,从而抑制病毒复制；之后也发现APOBEC3G以非脱氨形式抑制病毒复制。病毒侵染因子(Vif)介导APOBEC3G蛋白泛素化及蛋白酶体降解,使得HIV-1对抗APOBEC3G介导的抗病毒作用。近来有研究者指出APOBEC3G能抑制HBV DNA复制及蛋白表达,但是目前对其抗HBV的作用机制还不是很清楚。本研究组建立了APOBEC3G稳定表达细胞株,观察APOBEC3G抑制HBV复制过程中参与的信号通路及细胞因子；我们用荧光能量共振转移(FRET)技术及表面离子共振实验(SPR)检查APOBEC3G与HBV核心抗原(HBc)之间是否有结合,并且观察RNA对它们之间相互作用的关系的影响；同时我们构建APOBEC3G不同突变体,观察它们对HBV复制的抑制作用并研究它们与HBc的关系。因此本研究共分为三个部分。第一部分NF-κB参与人APOBEC3G抗HBV过程之中研究目的证实APOBEC3G能抑制HBV DNA复制和蛋白表达,建立稳定过表达APOBEC3G的细胞株,观察是NF-κB否参与APOBEC3G抗病毒过程中,探讨APOBEC3G抗病毒机制。研究方法转染APOBEC3G到HepG2细胞以建立稳定过表达APOBEC3G的细胞株,用PCR及western验证细胞株是否构建成功,同时转染pcDNA3.1 (-)-myc-his作为对照；用RT-CES系统实时检测细胞的生长状态；转染pEGFPN-1到构建成功的细胞株中,用流式细胞仪及荧光显微镜检测过表达APOBEC3G是否影响细胞接受外来质粒的能力；转染1.3拷贝的HBV质粒,用荧光定量PCR检测细胞中HBVDNA水平,用放射免疫法检测上清中HBs Ag及HBe Ag的表达量的变化；将pNF-KB-Luc质粒单独或与1.3拷贝的HBV质粒共转染到构建成功的细胞后,检测荧光素酶活性；转染1.3拷贝的HBV质粒到构建成功的细胞后,用含有IKKP抑制剂(IMD-0354)的培养基培养转染,荧光定量PCR测HBV DNA水平的变化；设计引物,用相对荧光定量PCR检测成功构建的细胞株中IL-6、IL-8、IL-1β、IL-1α的水平。研究结果1.从RNA及蛋白水平证实稳定表达APOBEC3G的HepG2细胞株构建成功；2.稳定过表达的APOBEC3G能抑制HBV DNA复制及HBs Ag及HBe Ag的表达；3. APOBEC3G不影响HepG2细胞的生长,RT-CES系统实时检测发现过表达APOBEC3G的HepG2细胞的生长状态与对照没有差异；4. APOBEC3G不影响HepG2细胞接受其他质粒,流式结果显示过表达APOBEC3G的HepG2细胞的pEGFPN-1的转染效率及荧光强度与对照组没有差异；用荧光显微镜观察得到同样结果；5. APOBEC3G能激活NF-κB,如果伴随HBV的感染NF-κB更是被显著激活;6.阻断NF-κB的激活能抑制APOBEC3G抗病毒作用,用含有IKKβ抑制剂(IMD-0354)的培养基培养过表达APOBEC3G细胞时,APOBEC3G对HBVDNA的抑制效率只有42%,不加IMD-0354时APOBEC3G对HBV DNA的抑制达到90%；7. APOBEC3G能上调IL-6、IL-8及IL在HepG2细胞中的表达;但对IL-1α的表达没有影响；研究结论1. APOBEC3G能抑制HBV DNA复制及HBs Ag及HBe Ag的表达；2. NF-κB参与APOBEC3G对HBV DNA的抑制过程中；3. APOBEC3G可能通过激活NF-κB再诱导IL-6、IL-8、IL-1β等炎症因子从而抗病毒。第二部分APOBEC3G能直接结合HBV核心抗原研究目的观察APOBEC3G与HBV核心抗原(HBc)之间的关系,了解它们是直接结合还是需要RNA或其他细胞成分的介导,探讨APOBEC3G抗HBV病毒的作用机制。研究方法构建pA3G-CFP及pHBc-YFP融合质粒,用激光扫描共聚焦显微镜观察pA3G-CFP和pHBc-YFP在HepG2细胞中的定位；共转染pA3G-CFP及pHBc-YFP到HepG2细胞中,用荧光共振能量转移(FRET)成像,再计算FR值,观察细胞中APOBEC3G是否能结合HBc；构建带有HA标签pA3G-HA及HBc聚集缺陷突变体pHBc-Y132A-HA,表达并纯化APOBEC3G及HBc-Y132A-HA蛋白,用BIAcore3000检测纯的APOBEC3G与HBc-Y132A-HA蛋白是否有相互作用,同时加入HepG2细胞RNA及HepG2.2.15细胞RNA看对它们的结合是否有影响。研究结果1.成功构建pA3G-CFP、pHBc-YFP、pA3G-HA及pHBc-Y132A-HA等质粒；2.用激光扫描共聚焦显微镜观察到APOBEC3G散点状分布在胞浆中,而HBc则在胞浆胞核中均有分布,两个蛋白在胞浆中有共定位；3.三通道FRET在FRET通道检测到APOBEC3G-CFP与HBc-YFP之间有FRET信号,FR值与阴性对照组相比有显著差异；同样荧光受体漂白实验也检测到APOBEC3G-CFP与HBc-YFP之间有FRET信号；4.成功获得APOBEC3G-HA及HBc-Y132A-HA蛋白,BIAcore3000检测到APOBEC3G-HA与HBc-Y132A-HA之间有SPR信号；5.加入HepG2细胞RNA及HepG2.2.15细胞RNA后,APOBEC3G-HA与HBc-Y132A-HA之间的SPR信号没有显著改变。研究结论1. APOBEC3G与HBc在HepG2细胞中有相互作用；2. APOBEC3G能与HBc直接结合,而不依赖与细胞中的其他成分,细胞RNA及病毒RNA对他们的结合没有影响。第三部分APOBEC3G不同结构域对HBV的抑制作用及机制研究研究目标研究APOBEC3G不同结构域对HBV DNA复制的影响,寻找APOBEC3G对HBV作用的功能域；寻找APOBEC3G与HBc的结合域。研究方法构建APOBEC3G功能域缺失体pCD1、pCD2和活性中心缺失体pDE12,将构建好的质粒分别与1.3拷贝HBV质粒共转到HepG2细胞中,用荧光定量PCR检测它们对HBV DNA的抑制效果；构建pCD1、pCD2、pDE12与CFP的融合质粒pCD1-CFP、pCD2-CFP、pDE12-CFP,将荧光蛋白融合质粒分别转入HepG2细胞中,激光扫描共聚焦显微镜观察CD1、CD2和DE12在细胞中的分布；将pCD1-CFP、pCD2-CFP、pDE12-CFP分别与pHBc-YFP共转到HepG2细胞中,用荧光受体漂白实验观察它们与HBc的关系。研究结果1.成功构建APOBEC3G功能域缺失体pCD1、pCD2及pDE12及pCD1-CFP、pCD2-CFP、pDE12-CFP;2.用激光扫描共聚焦显微镜发现CD1分布在胞浆中,而CD2在胞浆胞核中都有表达,DE12则分布在核周的胞浆中,且CD1和DE12有聚集现象；3.CD1和CD2均能显著抑制HBV DNA的复制,且CD2的抑制效果比全长APOBEC3G的抑制效果更显著,两个活性中心都去掉的DE12的抑制作用则相对较弱；4.荧光受体漂白实验检测到CD1-CFP、CD2-CFP与HBc-YFP之间都有FRET信号,但是CD2-CFP与HBc-YFP之间的信号比CD1-CFP与HBc-YFP之间的信号弱,且都比全长APOBEC3G与HBc之间的信号弱,DE12-CFP与HBc-YFP之间没有检测到FRET信号。研究结论1. APOBEC3G的两个功能域都能抑制HBV的复制,这说明APOBEC3G的抑制机制不是单一的；2. APOBEC3G的两个功能域都能与HBc结合,但是N端结构是主要结合域。全文结论1.APOBEC3G能显著抑制HBV DNA的复制及蛋白表达,且NF-κB可能参与APOBEC3G抗HBV过程之中；2.APOBEC3G和HBV核心抗原具有相互作用,且不依赖其他细胞蛋白及RNA；3.APOBEC3G的两个不同功能域APOBEC3G-CD1和APOBEC3G-CD2均有抑制HBV复制的作用,同时去掉后没有抑制功能,证明活性功能域是主要的作用结构,且说明APOBEC3G的抗病毒机制不是单一的。更多还原

【Abstract】 Hepatitis B virus (HBV) is a DNA virus that can cause acute and chronic liver disease. The outcome of HBV infection and the degree of liver disease is the result of complicated interactions between the virus and the immune response of the host, the innate immune system is the first defense against virus by a host.Apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 protein G (APOBEC3G)is a novel component of the innate immune system, which is a potent inhibitor of infection by a wide range of retroviruses. Early reports have shown that APOBEC3G is packaged into human immunodeficiency virus (HIV-1) virions through binding Gag, which deaminate cytidine bases in single-stranded DNA formed during reverse transcription of the RNA template. Recently, it was reported that APOBEC3G can restrict the replication of HBV and reduce the expression of HBs Ag and HBe Ag, but the exact mechanism of APOBEC3G anti-HBV is unknown.In this study, we constructed stable expressing-APOBEC3G cell line, and detected whether NF-κB involved in the process of APOBEC3G-mediated antiviral activity. We used the fluorescence resonance energy transfer (FRET) approach in living cells and surface plasmon resonance (SPR) technology to explore the relationship between APOBEC3G and HBV core protein (HBc). We constructed different mutants of APOBEC3G and investigated the effect of they mediated antiviral activity against HBV in cell cultures. The interaction between the mutants and HBc was also detected.This study includes three parts. PartⅠHuman APOBEC3G inhibits hepatitis B virus replication and NF-κB involves in the process of antiviral activityObjectivesTo clarify that APOBEC3G can suppress the replication of HBV DNA and the expression of HBs Ag and HBe Ag. To construct stable expressing-APOBEC3G cell line, and to detect whether NF-κB involves in the process of APOBEC3G-mediated antiviral activity, to study the mechanism of APOBEC3G anti-HBV.MethodsWe transfected the HepG2 cells with APOBEC3G plasmid (pcDNA-APO3G) and pcDNA3.1(-)-myc-his was transfected as control, named HepG2A and HepG2-3.1 cells. To confirm the expression of APOBEC3G in transfected HepG2 cells, RT-PCR and western blotting were carried out. RT-CES system was used to detect the status of the HepG2A and HepG2-3.1 cells. HepG2A and HepG2-3.1 cells were then transfected with GFP-expressing plasmid (pEGFPN-1) respectively. The transfected efficienfy was analyzed by fluorescent microscope and FACS. The HepG2A and HepG2-3.1 cells were transfected with 1.3 copy wild-type HBV genome vector (p1.37). Viral replicative DNA intermediates were analyzed by real-time and the expression of HBsAg and HBeAg was detected via radioimmunoassay. The HepG2A and HepG2-3.1 cells were transfected with pNF-KB-Luc alone, or together with p1.37, luciferase activities were measured. The HepG2A and HepG2-3.1 cells were transfected with p1.37, cells were then incubated with IMD-0354 ((1μM) or without, and HBV DNA level was detected by real time PCR. The expression of IL-6, IL-8, IL-1βand IL-1αwas quantified by real-time RT-PCR in HepG2A and HepG2-3.1 cells.Results1. A stable APOBEC3G-expressing cell line was successfully established. The 298bp band was brighter in HepG2A cells than that in HepG2 cells. The molecular weight of APOBEC3G was detected about 46KD in HepG2A cells.2. The level of HBV DNA and the expression of HBs Ag and HBe Ag were dramatically reduced in HepG2 cells cotransfected with pcDNA-APO3G and p1.37, but the inhibition efficiency was less than that of stable overexpressing APOBEC3G.3. The status of the HepG2 cells was not affected by overexpressing APOBEC3G which was monitored by real-time cell electronic sensing system (RT-CES system).4. There was no difference in average GFP-positive cell ratio between HepG2-3.1 and HepG2A cells by fluorescent microscope and FACS.5. NF-κB was activated by APOBEC3G and this activation was strongly enhanced by co-expression of HBV.6. The antiviral effect of APOBEC3G was strongly suppressed after treatment with IMD-0354. A reduction of 90% with HBV DNA was observed in HepG2A, while cells cultured with IMD-0354, a reduction of 42% with HBV DNA was observed.7. The levels of IL-6, IL-8, IL-1βwere upregluated by APOBEC3G, but the expression of IL-1αwas not affected.Conclusions1. APOBEC3G can inhibit the replication of HBV DNA and the expression of HBs Ag and HBe Ag.2. NF-κB involves in the process of antiviral activity of APOBEC3G.3. APOBEC3G may be through activating NF-κB to induce inflammatory mediators to inhibit the replication of HBV.Part II APOBEC3G directly binds hepatitis B virus core protein in cell and cell free systemsObjectivesTo explore the relationship between APOBEC3G and the HBV core protein (HBc), and examine whether RNA or other cellular protein affects the APOBEC3G-HBc interaction.MethodsAPOBEC3G and HBc coding sequence were amplified by PCR, the PCR products were inserted into HindⅢ/Kpnl restriction sites of pECFP-N1 or pEYFP-N1, named pA3G-CFP and pHBc-YFP. Plasmids pA3G-CFP and pHBc-YFP were prepared and transiently cotransfected into HepG2 cells. The localization of the APOBEC3G-CFP and HBc-YFP fusion proteins were detected in cotransfected HepG2 cells by confocal fluorescence microscopy. To determine whether APOBEC3G interacted with HBc in living cells, the FRET approach was used. APOBEC3G with a haemagglutinin (HA) tag at the C-terminus (pA3G-HA) was constructed by cloning APOBEC3G into the HindⅢand EcoRI restriction sites of pcDNA3.1 (+). To construct an assembly defective mutant of HBc, Y132A mutation was completed by using overlap extension PCR. The final PCR products of HBc-Y132A-HA was digested with HindⅢ/EcoRI and inserted into pcDNA3.1 (+), yield pHBc-Y132A-HA. pA3G-HA or pHBc-Y132A-HA was transfected into 293T cells. After 48 hours, cells were harvested and HA-fusion proteins were purified by EZviewa Red Anti-HA Affinity Gel. The purified HBc-Y132A-HA protein was immbolized on CM5 sensor chips, surface plasmon resonance (SPR) measurement was carried out in cell-free condition. To investigate whether RNA affects the A3G-HBc interaction, cellular RNA or virus RNA was added to the APOBEC3G protein solution before flow through the SPR chip.Results1. Plasmids pA3G-CFP, pHBc-YFP, pA3G-HA and pHBc-Y132A-HA were successfully construced.2. Localization of the APOBEC3G-CFP and HBc-YFP fusion proteins were detected in cotransfected HepG2 cells by confocal fluorescence microscopy. Very bright and punctuated APOBEC3G-CFP was observed in the cytoplasm while HBc-YFP was not only distributed evenly in the cytoplasm, but also in the nucleus. In a number of APOBEC3G-CFP and HBc-YFP co-expressing cells, extensive co-localization was observed.3. We detected FRET signal between APOBEC3G-CFP and HBc-YFP in FRET tube. We also measured a FRET signal in cytoplasm when APOBEC3G-CFP and HBc-YFP were co-expressed in HepG2 cells.4. APOBEC3G-HA and HBc-Y132A-HA proteins were obtained. And there were significant SPR signals generated between the APOBEC3G-HA and HBc-Y132A-HA proteins.5. The signal obtained from APOBEC3G-HA and HBc-Y132A-HA was not affected when total RNA of HepG2 or HepG2.2.15 cells was added.Conclusions1. APOBEC3G can bind to HBc in the cytoplasm of HepG2 cells.2. APOBEC3G can bind to HBc in cell free systems, this binding was neither enhanced by the HepG2 cell RNA nor the virus RNA.Part III Different domains of APOBEC3G inhibits hepatitis B virus replicationObjectivesTo investigate the effect of APOBEC3G different domains mediated antiviral activity against HBV in cell cultures and find the binding domain of APOBEC3G to HBc.MethodsN-terminal and C-terminal cytosine deaminase domain expression plasmids and both active sites deficient plasmids were constructed, named as pCD1, pCD2 and pDE12. HepG2 cells were cotransfected with pCD1, pCD2, pDE12 and 1.37. Viral replicative DNA intermediates were analyzed by real-time PCR. Plasmids pCD1-CFP, pCD2-CFP, pDE12-CFP was constructed and transiently transfected into HepG2 cells, respectively. The localization of the fluorescent fusion proteins were detected in transfected HepG2 cells by confocal fluorescence microscopy. pCD1-CFP, pCD2-CFP, pDE12-CFP and pHBc-YFP were cotransfected into HepG2 cells, acceptor photobleaching method was used to detect the relationship between them.Results1.Plasmids pCD1, pCD2, pDE 12 and pCD 1-CFP, pCD2-CFP and pDE 12-CFP were successfully construced.2. Localization of CD1-CFP, CD2-CFP and DE12-CFP fusion proteins were detected in transfected HepG2 cells by confocal fluorescence microscopy. Very bright and punctuated CD1-CFP was observed in the cytoplasm while CD2-CFP was not only distributed evenly in the cytoplasm, but also in the nucleus. DE12-CFP was detected in perinuclear.3. The N-terminal or C-terminal cytosine deaminase domain alone could inhibit HBV replication in HepG2 cells. The inhibition efficiency of CD2 was even more than that of APOBEC3G. DE12 has little inhibitory effect to HBV.4. Fret signal was obtained between CD1-CFP, CD2-CFP and HBc-YFP respectively, but the signal between CD1-CFP and HBc-YFP was stronger. There was no FRET signal between DE12-CFP and HBc-YFP.Conclusions1. The N-terminal or C-terminal cytosine deaminase domain alone can inhibit HBV DNA replication.2. The N-terminal or C-terminal cytosine deaminase domain can bind to HBc, but the N-terminal domain is the main structure.Conclusion of full text1.APOBEC3G can inhibit the replication of HBV DNA and the expression of HBs Ag and HBe Ag, and NF-κB may involve in the process of antiviral activity of APOBEC3G.2. APOBEC3G can bind to HBc in cell and cell free systems, and APOBEC3G imay be incorporated into HBV viral particles via direct binding with HBc protein.3. The N-terminal or C-terminal cytosine deaminase domain alone can inhibit HBV DNA replication, the mechanism of APOBEC3G antiretroviral action is not single.更多还原