乙型肝炎病毒载体表达的APOBEC3C抗病毒作用研究
|
摘要
乙型肝炎病毒（hepatitis B virus，HBV）感染的主要临床表现是慢性乙型肝炎，迄今慢性乙型肝炎的治疗效果很不理想，所以科研人员都在积极探索新的治疗策略。Natsoulis等于90年代初率先提出了一种全新的抗病毒策略：利用衣壳蛋白靶向灭活病毒（capsid targeted viral inactivation，CTVI），他们将这个方法应用于治疗逆转录病毒的实验研究，这个方法的主要原理是构建抗病毒蛋白与衣壳蛋白重组形成的融合蛋白，病毒组装时很多衣壳蛋白单体聚合形成衣壳蛋白，融合蛋白可以被病毒当做真的衣壳蛋白单体被组装到病毒核心颗粒中，这样融合蛋白携带的抗病毒蛋白就可以直接作用于核心颗粒内部的病毒核酸，干扰核酸合成使之发生变异失去作用或直接破坏清除病毒核酸，从而达到控制病毒复制的目标。
2001年Beterams和Nassal将这个方法扩展到HBV的实验研究。他们在HBV核心蛋白羧基端通过linker连接核酸酶(staphylococcal nuclease，SN)。构建SN与核心蛋白的融合蛋白，这种被命名为Core-SN的融合蛋白被装配到病毒核心颗粒中。在与HBV质粒共转染人肝癌细胞后，提取上清液检测HBV DNA，发现DNA滴度下降了95%，实验显示Core-SN对核心蛋白和RNA形成核心颗粒没有影响，然而干扰了核心颗粒内部DNA的合成。这个实验也有不足之处：钙离子浓度高的情况下SN才能发挥功能，然而钙离子浓度在细胞内比较低，也就是说SN对细胞内的病毒没有作用，只是对分泌到细胞外的病毒发挥作用，当然就更不能阻止细胞质内的HBV DNA进入细胞核转化为cccDNA，所以我们应当寻找能在细胞内直接发挥抗HBV作用的物质。
载脂蛋白B mRNA编辑酶催化多肽（apolipoprotein B mRNA-editing enzyme-catalyticpolypeptide，APOBEC）是近年来新发现的具有脱氨基作用的一类酶，可以使腺嘌呤（adenine，A）或胞嘧啶（cytosine，C）脱氨基分别转化为次黄嘌呤(inosine, I)或尿嘧啶（uracil，U），即A→I或C→U。2007年Thomas报道，APOBEC3C(A3C)对HBV具有抑制作用，HBV核心蛋白与A3C复合体非常稳定，A3C可以使多数的新合成HBVDNA基因组出现很多鸟嘌呤（guanine，G）→腺嘌呤突变，即G→A。这些研究结果表明HBV非常容易受到内源性人脱氨酶的影响，并提示A3C是机体固有的抗HBV宿主反应因子。然而A3C不容易进入核心颗粒内部。为此我们利用核衣壳导向的病毒灭活策略构建核心蛋白与A3C的融合蛋白，在核心蛋白组装时将A3C带进核心颗粒内部以便其更有效的发挥抗病毒作用。
本实验分为两部分：第一部分我们研究A3C蛋白独立的抗HBV作用和机制。在第二部分我们利用核衣壳导向的病毒灭活策略构建核心蛋白与A3C的融合蛋白，观察Core-A3C融合蛋白的抗HBV作用和机制。主要方法是应用电化学发光免疫法（electrochemiluminescence immunoassay，ECLIA）检测细胞培养上清中HBsAg和HBeAg的表达水平；应用Southern blotting方法检测细胞裂解液和上清液中HBV DNA观察抗病毒作用及pCH-LJ3-A3C在辅助质粒存在下的复制能力；应用3D-PCR技术扩增核心蛋白相关的HBV DNA，测定HBV DNA序列，研究Core-A3C融合蛋白抑制病毒机制；应用免疫细胞化学方法研究融合蛋白在细胞中的定位。
主要研究结果如下：
第一部分：复制缺损型HBV载体表达A3C抗HBV作用。应用PCR技术扩增目的基因A3C，经过Xho I和BSP1407I双酶切替换pCH-LJ3-hrGFP中的hrGFP基因，构建质粒pCH-LJ3-A3C，质粒经酶切和测序鉴定正确。质粒pCH-LJ3-A3C与野生型HBV质粒pCH-3093共转染HepG2细胞系。首先，应用ECLIA法检测细胞培养上清中HBsAg和HBeAg的表达水平。结果显示pCH-LJ3-A3C组HBsAg表达水平为对照组的104.4％%±2.25%；HBeAg表达水平为对照组的98.65%±0.85%，与对照组相比，均无统计学差异。随后，应用Southern blotting方法检测细胞裂解液和上清液HBV DNA，观察pCH-LJ3-A3C的抗病毒作用。结果显示pCH-LJ3-A3C可以抑制细胞浆内病毒DNA，使HBV DNA减少30%，pCH-LJ3-A3C可以抑制上清液中子代病毒颗粒使HBV DNA减少39%。之后，为了研究pCH-LJ3-A3C能否在辅助质粒帮助下恢复复制能力，pCH-LJ3-A3C与pCH-3142共转染HepG2细胞系，Southern blotting方法检测细胞裂解液和上清液HBVDNA。结果显示裂解液和上清液均检出病毒DNA。最后，为了研究其抑制病毒机制，应用3D-PCR技术扩增核心颗粒有关的HBV DNA，与pGEM-T测序载体连接，每组选取50个克隆测序。结果回报pCH-LJ3-A3C对新合成的HBV DNA发挥编辑功能产生了G→A的变异，总共36个克隆出现G→A的变异，G→A变异总数目达982。
第二部分：HBV载体表达核心蛋白与A3C融合蛋白抗HBV作用。应用PCR技术扩增目的基因A3C，经过BstE II和Acor13H I双酶切，插入载体质粒pdssX中，构建质粒pCH-Core-A3C，质粒经酶切和测序鉴定正确。pCH-Core-A3C与野生型HBV质粒pCH-3093共转染HepG2细胞系。首先，应用ECLIA法检测细胞培养上清中HBsAg和HBeAg的表达水平。结果显示pCH-Core-A3C组HBsAg表达水平为对照组的97.82%±1.37%，HBeAg表达水平为对照组的97.16%±0.86%，与对照组相比无统计学差异。随后，应用Southern blotting方法检测细胞裂解液和上清液HBV DNA，观察pCH-Core-A3C的抗病毒作用。结果显示pCH-Core-A3C可以抑制细胞浆内病毒DNA，使HBV DNA减少84%，pCH-Core-A3C可以抑制上清液中子代病毒颗粒使HBV DNA减少91%。之后，为了研究其抑制病毒机制，应用3D-PCR技术扩增核心颗粒有关的HBV DNA，与pGEM-T测序载体连接，每组选取50个克隆测序。结果回报pCH-Core-A3C对新合成的HBV DNA发挥编辑功能，产生了大量的G→A的变异，总共48个克隆出现G→A的变异，G→A变异总数目达2416。最后，应用免疫细胞化学方法研究融合蛋白在细胞中的定位。结果显示Core-A3C融合蛋白主要定位于细胞浆。
综合前两部分研究我们得出主要研究结论如下：
1．成功构建了HBV载体表达核心蛋白与A3C融合蛋白质粒pCH-Core-A3C；复制缺损型HBV载体表达A3C质粒pCH-LJ3-A3C。
2．pCH-LJ3-A3C和pCH-Core-A3C质粒对细胞培养上清中HBsAg和HBeAg的表达水平没有影响。
3．pCH-Core-A3C转染HepG2细胞系，细胞免疫化学显示Core-A3C融合蛋白主要定位于细胞浆。
4．pCH-LJ3-A3C在辅助质粒pCH-3142提供包装时形成了完整的HBV颗粒。
5．与野生型HBV质粒pCH-3093共转染HepG2细胞系，Southern blotting方法检测发现pCH-LJ3-A3C和pCH-Core-A3C均可以抑制细胞裂解液和细胞上清液HBVDNA，且pCH-Core-A3C抑制病毒复制作用明显优于pCH-LJ3-A3C。
6．应用3D-PCR技术扩增核心颗粒有关的HBV DNA并克隆至pGEM-T载体，每组选取50个克隆测序，pCH-LJ3-A3C和pCH-Core-A3C均对新合成的HBV DNA发挥编辑功能产生了G→A的变异，且pCH-Core-A3C的编辑作用明显优于pCH-LJ3-A3C。
总之，我们构建了表达Core-A3C的HBV载体，发现其对野生型HBV具有较强的的编辑作用，同时还能在辅助病毒存在时形成子代病毒，为下一步的动物试验及基因治疗奠定了良好的基础。将来在人体内应用时，有望实现“一次性治疗”获得持久的抗HBV作用。
Hepatitis B virus (HBV) infection causes acute and chronic hepatitis in humans.Treatment has largely been unsuccessful, and there is clearly an urgent need to identify newand better therapeutic agents. Natsoulis and Boeke (1991) developed a new anti-viral strategy,referred as the capsid targeted viral inactivation (CTVI). CTVI is conceptually a powerfulantiviral strategy that exploits viral structural proteins or virion-associated protein as carriersthat target a destructive enzyme specifically into progeny virions to achieve the purpose ofinhibiting virus replication.
Beterams and Nassal (2001) applied CTVI to the treatment of HBV. Calcium-dependentstaphylococcal nuclease (SN) was fused to the C-terminus of HBV capsid protein to yield achimeric protein, Core-SN. When the fusion protein was co-transfected into human hepatomacells with HBV in a ratio of1:10(<1Core-SN protein per10wild-type core proteins),>95%reduction in enveloped particles was reported. However, SN requires particles to reach a Ca2+rich environment. Intracellular calcium concentration is too low for activation of Core-SN.Core-SN can only be activated when the virus is released into the extracellular medium andstarts to degrade HBV DNA. This can avoid the effects DNase on DNA inside cells. But thiscannot inhibit HBV DNA inside the cells gettingt more cDNA in nucleus. Therefore it isnecessary to use directly the protein that has destroyed HBV DNA inside the cells.
Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide (APOBEC) proteinscatalyse the hydrolytic deamination of cytosine, where cytosine (C) is converted to uracil (U)by the addition of water and the removal of an amine group. Thomas et al (2007) reported thedramatic effect of APOBEC3C (A3C) on HBV. The HBV core protein and A3C were verystable and can induced hypermutation in the genome of HBV, where guanine (G) is convertedto adenine (A). This showed that HBV were susceptible to the editing effect of A3C, andindicated that A3C can elicit an anti-HBV host response. But A3C cannot enter into thenucleus.
In present study, we first observed the anti-HBV effects of the replicating-defective HBV vector plasmid expressing A3C (pCH-LJ3-A3C). Then, we tried to apply viral capsid-orientedCTVI to construct the nucleocapsid protein and A3C fusion protein (pCH-Core-A3C) andthen made a detailed analysis of the inhibitory effect of A3C on hepatitis B virus replicationin cell culture. The main methods are as follows: The levels of HBsAg and HBeAg from inthe culture supernatant were measured by ECLIA; viral DNA were extracted from the culturesupernatant and cytoplasmic lysates and visualized by Southern blotting analysis; HBV DNAextracted from cell lysates were analyzed by3D-PCR; the subcellular localization of thefusion protein in the cell was analyzed by cellular immunochemical method; pCH-LJ3-A3Cwas co-transfected with the helper construct pCH-3142that devoid of the encapsidation signalε, the viral DNA were extracted and visualized by Southern blotting analysis.
The main results of our study were listed as follows:
Part I: Construction of replicating-defective HBV vector plasmid expressing A3C andantiviral effects. A3C gene was amplified by PCR and was inserted into pCH-LJ3-hrGFPafter digestion by XhoI and BSP1407I, replaced the hrGFP gene of the pCH-LJ3-hrGFP toconstruct pCH-LJ3-A3C. The sequence was confirmed by DNA sequencing. Plasmid pCH-LJ3-A3C was cotransfected with wild-type HBV expressing plasmid (pCH-3093) into HepG2Cells. Firstly, the levels of HBsAg and HBeAg from in the culture supernatant were measuredby ECLIA. The results showed that pCH-LJ3-A3C had no effect on the expression of HBsAg(104.4％±2.25%) and HBeAg (98.65%±0.85%), respecterly. Secondly, as to elucidateantivirus function mediated by pCH-LJ3-A3C, pCH-LJ3-A3C was cotransfected into HepG2cells together with a construct harboring the wild-type HBV genome. Viral DNA wasextracted from the culture supernatant and cytoplasmic lysates and visualized by Southernblotting analysis. The results showed that accumulation of HBV replicative intermediates incytoplasmic lysates were reduced by30%with pCH-LJ3-A3C; release of viral particles intothe culture supernatant was reduced by39%with pCH-LJ3-A3C. Thirdly, as to investigatereplication and encapsidation capabilities of pCH-LJ3-A3C, pCH-LJ3-A3C was cotransfectedwith the helper construct (pCH-3142). The results showed that with aid of pCH-3142, thecapabilities of replication and encapsidation could be rescued. The progeny “therapeutic”HBV particles with A3C can be generated to secrete into the extracellular supernatant. Endlly,to determine the editing function of HBV DNA in viral nucleocapsids mediated by A3Cprotein, HBV DNA extracted from cell lysate was analyzed by3D-PCR. The PCR products were ligated into the pGEM-T cloning vector and transformed into TOP10competent E. colicells. The transformed cells were stored overnight, and this suspension was applied directlyfor DNA sequencing. Fifty individual clones were selected for HBV DNA sequence analysis.36clones displayed G-to-A mutations, while total number of G-to-A mutations was982.
Part II: Construction of recombinant HBV vector plasmid expressing Core-A3C fusionprotein and its antiviral effects. A3C gene was amplified by PCR and was inserted into pdssXafter digestion by BstEII and Acor13HI to construct plasmid pCH-Core-A3C.The sequencewas confirmed by DNA sequencing. Plasmid pCH-Core-A3C was cotransfected withwild-type HBV expressing plasmid (pCH-3093) into HepG2Cells. Firstly, the levels ofHBsAg and HBeAg from in the culture supernatant were measured by ECLIA. The resultsshowed that pCH-Core-A3C had no effect on the expression of HBsAg (97.82%±1.37%) andHBeAg (97.16%±0.86%), respectively. Secondly, as to elucidate antivirus function ofpCH-Core-A3C, pCH-Core-A3C was cotransfected into HepG2cells together with aconstruct harboring the wild-type HBV genome. Viral DNA was extracted from the culturesupernatant and cytoplasmic lysates and visualized by Southern blotting analysis. The resultsshowed that accumulation of HBV replicative intermdiates in cytoplasmic lysates werereduced by84%with pCH-Core-A3C; release of viral particles into the culture supernatantwas reduced by91%with pCH-Core-A3C. Thirdly, to investigate the editing function ofHBV DNA in viral nucleocapsids mediated by pCH-Core-A3C, HBV DNA extracted fromcell lysate was analyzed by3D-PCR. The PCR products were ligated into the pGEM-Tcloning vector and transformed into TOP10competent E. coli cells. The transformed cellswere stored overnight, and this suspension was applied directly for DNA sequencing. Fiftyindividual clones were selected for HBV DNA sequence analysis.48clones displayingG-to-A mutations, while total number of G-to-A mutations was2416. Finally, the subcellularlocalization of the fusion protein in the cell was analyzed by cellular immunochemicalmethods, the results showed that the Core-A3C fusion protein was mainly localized in thecytoplasm.
From these results, we drawed conclusions as follows:
1．Recombinant HBV vector expressing Core-A3C fusion protein plasmid pCH-Core-A3C was successfully constructed. Replicating-defective HBV vector expressing A3Cplasmid pCH-LJ3-A3C was successfully constructed.
2．The levels of HBsAg and HBeAg from in the culture supernatant were measured byECLIA. The results showed that pCH-LJ3-A3C and pCH-Core-A3C had no effect on theexpression of HBsAg and HBeAg.
3．The subcellular localization of the fusion protein in the cell was demonstrated bycellular immunochemical methods: the Core-A3C fusion protein was localized in thecytoplasm.
4．The capabilities of replication and encapsidation in these replicating-defective HBVvector expressing A3C plasmid pCH-LJ3-A3C could be rescued with aid of the homologoushelper construct pCH-3142that devoid of encapsidation signal ε.
5．Accumulation of HBV replicative intermdiates in cytoplasmic lysates and release ofviral particles into the culture supernatant were reduced by pCH-LJ3-A3C andpCH-Core-A3C, respectively. Compared with pCH-LJ3-A3C, the pCH-Core-A3C plasmidencoding Core-A3C chimerical protein substantially inhibited HBV production intracellularlyand extracellularly.
6．HBV DNA extracted from cell lysate was analyzed by3D-PCR, fifty individualclones were selected for HBV DNA sequence analysis. Compared with pCH-LJ3-A3C, theediting function of pCH-Core-A3C on HBV genomes (total number of G-to-A mutations) wasmore potent.
In conclusion, we constructed a new fusion protein with strong antiviral action. Thefusion protein can be assembled into HBV capsids with viral proteins. The editing function ofpCH-Core-A3C on HBV genomes was more potent. With aid of pCH-3142, the progeny“therapeutic” HBV particles with A3C can be generated and secreted into the extracellularmedium. The new A3C fusion protein could be regarded as an excellent candidate forfulfilling gene therapy strategies against HBV. It is expected to achieve “One-time treatment”for long-lasting anti-HBV effect in vivo.
2001年Beterams和Nassal将这个方法扩展到HBV的实验研究。他们在HBV核心蛋白羧基端通过linker连接核酸酶(staphylococcal nuclease，SN)。构建SN与核心蛋白的融合蛋白，这种被命名为Core-SN的融合蛋白被装配到病毒核心颗粒中。在与HBV质粒共转染人肝癌细胞后，提取上清液检测HBV DNA，发现DNA滴度下降了95%，实验显示Core-SN对核心蛋白和RNA形成核心颗粒没有影响，然而干扰了核心颗粒内部DNA的合成。这个实验也有不足之处：钙离子浓度高的情况下SN才能发挥功能，然而钙离子浓度在细胞内比较低，也就是说SN对细胞内的病毒没有作用，只是对分泌到细胞外的病毒发挥作用，当然就更不能阻止细胞质内的HBV DNA进入细胞核转化为cccDNA，所以我们应当寻找能在细胞内直接发挥抗HBV作用的物质。
载脂蛋白B mRNA编辑酶催化多肽（apolipoprotein B mRNA-editing enzyme-catalyticpolypeptide，APOBEC）是近年来新发现的具有脱氨基作用的一类酶，可以使腺嘌呤（adenine，A）或胞嘧啶（cytosine，C）脱氨基分别转化为次黄嘌呤(inosine, I)或尿嘧啶（uracil，U），即A→I或C→U。2007年Thomas报道，APOBEC3C(A3C)对HBV具有抑制作用，HBV核心蛋白与A3C复合体非常稳定，A3C可以使多数的新合成HBVDNA基因组出现很多鸟嘌呤（guanine，G）→腺嘌呤突变，即G→A。这些研究结果表明HBV非常容易受到内源性人脱氨酶的影响，并提示A3C是机体固有的抗HBV宿主反应因子。然而A3C不容易进入核心颗粒内部。为此我们利用核衣壳导向的病毒灭活策略构建核心蛋白与A3C的融合蛋白，在核心蛋白组装时将A3C带进核心颗粒内部以便其更有效的发挥抗病毒作用。
本实验分为两部分：第一部分我们研究A3C蛋白独立的抗HBV作用和机制。在第二部分我们利用核衣壳导向的病毒灭活策略构建核心蛋白与A3C的融合蛋白，观察Core-A3C融合蛋白的抗HBV作用和机制。主要方法是应用电化学发光免疫法（electrochemiluminescence immunoassay，ECLIA）检测细胞培养上清中HBsAg和HBeAg的表达水平；应用Southern blotting方法检测细胞裂解液和上清液中HBV DNA观察抗病毒作用及pCH-LJ3-A3C在辅助质粒存在下的复制能力；应用3D-PCR技术扩增核心蛋白相关的HBV DNA，测定HBV DNA序列，研究Core-A3C融合蛋白抑制病毒机制；应用免疫细胞化学方法研究融合蛋白在细胞中的定位。
主要研究结果如下：
第一部分：复制缺损型HBV载体表达A3C抗HBV作用。应用PCR技术扩增目的基因A3C，经过Xho I和BSP1407I双酶切替换pCH-LJ3-hrGFP中的hrGFP基因，构建质粒pCH-LJ3-A3C，质粒经酶切和测序鉴定正确。质粒pCH-LJ3-A3C与野生型HBV质粒pCH-3093共转染HepG2细胞系。首先，应用ECLIA法检测细胞培养上清中HBsAg和HBeAg的表达水平。结果显示pCH-LJ3-A3C组HBsAg表达水平为对照组的104.4％%±2.25%；HBeAg表达水平为对照组的98.65%±0.85%，与对照组相比，均无统计学差异。随后，应用Southern blotting方法检测细胞裂解液和上清液HBV DNA，观察pCH-LJ3-A3C的抗病毒作用。结果显示pCH-LJ3-A3C可以抑制细胞浆内病毒DNA，使HBV DNA减少30%，pCH-LJ3-A3C可以抑制上清液中子代病毒颗粒使HBV DNA减少39%。之后，为了研究pCH-LJ3-A3C能否在辅助质粒帮助下恢复复制能力，pCH-LJ3-A3C与pCH-3142共转染HepG2细胞系，Southern blotting方法检测细胞裂解液和上清液HBVDNA。结果显示裂解液和上清液均检出病毒DNA。最后，为了研究其抑制病毒机制，应用3D-PCR技术扩增核心颗粒有关的HBV DNA，与pGEM-T测序载体连接，每组选取50个克隆测序。结果回报pCH-LJ3-A3C对新合成的HBV DNA发挥编辑功能产生了G→A的变异，总共36个克隆出现G→A的变异，G→A变异总数目达982。
第二部分：HBV载体表达核心蛋白与A3C融合蛋白抗HBV作用。应用PCR技术扩增目的基因A3C，经过BstE II和Acor13H I双酶切，插入载体质粒pdssX中，构建质粒pCH-Core-A3C，质粒经酶切和测序鉴定正确。pCH-Core-A3C与野生型HBV质粒pCH-3093共转染HepG2细胞系。首先，应用ECLIA法检测细胞培养上清中HBsAg和HBeAg的表达水平。结果显示pCH-Core-A3C组HBsAg表达水平为对照组的97.82%±1.37%，HBeAg表达水平为对照组的97.16%±0.86%，与对照组相比无统计学差异。随后，应用Southern blotting方法检测细胞裂解液和上清液HBV DNA，观察pCH-Core-A3C的抗病毒作用。结果显示pCH-Core-A3C可以抑制细胞浆内病毒DNA，使HBV DNA减少84%，pCH-Core-A3C可以抑制上清液中子代病毒颗粒使HBV DNA减少91%。之后，为了研究其抑制病毒机制，应用3D-PCR技术扩增核心颗粒有关的HBV DNA，与pGEM-T测序载体连接，每组选取50个克隆测序。结果回报pCH-Core-A3C对新合成的HBV DNA发挥编辑功能，产生了大量的G→A的变异，总共48个克隆出现G→A的变异，G→A变异总数目达2416。最后，应用免疫细胞化学方法研究融合蛋白在细胞中的定位。结果显示Core-A3C融合蛋白主要定位于细胞浆。
综合前两部分研究我们得出主要研究结论如下：
1．成功构建了HBV载体表达核心蛋白与A3C融合蛋白质粒pCH-Core-A3C；复制缺损型HBV载体表达A3C质粒pCH-LJ3-A3C。
2．pCH-LJ3-A3C和pCH-Core-A3C质粒对细胞培养上清中HBsAg和HBeAg的表达水平没有影响。
3．pCH-Core-A3C转染HepG2细胞系，细胞免疫化学显示Core-A3C融合蛋白主要定位于细胞浆。
4．pCH-LJ3-A3C在辅助质粒pCH-3142提供包装时形成了完整的HBV颗粒。
5．与野生型HBV质粒pCH-3093共转染HepG2细胞系，Southern blotting方法检测发现pCH-LJ3-A3C和pCH-Core-A3C均可以抑制细胞裂解液和细胞上清液HBVDNA，且pCH-Core-A3C抑制病毒复制作用明显优于pCH-LJ3-A3C。
6．应用3D-PCR技术扩增核心颗粒有关的HBV DNA并克隆至pGEM-T载体，每组选取50个克隆测序，pCH-LJ3-A3C和pCH-Core-A3C均对新合成的HBV DNA发挥编辑功能产生了G→A的变异，且pCH-Core-A3C的编辑作用明显优于pCH-LJ3-A3C。
总之，我们构建了表达Core-A3C的HBV载体，发现其对野生型HBV具有较强的的编辑作用，同时还能在辅助病毒存在时形成子代病毒，为下一步的动物试验及基因治疗奠定了良好的基础。将来在人体内应用时，有望实现“一次性治疗”获得持久的抗HBV作用。
Hepatitis B virus (HBV) infection causes acute and chronic hepatitis in humans.Treatment has largely been unsuccessful, and there is clearly an urgent need to identify newand better therapeutic agents. Natsoulis and Boeke (1991) developed a new anti-viral strategy,referred as the capsid targeted viral inactivation (CTVI). CTVI is conceptually a powerfulantiviral strategy that exploits viral structural proteins or virion-associated protein as carriersthat target a destructive enzyme specifically into progeny virions to achieve the purpose ofinhibiting virus replication.
Beterams and Nassal (2001) applied CTVI to the treatment of HBV. Calcium-dependentstaphylococcal nuclease (SN) was fused to the C-terminus of HBV capsid protein to yield achimeric protein, Core-SN. When the fusion protein was co-transfected into human hepatomacells with HBV in a ratio of1:10(<1Core-SN protein per10wild-type core proteins),>95%reduction in enveloped particles was reported. However, SN requires particles to reach a Ca2+rich environment. Intracellular calcium concentration is too low for activation of Core-SN.Core-SN can only be activated when the virus is released into the extracellular medium andstarts to degrade HBV DNA. This can avoid the effects DNase on DNA inside cells. But thiscannot inhibit HBV DNA inside the cells gettingt more cDNA in nucleus. Therefore it isnecessary to use directly the protein that has destroyed HBV DNA inside the cells.
Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide (APOBEC) proteinscatalyse the hydrolytic deamination of cytosine, where cytosine (C) is converted to uracil (U)by the addition of water and the removal of an amine group. Thomas et al (2007) reported thedramatic effect of APOBEC3C (A3C) on HBV. The HBV core protein and A3C were verystable and can induced hypermutation in the genome of HBV, where guanine (G) is convertedto adenine (A). This showed that HBV were susceptible to the editing effect of A3C, andindicated that A3C can elicit an anti-HBV host response. But A3C cannot enter into thenucleus.
In present study, we first observed the anti-HBV effects of the replicating-defective HBV vector plasmid expressing A3C (pCH-LJ3-A3C). Then, we tried to apply viral capsid-orientedCTVI to construct the nucleocapsid protein and A3C fusion protein (pCH-Core-A3C) andthen made a detailed analysis of the inhibitory effect of A3C on hepatitis B virus replicationin cell culture. The main methods are as follows: The levels of HBsAg and HBeAg from inthe culture supernatant were measured by ECLIA; viral DNA were extracted from the culturesupernatant and cytoplasmic lysates and visualized by Southern blotting analysis; HBV DNAextracted from cell lysates were analyzed by3D-PCR; the subcellular localization of thefusion protein in the cell was analyzed by cellular immunochemical method; pCH-LJ3-A3Cwas co-transfected with the helper construct pCH-3142that devoid of the encapsidation signalε, the viral DNA were extracted and visualized by Southern blotting analysis.
The main results of our study were listed as follows:
Part I: Construction of replicating-defective HBV vector plasmid expressing A3C andantiviral effects. A3C gene was amplified by PCR and was inserted into pCH-LJ3-hrGFPafter digestion by XhoI and BSP1407I, replaced the hrGFP gene of the pCH-LJ3-hrGFP toconstruct pCH-LJ3-A3C. The sequence was confirmed by DNA sequencing. Plasmid pCH-LJ3-A3C was cotransfected with wild-type HBV expressing plasmid (pCH-3093) into HepG2Cells. Firstly, the levels of HBsAg and HBeAg from in the culture supernatant were measuredby ECLIA. The results showed that pCH-LJ3-A3C had no effect on the expression of HBsAg(104.4％±2.25%) and HBeAg (98.65%±0.85%), respecterly. Secondly, as to elucidateantivirus function mediated by pCH-LJ3-A3C, pCH-LJ3-A3C was cotransfected into HepG2cells together with a construct harboring the wild-type HBV genome. Viral DNA wasextracted from the culture supernatant and cytoplasmic lysates and visualized by Southernblotting analysis. The results showed that accumulation of HBV replicative intermediates incytoplasmic lysates were reduced by30%with pCH-LJ3-A3C; release of viral particles intothe culture supernatant was reduced by39%with pCH-LJ3-A3C. Thirdly, as to investigatereplication and encapsidation capabilities of pCH-LJ3-A3C, pCH-LJ3-A3C was cotransfectedwith the helper construct (pCH-3142). The results showed that with aid of pCH-3142, thecapabilities of replication and encapsidation could be rescued. The progeny “therapeutic”HBV particles with A3C can be generated to secrete into the extracellular supernatant. Endlly,to determine the editing function of HBV DNA in viral nucleocapsids mediated by A3Cprotein, HBV DNA extracted from cell lysate was analyzed by3D-PCR. The PCR products were ligated into the pGEM-T cloning vector and transformed into TOP10competent E. colicells. The transformed cells were stored overnight, and this suspension was applied directlyfor DNA sequencing. Fifty individual clones were selected for HBV DNA sequence analysis.36clones displayed G-to-A mutations, while total number of G-to-A mutations was982.
Part II: Construction of recombinant HBV vector plasmid expressing Core-A3C fusionprotein and its antiviral effects. A3C gene was amplified by PCR and was inserted into pdssXafter digestion by BstEII and Acor13HI to construct plasmid pCH-Core-A3C.The sequencewas confirmed by DNA sequencing. Plasmid pCH-Core-A3C was cotransfected withwild-type HBV expressing plasmid (pCH-3093) into HepG2Cells. Firstly, the levels ofHBsAg and HBeAg from in the culture supernatant were measured by ECLIA. The resultsshowed that pCH-Core-A3C had no effect on the expression of HBsAg (97.82%±1.37%) andHBeAg (97.16%±0.86%), respectively. Secondly, as to elucidate antivirus function ofpCH-Core-A3C, pCH-Core-A3C was cotransfected into HepG2cells together with aconstruct harboring the wild-type HBV genome. Viral DNA was extracted from the culturesupernatant and cytoplasmic lysates and visualized by Southern blotting analysis. The resultsshowed that accumulation of HBV replicative intermdiates in cytoplasmic lysates werereduced by84%with pCH-Core-A3C; release of viral particles into the culture supernatantwas reduced by91%with pCH-Core-A3C. Thirdly, to investigate the editing function ofHBV DNA in viral nucleocapsids mediated by pCH-Core-A3C, HBV DNA extracted fromcell lysate was analyzed by3D-PCR. The PCR products were ligated into the pGEM-Tcloning vector and transformed into TOP10competent E. coli cells. The transformed cellswere stored overnight, and this suspension was applied directly for DNA sequencing. Fiftyindividual clones were selected for HBV DNA sequence analysis.48clones displayingG-to-A mutations, while total number of G-to-A mutations was2416. Finally, the subcellularlocalization of the fusion protein in the cell was analyzed by cellular immunochemicalmethods, the results showed that the Core-A3C fusion protein was mainly localized in thecytoplasm.
From these results, we drawed conclusions as follows:
1．Recombinant HBV vector expressing Core-A3C fusion protein plasmid pCH-Core-A3C was successfully constructed. Replicating-defective HBV vector expressing A3Cplasmid pCH-LJ3-A3C was successfully constructed.
2．The levels of HBsAg and HBeAg from in the culture supernatant were measured byECLIA. The results showed that pCH-LJ3-A3C and pCH-Core-A3C had no effect on theexpression of HBsAg and HBeAg.
3．The subcellular localization of the fusion protein in the cell was demonstrated bycellular immunochemical methods: the Core-A3C fusion protein was localized in thecytoplasm.
4．The capabilities of replication and encapsidation in these replicating-defective HBVvector expressing A3C plasmid pCH-LJ3-A3C could be rescued with aid of the homologoushelper construct pCH-3142that devoid of encapsidation signal ε.
5．Accumulation of HBV replicative intermdiates in cytoplasmic lysates and release ofviral particles into the culture supernatant were reduced by pCH-LJ3-A3C andpCH-Core-A3C, respectively. Compared with pCH-LJ3-A3C, the pCH-Core-A3C plasmidencoding Core-A3C chimerical protein substantially inhibited HBV production intracellularlyand extracellularly.
6．HBV DNA extracted from cell lysate was analyzed by3D-PCR, fifty individualclones were selected for HBV DNA sequence analysis. Compared with pCH-LJ3-A3C, theediting function of pCH-Core-A3C on HBV genomes (total number of G-to-A mutations) wasmore potent.
In conclusion, we constructed a new fusion protein with strong antiviral action. Thefusion protein can be assembled into HBV capsids with viral proteins. The editing function ofpCH-Core-A3C on HBV genomes was more potent. With aid of pCH-3142, the progeny“therapeutic” HBV particles with A3C can be generated and secreted into the extracellularmedium. The new A3C fusion protein could be regarded as an excellent candidate forfulfilling gene therapy strategies against HBV. It is expected to achieve “One-time treatment”for long-lasting anti-HBV effect in vivo.
引文
1. Ganem D, Prince AM. Hepatitis B virus infection natural history and clinicalconsequences. N Engl J Med,2004;350:1118-1129.
2. Liang X, Bi S, Yang W, et al. Epidemiological serosurvey of hepatitis B inChina-decliningHBV prevalence due to hepatitis B vaccination. Vaccine,2009;27:6550-6557.
3. Liang X, Bi S, Yang W, et al. Evaluation of the impact of hepatitis B vaccinationamongchildren born during1992-2005in China. J Infect Dis,2009;200:39-47.
4. Lu FM, Zhuang H. Management of hepatitis B in China [J]. Chin Med J (Engl),2009;122:3-4.
5. Rijckborst V, Janssen HL. The Role of Interferon in Hepatitis B Therapy. Curr Hepat Rep,2010;9:231-238.
6. Chen YC, Chu CM, Yeh CT, et al. Natural course following the onset of cirrhosisinpatients with chronic hepatitis B: a long-term follow-up study. Hepatol Int,2007;1:267-273.
7. Hsu YS, Chien RN, Yeh CT, et al. Long-term outcome after spontaneousHBeAgseroconversion in patients with chronic hepatitis B. Hepatology,2002;35:1522-1527.
8. van Zonneveld M,van Nunen AB, Niesters HG, et al. Lamivudine treatmentduringpregnancy to prevent perinatal transmission of hepatitis B virus infection. J ViralHepat,2003;10:294-297.
9. Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patientswith chronic hepatitis B. Gastroenterology,2003;125:1714-1722.
10.姚光弼,崔振宇,姚集鲁,等.国产拉米夫定治疗2200例慢性乙型肝炎的Ⅳ期临床试验.中华肝脏病杂志,2003;11:103-108.
11.姚光弼,王宝恩,崔振宇,等.拉米夫定治疗慢性乙型肝炎三年疗效观察.中华内科杂志,2003;42:382-387.
12. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy with adefovirdipivoxil for HBeAg-negative chronic hepatitis B for up to5years. Gastroenterology,2006;131:1743-1751.
13. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy with adefovirdipivoxil for HBeAg-negative chronic hepatitis B. N Engl J Med,2005;352:2673-2681.
14. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Adefovir dipivoxil for thetreatment of hepatitis B e antigen-negative chronic hepatitis B. N Engl J Med,2003;348:800-807.
15. Hou J, Yin YK, Xu D, et al. Telbivudine versus lamivudine in Chinese patients withchronic hepatitis B: Results at1year of a randomized, double-blind trial. Hepatology,2008;47:447-454.
16. Natsoulis G, Boeke JD. New antiviral strategy using capsid nuclease fusionproteins.Nature,1991;352:632-635.
17. Beterams G, Nassal M. Significant interference with hepatitisB virus replication by acore-nuclease fusion protein. J Biol Chem,2001;276:8875-8883.
18. Baumert TF, R sler C, Malim MH, et al. Hepatitis B virus DNA is subject to extensiveediting by the human deaminase APOBEC3C. Hepatology,2007;46:682–689.
19. Jarmuz A, Chester A, Bayliss J, et al. An anthropoid-specific locus of orphan C to URNA-editing enzymes on chromosome22. Genomics,2002;79:285-296.
20. OhAinle M, Kerns JA, Malik HS, et al. Adaptive evolution and antiviral activity of theconserved mammalian cytidine deaminase APOBEC3H. J Virol,2006;80:3853-3862.
21. Yu Q, Konig R, Pillai S, et al. Single-strand specificity of APOBEC3Gaccounts forminus-strand deamination of the HIV genome. Nat St ruct Mol Biol,2004;11:435-442.
22. Hultquist JF, Lengyel JA, Refsland EW, et al. Human and Rhesus APOBEC3D,APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity torestrict Vif-deficient HIV-1. J Virol,2011;85:11220-11234.
23. Zielonka J, Bravo IG, Marino D, et al.Restriction of equine infectious anemia virus byequine APOBEC3cytidine deaminases. J Virol,2009;83:7547-7559.
24. Dang Y, Wang X, Esselman WJ, et al. Identification of APOBEC3DE as anotherantiretroviral factor from the human APOBEC family. J Virol,2006;80:10522-10533.
25. Delebecque F, Suspene R, Calattini S, et al. Restriction of foamy viruses by APOBECcytidine deaminases. J Virol,2006;80:605-614.
26. Esnault C, Millet J, Schwartz O, et al. Dual inhibitory effects of APOBEC familyproteins on retrotransposition of mammalian endogenous retroviruses. Nucleic Acids Res,2006;34:1522-1531.
27. Schumann GG. APOBEC3proteins: major players in intracellular defence againstLINE-1-mediated retrotransposition.Biochem Soc Trans,2007;35:637-642.
28. Bonvin M, Achermann F, Greeve I, et al. Interferon-inducible expression of APOBEC3editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.Hepatology,2006;43:1364-1374.
29. Gunter S, Sommer G, Plikat U, et al. Naturally occurring hepatitis B virus genomesbearing the hallmarks of retroviral G to A hypermutation. Virology,1997;18:104–108.
30. R sler C, K ck J, Malim MH, et al. Comment on ‘Inhibition of hepatitis B virusreplication’ by APOBEC3G. Science,2004;305:1403a.
31. Holmes RK, Malim MH, Bishop KN. APOBEC-mediated viral restriction:not simplyediting?. Trends Biochem Sci,2007;32:118-128.
32. Turelli P, Mangeat B, Jost S, et al. Inhibition of hepatitis B virus replication byAPOBEC3G. Science,2004;303:1829.
33. Günther S, Sommer G, Plikat U, et al. Naturally occurring hepatitisB virus genomesbearing the hallmarks of retroviral G-A hypermutation. Virology,1997;235:104-108.
34. Muller V, Bonhoeffer S. Guanine-adenine bias:a general property of retroid viruses that isunrelated to host-induced hypermutation. Trends Genet,2005;21:264-268.
35. Noguchi C, Ishino H, Tsuge M, et al. G to A hypermutation of hepatitis B virus.Hepatology,2005;41:626–633.
36. Jarmuz A, hester A, Baylls J, et al. An anthropoid-specific locus of orphan C to URNA-editing enzymes on chromosome22. Genomics,2002;79:285-296.
37. Yu Q, Chen D, K nig R, et al. APOBEC3B and APOBEC3C are potent inhibitors ofsimian immunodeficiency virus replication. J Biol Chem,2004;279:53379-53386.
38. Protzer U, Nassal M, Chiang PW, et al. Interferon gene transfer by a hepatitis B virusvector efficiently suppresses wild-type virus infection. Proc Natl Acad Sci,1999;96:10818-10823.
39.孙殿兴，胡大荣.抗乙型肝炎病毒分子治疗新策略.中华肝脏病杂志，2001;9:315-316.
40.慢性乙型肝炎防治指南(2010年版).肝脏，2011;16:2-15.
41. Okui N, Sakuma R, Kobayashi N, et al. Packageable antiviral therapeutics against humanimmunodeficiency virus type1: virion-targeted virus inactivation by incorporation of asingle-chain antibody against viral integrase into progeny virions. Hum Gene Ther,2000;11:537-546.
42. Schwartz S, Felber BK, Pavlakis GN. Distinct RNA sequences in the gag region ofhuman immunodeficiency virus type1decrease RNA stability and inhibit expression inthe absence of Rev protein. J Virol,1992;66:150-159.
43. Wu X, Liu H, Xiao H, et al.Targeting foreign proteins to human immuno-deficiencyvirus particles via fusion with Vpr and Vpx. J Virol,1995;69:3389-3398.
44. Wu X, Liu H, Xiao H, et al. Inhibition of human and simian immunodeficiency virusprotease function by targeting Vpx-protease-mutant fusion protein into viral particles. JVirol,1996;70:3378-3384.
45. Wang CT, Stegeman-Olsen J, Zhang Y, et al. Assembly of HIV GAG-BgalactoSidase-fusion proteins into virus particles. Virology,1994;200:524-534.
46. Natsoulis G, Seshaiah P,Federspie MJ.Targeting of a nuclease tomurine leukemia viruscapsid inhibits viral multiplication. ProcNatl Acad Sci USA,1995;92:364-368.
47. Beterams G, Bottcher B, Nassal M. Packing of up to240subunits of a17kDa nucleaseinto the interior of recombinant hepatitis B virus capsids. FEBS Lett,2000;481:169-176.
48.秦成峰，秦鄂德，于曼等.登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用.中国生物化学与分子生物学报，2004;20:757-761.
49. Qin CF, Qin ED. Capsid-targeted viral inactivation can destroy dengue2virus fromwithin in vitro. Arch Virol,2006;151:379-385.
50. Qin CF, Qin E, Yu M, et al. Therapeutic effects of dengue2virus capsid protein andstaphylococcal nuclease fusion protein on dengue-infected cell cultures. ArchVirol,2005;150:659-669.
51. Qin CF, Qin ED. Development of cell lines stably expressing staphylococcal nucleasefused to dengue2virus capsid protein for CTVI. Acta Biochim Biophys Sin (Shanghai),2004;36:577-582.
52. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev,2000;64:51-68.
53. Ning B, Shih C. Nucleolar localization of human hepatitis B virus capsid protein. J Virol,2004;78:13653-13668.
54. Hu J, Toft DO, Seeger C. Hepadnavirus assembly and reverse transcription require amulti-component chaperone complex which is incorporated into nucleocapsids. EMBO J,1997;16:59-68.
55. Crowther RA, Kiselev NA, Bottcher B, et al. Three-dimensional structure of hepatitis Bvirus core particles determined by electron cryomicroscopy. Cell,1994;77:943-950.
56. Pumpens P, Grens E. Hepatitis B core particles as a universal display model: astructure-function basis for development. FEBS Letter,1999;442:1-6.
57. Kremer M, Bittner A, Schnierle BS. Human APOBEC3G in corporation into murineleukemia virus particles. Virology,2005;337:175-182.
58.秦成峰，秦鄂德.组装抗病毒疗法:抗病毒感染的新策略.军事医学科学院院刊，2004；8:388-390.
59. Ma L, Jones CT, Groesch TD, et al.Solution structure of dengue virus capsid proteinreveals another fold. Proc Natl Acad Sci U S A.2004;101:3414-3419.
60. Suspene R, Henry M, Guillot S, et al. Recovery of APOBEC3G-edited humanimmunodeficiency virus G to A hypermutants by differential DNA denaturation PCR. JGen Virol,2005;86:125-129.
61. Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNAstrands by APOBEC3cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A,2005;102:8321–8326.
62. Nguyen DH, Gummuluru S, Hu J. Deamination-independent inhibition of hepatitis Bvirus reverse transcription by APOBEC3G. J Virol,2007;81:4465–4472.
63. Tasaki T, Taharaguchi S, Kobayashi T, et al. Inhibition of pseudorabies virus replicationby a dominant-negative mutant of early protein0expressed in a tetracycline-regulatedsystem. Vet Microbiol,2001;78:195-203.
64. Von Weizsacker F, Kock J, Wieland S, et al. Dominant negative mutants of the duckhepatitis B virus core protein interfere with RNA pregenome packaging and viral DNAsynthesis. Hepatology,1999;30:308-315.
65. Koschel M, Oed D, Gerelsaikhan T, et al. Hepatitis B virus core gene mutations whichblock nucleocapsid envelopment. J Virol,2000;74:1-7.
66. Scaglioni P, Melegari M, Takahashi M. Use of dominant negative mutants of thehepadnaviral core protein as antiviral agents. Hepatology,1996;24:1010-1017.
67. von Weizsacker F, Wieland S, Blum HE. Inhibition of viral replication by geneticallyengineered mutants of the duck hepatitis B virus core protein. Hepatology,1996;24:294-299.
68. Desai P, Person S. Incorporation of the green fluorescent protein into the herpes simplexvirus type1capsid. J Virol,1998;72:7563-7568.
69. McNamee EE, Taylor TJ, Knipe DM. A dominant-negative herpesvirus protein inhibitsintranuclear targeting of viral proteins: effects on DNA replication and late geneexpression. J Virol,2000;74:10122-10131.
70. Posada R, Pettoello-Mantovani M, Sieweke M, et al. Suppression of HIV type1replication by a dominant-negative Ets-1mutant. AIDS Res Hum Retroviruses,2000;16:1981-1989.
71. Szilvay AM, B e SO, Kalland KH. Co-expression of a trans-dominant negative mutant ofthe human immunodeficiency virus type1(HIV-1) Rev protein affects theRev-dependent splicing pattern and expression of HIV-1RNAs. J Gen Virol,1999;80:1965-1974.
72. Scaglioni P, Melegari M, Takahashi M. Use of dominant negative mutants of thehepadnaviral core protein as antiviral agents. Hepatology,1996;24:1010-1017.
73. Zhang W, Zhang X, Tian C, et al. Cytidine deaminase APOBEC3B interacts withheterogeneous nuclear ribonucleoprotein K and suppresses hepatitis B virus expression.Cell Microbiol,2008;10:112-121.
1. Ganem D, Prince AM. Hepatitis B virus infection natural history and clinicalconsequences. N Engl J Med,2004;350:1118-1129.
2. Guidotti LG, Rochford R, Chung J, et al. Viral clearance without destruction of infectedcells during acute HBV infection. Science,1999;284:825-828.
3. Thimme R, Wieland S, Steiger C, et al. CD8+T cells mediate viral clearance and diseasepathogenesis during acute hepatitis B virus infection. J Virol,2003;77:68-76.
4. Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of alpha/beta interferoneliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. J Virol,2000;74:4165-4173.
5. Navaratnam N, Fujino T, Bayliss J, et al. Escherichia coli cytidine deaminase provides amolecular model for ApoB RNA editing and a mechanism for RNA substraterecognition.J Mol Biol,1998;275:695-714.
6. Betts L, Xiang S, Short SA, et al. Cytidine deaminase. The2.3A crystal structure of anenzyme: transition-state analog complex. J Mol Biol,1994;235:635-656.
7. Johansson E, Mejlhede N, Neuhard J, et al. Crystal structure of the tetrameric cytidinedeaminase from Bacillus subtilis at2.0A resolution. Biochemistry,2002;41:2563-2570.
8. Xie K, Sowden MP, Dance GS, et al. The structure of a yeast RNA-editing deaminaseprovides insight into the fold and function of activation-induced deaminase andAPOBEC-1. Proc Natl Acad Sci U S A,2004;101:8114-8119.
9. Chung SJ, Fromme JC, Verdine GL. Structure of human cytidine deaminase bound to apotent inhibitor. J Med Chem,2005;48:658-660.
10.吴小霞，马义才. APOBEC家族的防御机制.现代预防医学,2006;33:41-43.
11. Smith HC, Gott JM, Hanson MR. A guide to RNA editing. RNA,1997;3:1105-1123.
12. Powell LM, Wallis SC, Pease RJ, et al. A novel form of tissue specifi cRNA processingproduces apolipoprotein-B48in intestine. Cell,1987;50:831-840.
13. Chen SH, Habib G, Yang CY, et al. Apolipoprotein B-48is the product of a messengerRNA with an organ-specific in frame stop codon. Science,1987;238:363-366.
14. Teng B, Burant CF, Davidson NO. Molecular cloning of an apolipoprotein BmessengerRNA editing protein. Science,1993;260:1816-1819.
15. Anant S, Mac Ginnitie AJ, Davidson NO. APOBEC-1, the catalytic subunit of themammalian apolipoprotein B mRNA editing enzyme, is a novel RNA-binding protein. JBiol Chem,1995;270:14762-14767.
16.李卫中，洪敏，李琦涵. APOBEC家族：介导天然抗病毒免疫的新型宿主细胞因子.病毒学报,2006;22:74-77.
17. Navaratnam N, Bhattacharya S, Fujino T, et al. Evolutionary origins of apoB mRNAediting: catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif atits active site. Cell,1995;81:187-195.
18. Mehta A, Kinter MT, Sherman NE, et al. Molecular cloning of apobec-1complementation factor, a novel RNA-binding protein involved in the editing ofapolipoprotein B mRNA. Mol Cell Biol,2000;20:1846-1854.
19. Blanc V, Henderson JO, Kennedy S, et al. Mutagenesis of apobec-1complementationfactor reveals distinct domains that modulate RNA binding, protein-protein interactionwith apobec-1, and complementation of C to U RNA-editing activity. J Biol Chem,2001;276:46386-46393.
20. Lau PP, Zhu HJ, Baldini HA, et al. Dimeric structure of a human apo B mRNA editingprotein and cloning and chromosomal localization of its gene. Proc Natl Acad Sci USA,1994;91:8522-8526.
21. Galloway CA, Kumar A, Krucinska J, et al. APOBEC-1complementation factor (ACF)forms RNA-dependent multimers. Biochem Biophys Res Commun,2010;398:38-43.
22. Sowden MP, Ballatori N, Jensen KL, et al. The editosome for cytidine to uridine mRNAediting has a native complexity of27S: identification of intracellular domainscontaining active and inactive editing factors. J Cell Sci,2002;115:1027-1039.
23. Navaratnam N, Patel D, Shah RR, et al. An additional editing site is present inapolipoprotein B mRNA. Nucleic Acids Re,1991;19:1741-1744.
24. Backus JW, Schock D, Smith HC. Only cytidines5' of the apolipoprotein B mRNAmooring sequence are edited. Biochim Biophys Acta,1994;1219:1-14.
25. Skuse GR, Cappione AJ, Sowden M, et al. The neurofibromatosis type I messenger RNAundergoes base modification RNA editing. Nucleic Acids Res,1996;24:478-485.
26. Sowden M, Hamm JK, pinelli S, et al. Determinants involved in regulating theproportion of edited apolipoprotein B RNAs. RNA,1996;2:274-288.
27. Rosenberg BR, Hamilton CE, Mwangi MM, et al. Transcriptome-wide sequencingreveals numerous APOBEC1mRNA-editing targets in transcript3' UTRs. NatStruct Mol Biol,2011;18:230-236.
28. Lehmann DM, Galloway CA, Macelrevey C, et al. Functional characterization ofAPOBEC-1complementation factor phosphorylation sites. Biochim Biophys Acta,2007;1773:408-418.
29. Lehmann DM, Galloway CA, Sowden MP, et al. Metabolic regulation of apoB mRNAediting is associated with phosphorylation of APOBEC-1comple-mentation factor.Nucleic Acids Res,2006;34:3299-3308.
30. Mukhopadhyay D, Anant S, Lee RM, et al. C→U editing of neurofibromatosis1mRNAoccurs in tumors that express both the type II transcript and apobec-1, the catalytic subunitof the apolipoprotein B mRNA-editing enzyme. Am J Hum Genet,2002;70:38-50.
31. Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequentlylocated at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U SA,2004;101:2999-3004.
32. Smith HC, Mark P, Kefang X, et al. In: Grosjean H, editor. Structure and Function ofMammalian Cytidine Deaminases that Mediate Expressed Sequence Diversification. NY:Springer-Verlag,2005; p:1610-2145.
33. Hirano K, Young SG, Farese Jr RV, et al. Targeted disruption of the mouse apobec-1gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48. J BiolChem,1996;271:9887-9890.
34. Nakamuta M, Chang BHJ, Zsigmond E, et al. Complete phenotypic characterization ofthe apobec-1knockout mice with a wild-type genetic background and a humanapolipoprotein B transgenic background, and restoration of apolipoprotein B mRNAediting by somatic gen transfer of Apobec-1. J Biol Chem,1996;271:25981-25988.
35. Yamanaka S, Poksay KS, Driscoll DM, et al. Hyperediting of multiple cytidines ofapolipoprotein B mRNA by APOBEC-1requires auxiliary protein(s) but not a mooringsequence motif. J Biol Chem,1996;271:11506-11510.
36. Yamanaka S, Poksay KS, Arnold KS, et al. A novel translational repressor mRNA isedited extensively in livers containing tumors caused by the transgene expression of theapoB mRNA-editing enzyme. Genes Dev,1997;11:321-333.
37. Yamanaka S, Balestra M, Ferrell L, et al. Apolipoprotein B mRNA editing proteininduces hepatocellular carcinoma and dysplasia in transgenic animals. Proc Natl Acad SciUSA,1995;92:8483-8487.
38. Harris RS, Petersen-Mahrt SK, Neuberger MS. RNA editing enzyme APOBEC1andsome of its homologs can act as DNA mutators. Mol Cell,2002;10:1247-1253.
39. Petersen-Mahrt SK, Neuberger MS. In vitro deamination of cytosine to uracil insingle-stranded DNA by apolipoprotein B editing complex catalytic subunit1(APOBEC1). J Biol Chem,2003;278:19583-19586.
40. Gee P, Ando Y, Kitayama H, et al. APOBEC1-mediated editing and attenuation ofHSV-1DNA implicates an antiviral role in neurons during encephalitis. J Virol,2011;85:9726-9736.
41. Bishop KN, Holmes RK, Sheehy AM, et al. APOBEC-mediated editing of viral RNA.Science,2004;305:645.
42. Kracker S, Durandy A. Insights into the B cell specific process of immunoglobulin classswitch recombination. Immunol Lett,2011;138:97-103.
43. Di Noia JMNM. Molecular mechanisms of antibody somatic hypermutation. Annu RevBiochem,2007;76:1-22.
44. Muramatsu M, Anant SV, Sugai S, et al.Specific expression of activation-inducedcytidine deaminase (AID), a novel member of the RNA-editing deaminase family ingerminal center B cells. J Biol Chem,1999;274:18470-18476.
45. Martin ASM. Antibody alterations. Nature,2001;412:870-871.
46. Papavasiliou FNSD. Somatic hypermutation of immunoglobulin genes: mergingmechanisms for genetic diversity. Cell,2002;109:S35-44.
47. Odegard Vh SD. Targeting of omatic hypermutation.Nat Rev Immunol,2006;6:573-583.
48. Arakawa HHJ, Buerstedde M. Requirement of the activation-induced deaminase (AID)gene for immunoglobulin gene conversion. Science,2001;295:1301.
49. Fugmann SD, Schatz DG. Immunology. One AID to unite them all. Science,2002;295:1244-1245.
50. Petersen-Mahrt SK, Harris RS, Neuberger MS. AID mutates E. coli suggesting a DNAdeamination mechanism for antibody diversification. Nature,2002;418:99-103.
51. Revy PMT, Levy Y, Geissmann F, et al. Activation-induced cytidine deaminase (AID)deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2).Cell,2000;102:565-575.
52. Muramatsu M, Fagarasan KK, Yamada S, et al. Class switch recombination andhypermutation require activation-induced cytidine deaminase (AID), a potential RNAediting enzyme. Cell,2000;102:553-563.
53. Okazaki IM, Honjo KAT. Role of AID in tumorigenesis. Adv Immunol,2007;94:245-273.
54. McCarthy HWW, Barron LL, Cromwell CC, et al. High expression of activation-induced cytidine deaminase (AID) and splice variants is a distinctive feature ofpoor-prognosis chronic lymphocytic leukemia. Blood,2003;101:4903-4908.
55. Marantidou F, Stalika DA, Korkolopoulou E, et al. Activation-induced cytidinedeaminase splicing patterns in chronic lympho-cytic leukemia. Blood Cells MolDis,2010;44:262-267.
56. Albesiano E, Damle MB, Allen RN, et al. Activation-induced cytidine deaminase inchronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic,variably sized fraction of the clone. Blood,2003;102:3333-3339.
57. Wu XD, Chang SK, Nowakowski GS, et al. Alternative splicing regulatesactivation-induced cytidine deaminase (AID): implications for suppression of AIDmutagenic activity in normal and malignant B cells. Blood,2008;112:4675-4682.
58. van Maldegem FJR, van Dijk R, Bende RJ, et al. Activation-induced cytidine deaminasesplice variants are defective because of the lack of struc-tural support for the catalyticsite. J Immunol,2010;184:2487-2491.
59. Endo Y, Marusawa H, Kinoshita K, et al.Expression of activation-induced cytidinedeaminase in human hepatocytes via NF-kappaB signaling. Oncogene,2007;26:5587-5595.
60. Sayegh CEQM, Agata Y, Murre C. E-proteins directly regulate expression ofactivation-induced deaminase in mature B cells. Nat Immunol,2003;4:586-593.
61. Jannek Hauser NS, Wallenius A, Baradaran S, et al. B-cell receptor activation inhibitsAID expression through calmodulin inhibition of E-proteins. Proc Natl Acad Sci USA,2008;105:1267-1272.
62. Ta VT, Catalan NH, Durandy N, et al. AID mutant anal-yses indicate requirement forclass-switch-specific cofactors. Nat Immunol,2003;4:843-848.
63. Okuyama S, Marusawa H, Matsumoto T, et al. Excessive activity of apolipoprotein BmRNA editing enzyme catalytic polypeptide2(APOBEC2) contributes to liver and lungtumorigenesis. Int J Cancer,2012;130:1294-1301.
64. Prochnow C, Bransteitter R, Klein MG, et al. The APOBEC-2crystal structure andfunctional implications for the deaminase AID. Nature,2007;445:447-451.
65. Lada AG, Krick CF, Kozmin SG, et al. Mutator effects and mutation signatures of editingdeaminases produced in bacteria and yeast. Biochemistry (Mosc),2011;76:131-146.
66. Sato Y, Probst HC, Tatsumi R, et al. Deficiency in APOBEC2leads to a shift in musclefiber type, diminished body mass, and myopathy. J Biol Chem,2010;285:7111-7118.
67. OhAinle M, Kerns JA, Malik HS, et al. Adaptive evolution and antiviral activity of theconserved mammalian cytidine deaminase APOBEC3H. J Virol,2006;80:3853-3862.
68. Jarmuz A, Chester A, Bayliss J, et al. An anthropoid-specific locus of orphan C to URNA-editing enzymes on chromosome22. Genomics,2002;79:285-296.
69. Hersberger M, Patarroyo-White S, Qian X, et al. Regulatable liver expression of therabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide1(APOBEC-1) inmice lack ing endogenous APOBEC-1leads to aberrant hyper editing. Biochem J,2003;369:255-262.
70. Yu Q, Konig R, Pillai S, et al. Single-strand specificity of APOBEC3G accounts forminus-strand deamination of the HIV genome. Nat St ruct Mol Biol,2004;11:435-442.
71. Sheehy AM, Gaddis NC, Choi JD, et al. Isolation of a human gene that inhibits HIV-1infection and is suppressed by the viral Vif protein. Nature,2002;418:646-650.
72. Smith HC. APOBEC3G: a double agent in defense. Trends Biochem Sci,2011;36:239-244.
73. Mangeat B, Turelli P, Caron G, et al. Broad antiretroviral defence by human APOBEC3Gthrough lethal editing of nascent reverse transcripts. Nature,2003;424:99-103.
74. Zhang H, Yang B, Pomerantz RJ, et al. The cytidine deaminase CEM15induceshypermutation in newly synthesized HIV-1DNA. Nature,2003;424:94-98.
75. Hultquist JF, Lengyel JA, Refsland EW, et al. Human and Rhesus APOBEC3D,APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity torestrict Vif-deficient HIV-1. J Virol,2011;85:11220-11234.
76. Zielonka J, Bravo IG, Marino D, et al.Restriction of equine infectious anemia virus byequine APOBEC3cytidine deaminases. J Virol,2009;83:7547-7559.
77. Dang Y, Wang X, Esselman WJ, et al. Identification of APOBEC3DE as anotherantiretroviral factor from the human APOBEC family. J Virol,2006;80:10522-10533.
78. Delebecque F, Suspene R, Calattini S, et al. Restriction of foamy viruses by APOBECcytidine deaminases. J Virol,2006;80:605-614.
79. Henry M, Guetard D, Suspene R, et al. Genetic editing of HBV DNA by monodomainhuman APOBEC3cytidine deam-inases and the recombinant nature of APOBEC3G.PLoS One,2009;4:e4277.
80. Liddament MT, Brown WL, Schumacher AJ, et al. APOBEC3F properties andhypermutation preferences indicate activity against HIV-1in vivo.Curr Biol,2004;14:1385-1391.
81. Patenaude AMOA, Hu Y, Campo VH, et al. Active nuclear import and cytoplasmicretention of activation-induced deaminase. Nat Struct Mol Biol,2009;16:517-527.
82. Thielen BK, McNevin JP, McElrath MJ, et al. Innate immune signaling induces highlevels of TC-specific deaminase activity in primary monocyte-derived cells throughexpression of APOBEC3isoforms. J Biol Chem,2010;285:27753-27766.
83. Bulliard Y, Narvaiza I, Bertero A, et al. Structure-function analyses point to apolynucleotide-accommodating groove essential for APOBEC3A restriction activities. JVirol,2011;85:1765-1776.
84. Bonvin M, Achermann F, Greeve I, et al. Interferon-inducible expression of APOBEC3editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.Hepatology,2006;43:1364-1374.
85. Xu R, Zhang X, Zhang W, et al. Association of human APOBEC3cytidine deaminaseswiththe generation of hepatitis virus B x antigen mutants and hepatocellular carcinoma.Hepatology,2007;46:1810-1820.
86. Chiu YL, Witkowska HE, Hall SC, et al. High-molecular-mass APOBEC3G complexesrestrict Alu retrotransposition. Proc Natl Acad Sci USA,2006;103:15588-15593.
87. Schumann GG. APOBEC3proteins: major players in intracellular defence againstLINE-1-mediated retrotransposition.Biochem Soc Trans,2007;35:637-642.
88. Esnault C, Millet J, Schwartz O, et al. Dual inhibitory effects of APOBEC familyproteins on retrotransposition of mammalian endogenous retroviruses. Nucleic Acids Res,2006;34:1522-1531.
89. Schumacher AJ, Nissley DV, Harris RS. APOBEC3G hypermutates genomic DNA andinhibits Ty1retrotransposition in yeast. Proc Natl Acad Sci USA,2005;102:9854-9859.
90. Hulme AE, Bogerd HP, Cullen BR, et al. Selective inhibition of Alu retro-transpositionby APOBEC3G. Gene,2007;390:199-205.
91. Bogerd HP, Wiegand HL, Doehle BP, et al. APOBEC3A and APOBEC3B are potentinhibitors of LTR-retrotransposon function in human cells. Nucleic Acids Res,2006;34:89-95.
92. Chen H, Lilley CE, Yu Q, et al. APOBEC3A is a potent inhibitor of adeno-associatedvirus and retrotransposons. Curr Biol,2006;16:480-485.
93. Muckenfuss H, Hamdorf M, Held U, et al. APOBEC3proteins inhibit human LINE-1retrotransposition. J Biol Chem,2006;281:22161-22172.
94. Stenglein MD, Harris RS. APOBEC3B and APOBEC3F inhibit L1retrotrans-position bya DNA deamination-independent mechanism. J Biol Chem,2006;281:16837-16841.
95. Kinomoto M, Kanno T, Shimura M, et al. All APOBEC3family proteins differentiallyinhibit LINE-1retrotransposition. Nucleic Acids Res,2007;35:2955-2964.
96. Tan L, Sarkis PT, Wang T, et al. Sole copy of Z2-type human cytidine deaminaseAPOBEC3H has inhibitory activity against retrotransposons and HIV-1. FASEB J,2009;23:279-287.
97. Wichroski MJ, Robb GB, Rana TM. Human retroviral host restriction factors APOBEC3Gand APOBEC3F localize to mRNA processing bodies. PLoS Pathog,2006;2:e41.
98. Landry S, Narvaiza I, Linfesty DC, et al. APOBEC3A can activate the DNAdamageresponse and cause cell-cycle arrest. EMBO Rep,2011;12:444-450.
99. Rogozin IB, Basu MK, Jordan IK, et al. APOBEC4, a new member of theAID/APOBEC family of polynucleotide (deoxy) cytidine deaminases predicted bycomputational analysis. Cell Cycle,2005;4:1281-1285.
100.Gunter S, Sommer G, Plikat U, et al. Naturally occurring hepatitis B virus genomesbearing the hallmarks of retroviral G to A hypermutation. Virology,1997;18:104-108.
101.Turelli P, Mangeat B, Jost S, et al. Inhibition of hepatitis B virus replication byAPOBEC3G. Science,2004;303:1829.
102.R sler C, K ck J, Malim MH, et al. Comment on ‘Inhibition of hepatitis B virusreplication’ by APOBEC3G. Science,2004;305:1403a.
103.R sler C, K ck J, Kann M, et al. APOBEC-mediated interference with hepadnavirusproduction. Hepatology,2005;42:301-309.
104.Nguyen DH, Gummuluru S, Hu J. Deamination-independent inhibition of hepatitis Bvirus reverse transcription by APOBEC3G. J Virol,2007;81:4465-4472.
105.Noguchi C, Hiraga N, Mori N, et al. Dual effects of APOBEC3G on hepatitis B virus. JGen Virol,2007;88:432-440.
106.Bonvin M, Greeve J. Effects of point mutations in the cytidine deaminase domains ofAPOBEC3B on replication and hypermutation of hepatitis B virus in vitro. J Gen Virol,2007;88:3270-3274.
107.Suspene R, Henry M, Guillot S, et al. Recovery of APOBEC3G-edited humanimmunodeficiency virus G to A hypermutants by differential DNA denaturation PCR. JGen Virol,2005;86:125-129.
108.Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNAstrands by APOBEC3cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A,2005;102:8321-8326.
109.Baumert TF, R sler C, Malim MH, et al. Hepatitis B virus DNA is subject to extensiveediting by the human deaminase APOBEC3C. Hepatology,2007;46:682-689.
110.Zhang W, Zhang X, Tian C, et al. Cytidine deaminase APOBEC3B interacts withheterogeneous nuclear ribonucleoprotein K and suppresses hepatitis B virus expression.Cell Microbiol,2008;10:112-121.
111.Murakami Y, Saigo K, Takashima H, et al. Large scaled analysis of hepatitis B virus(HBV) DNA integration in HBV related hepatocellular carcinomas. Gut,2005;54:1162-1168.
112.Iavarone M, Trabuet JB, Delpuech O, et al. Characterization of hepatitis B virus Xprotein mutants in tumour and nontumour liver cells using laser capture microdissection.J Hepatol,2003;39:253-261.
113.Jost S, Turelli P, Mangeat B, et al. Induction of antiviral cytidine deaminases does notexplain the inhibition of hepatitis B virus replication by interferons. J Virol,2007;81:10588-10596.
2. Liang X, Bi S, Yang W, et al. Epidemiological serosurvey of hepatitis B inChina-decliningHBV prevalence due to hepatitis B vaccination. Vaccine,2009;27:6550-6557.
3. Liang X, Bi S, Yang W, et al. Evaluation of the impact of hepatitis B vaccinationamongchildren born during1992-2005in China. J Infect Dis,2009;200:39-47.
4. Lu FM, Zhuang H. Management of hepatitis B in China [J]. Chin Med J (Engl),2009;122:3-4.
5. Rijckborst V, Janssen HL. The Role of Interferon in Hepatitis B Therapy. Curr Hepat Rep,2010;9:231-238.
6. Chen YC, Chu CM, Yeh CT, et al. Natural course following the onset of cirrhosisinpatients with chronic hepatitis B: a long-term follow-up study. Hepatol Int,2007;1:267-273.
7. Hsu YS, Chien RN, Yeh CT, et al. Long-term outcome after spontaneousHBeAgseroconversion in patients with chronic hepatitis B. Hepatology,2002;35:1522-1527.
8. van Zonneveld M,van Nunen AB, Niesters HG, et al. Lamivudine treatmentduringpregnancy to prevent perinatal transmission of hepatitis B virus infection. J ViralHepat,2003;10:294-297.
9. Lok AS, Lai CL, Leung N, et al. Long-term safety of lamivudine treatment in patientswith chronic hepatitis B. Gastroenterology,2003;125:1714-1722.
10.姚光弼,崔振宇,姚集鲁,等.国产拉米夫定治疗2200例慢性乙型肝炎的Ⅳ期临床试验.中华肝脏病杂志,2003;11:103-108.
11.姚光弼,王宝恩,崔振宇,等.拉米夫定治疗慢性乙型肝炎三年疗效观察.中华内科杂志,2003;42:382-387.
12. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy with adefovirdipivoxil for HBeAg-negative chronic hepatitis B for up to5years. Gastroenterology,2006;131:1743-1751.
13. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy with adefovirdipivoxil for HBeAg-negative chronic hepatitis B. N Engl J Med,2005;352:2673-2681.
14. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Adefovir dipivoxil for thetreatment of hepatitis B e antigen-negative chronic hepatitis B. N Engl J Med,2003;348:800-807.
15. Hou J, Yin YK, Xu D, et al. Telbivudine versus lamivudine in Chinese patients withchronic hepatitis B: Results at1year of a randomized, double-blind trial. Hepatology,2008;47:447-454.
16. Natsoulis G, Boeke JD. New antiviral strategy using capsid nuclease fusionproteins.Nature,1991;352:632-635.
17. Beterams G, Nassal M. Significant interference with hepatitisB virus replication by acore-nuclease fusion protein. J Biol Chem,2001;276:8875-8883.
18. Baumert TF, R sler C, Malim MH, et al. Hepatitis B virus DNA is subject to extensiveediting by the human deaminase APOBEC3C. Hepatology,2007;46:682–689.
19. Jarmuz A, Chester A, Bayliss J, et al. An anthropoid-specific locus of orphan C to URNA-editing enzymes on chromosome22. Genomics,2002;79:285-296.
20. OhAinle M, Kerns JA, Malik HS, et al. Adaptive evolution and antiviral activity of theconserved mammalian cytidine deaminase APOBEC3H. J Virol,2006;80:3853-3862.
21. Yu Q, Konig R, Pillai S, et al. Single-strand specificity of APOBEC3Gaccounts forminus-strand deamination of the HIV genome. Nat St ruct Mol Biol,2004;11:435-442.
22. Hultquist JF, Lengyel JA, Refsland EW, et al. Human and Rhesus APOBEC3D,APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity torestrict Vif-deficient HIV-1. J Virol,2011;85:11220-11234.
23. Zielonka J, Bravo IG, Marino D, et al.Restriction of equine infectious anemia virus byequine APOBEC3cytidine deaminases. J Virol,2009;83:7547-7559.
24. Dang Y, Wang X, Esselman WJ, et al. Identification of APOBEC3DE as anotherantiretroviral factor from the human APOBEC family. J Virol,2006;80:10522-10533.
25. Delebecque F, Suspene R, Calattini S, et al. Restriction of foamy viruses by APOBECcytidine deaminases. J Virol,2006;80:605-614.
26. Esnault C, Millet J, Schwartz O, et al. Dual inhibitory effects of APOBEC familyproteins on retrotransposition of mammalian endogenous retroviruses. Nucleic Acids Res,2006;34:1522-1531.
27. Schumann GG. APOBEC3proteins: major players in intracellular defence againstLINE-1-mediated retrotransposition.Biochem Soc Trans,2007;35:637-642.
28. Bonvin M, Achermann F, Greeve I, et al. Interferon-inducible expression of APOBEC3editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.Hepatology,2006;43:1364-1374.
29. Gunter S, Sommer G, Plikat U, et al. Naturally occurring hepatitis B virus genomesbearing the hallmarks of retroviral G to A hypermutation. Virology,1997;18:104–108.
30. R sler C, K ck J, Malim MH, et al. Comment on ‘Inhibition of hepatitis B virusreplication’ by APOBEC3G. Science,2004;305:1403a.
31. Holmes RK, Malim MH, Bishop KN. APOBEC-mediated viral restriction:not simplyediting?. Trends Biochem Sci,2007;32:118-128.
32. Turelli P, Mangeat B, Jost S, et al. Inhibition of hepatitis B virus replication byAPOBEC3G. Science,2004;303:1829.
33. Günther S, Sommer G, Plikat U, et al. Naturally occurring hepatitisB virus genomesbearing the hallmarks of retroviral G-A hypermutation. Virology,1997;235:104-108.
34. Muller V, Bonhoeffer S. Guanine-adenine bias:a general property of retroid viruses that isunrelated to host-induced hypermutation. Trends Genet,2005;21:264-268.
35. Noguchi C, Ishino H, Tsuge M, et al. G to A hypermutation of hepatitis B virus.Hepatology,2005;41:626–633.
36. Jarmuz A, hester A, Baylls J, et al. An anthropoid-specific locus of orphan C to URNA-editing enzymes on chromosome22. Genomics,2002;79:285-296.
37. Yu Q, Chen D, K nig R, et al. APOBEC3B and APOBEC3C are potent inhibitors ofsimian immunodeficiency virus replication. J Biol Chem,2004;279:53379-53386.
38. Protzer U, Nassal M, Chiang PW, et al. Interferon gene transfer by a hepatitis B virusvector efficiently suppresses wild-type virus infection. Proc Natl Acad Sci,1999;96:10818-10823.
39.孙殿兴，胡大荣.抗乙型肝炎病毒分子治疗新策略.中华肝脏病杂志，2001;9:315-316.
40.慢性乙型肝炎防治指南(2010年版).肝脏，2011;16:2-15.
41. Okui N, Sakuma R, Kobayashi N, et al. Packageable antiviral therapeutics against humanimmunodeficiency virus type1: virion-targeted virus inactivation by incorporation of asingle-chain antibody against viral integrase into progeny virions. Hum Gene Ther,2000;11:537-546.
42. Schwartz S, Felber BK, Pavlakis GN. Distinct RNA sequences in the gag region ofhuman immunodeficiency virus type1decrease RNA stability and inhibit expression inthe absence of Rev protein. J Virol,1992;66:150-159.
43. Wu X, Liu H, Xiao H, et al.Targeting foreign proteins to human immuno-deficiencyvirus particles via fusion with Vpr and Vpx. J Virol,1995;69:3389-3398.
44. Wu X, Liu H, Xiao H, et al. Inhibition of human and simian immunodeficiency virusprotease function by targeting Vpx-protease-mutant fusion protein into viral particles. JVirol,1996;70:3378-3384.
45. Wang CT, Stegeman-Olsen J, Zhang Y, et al. Assembly of HIV GAG-BgalactoSidase-fusion proteins into virus particles. Virology,1994;200:524-534.
46. Natsoulis G, Seshaiah P,Federspie MJ.Targeting of a nuclease tomurine leukemia viruscapsid inhibits viral multiplication. ProcNatl Acad Sci USA,1995;92:364-368.
47. Beterams G, Bottcher B, Nassal M. Packing of up to240subunits of a17kDa nucleaseinto the interior of recombinant hepatitis B virus capsids. FEBS Lett,2000;481:169-176.
48.秦成峰，秦鄂德，于曼等.登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用.中国生物化学与分子生物学报，2004;20:757-761.
49. Qin CF, Qin ED. Capsid-targeted viral inactivation can destroy dengue2virus fromwithin in vitro. Arch Virol,2006;151:379-385.
50. Qin CF, Qin E, Yu M, et al. Therapeutic effects of dengue2virus capsid protein andstaphylococcal nuclease fusion protein on dengue-infected cell cultures. ArchVirol,2005;150:659-669.
51. Qin CF, Qin ED. Development of cell lines stably expressing staphylococcal nucleasefused to dengue2virus capsid protein for CTVI. Acta Biochim Biophys Sin (Shanghai),2004;36:577-582.
52. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev,2000;64:51-68.
53. Ning B, Shih C. Nucleolar localization of human hepatitis B virus capsid protein. J Virol,2004;78:13653-13668.
54. Hu J, Toft DO, Seeger C. Hepadnavirus assembly and reverse transcription require amulti-component chaperone complex which is incorporated into nucleocapsids. EMBO J,1997;16:59-68.
55. Crowther RA, Kiselev NA, Bottcher B, et al. Three-dimensional structure of hepatitis Bvirus core particles determined by electron cryomicroscopy. Cell,1994;77:943-950.
56. Pumpens P, Grens E. Hepatitis B core particles as a universal display model: astructure-function basis for development. FEBS Letter,1999;442:1-6.
57. Kremer M, Bittner A, Schnierle BS. Human APOBEC3G in corporation into murineleukemia virus particles. Virology,2005;337:175-182.
58.秦成峰，秦鄂德.组装抗病毒疗法:抗病毒感染的新策略.军事医学科学院院刊，2004；8:388-390.
59. Ma L, Jones CT, Groesch TD, et al.Solution structure of dengue virus capsid proteinreveals another fold. Proc Natl Acad Sci U S A.2004;101:3414-3419.
60. Suspene R, Henry M, Guillot S, et al. Recovery of APOBEC3G-edited humanimmunodeficiency virus G to A hypermutants by differential DNA denaturation PCR. JGen Virol,2005;86:125-129.
61. Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNAstrands by APOBEC3cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A,2005;102:8321–8326.
62. Nguyen DH, Gummuluru S, Hu J. Deamination-independent inhibition of hepatitis Bvirus reverse transcription by APOBEC3G. J Virol,2007;81:4465–4472.
63. Tasaki T, Taharaguchi S, Kobayashi T, et al. Inhibition of pseudorabies virus replicationby a dominant-negative mutant of early protein0expressed in a tetracycline-regulatedsystem. Vet Microbiol,2001;78:195-203.
64. Von Weizsacker F, Kock J, Wieland S, et al. Dominant negative mutants of the duckhepatitis B virus core protein interfere with RNA pregenome packaging and viral DNAsynthesis. Hepatology,1999;30:308-315.
65. Koschel M, Oed D, Gerelsaikhan T, et al. Hepatitis B virus core gene mutations whichblock nucleocapsid envelopment. J Virol,2000;74:1-7.
66. Scaglioni P, Melegari M, Takahashi M. Use of dominant negative mutants of thehepadnaviral core protein as antiviral agents. Hepatology,1996;24:1010-1017.
67. von Weizsacker F, Wieland S, Blum HE. Inhibition of viral replication by geneticallyengineered mutants of the duck hepatitis B virus core protein. Hepatology,1996;24:294-299.
68. Desai P, Person S. Incorporation of the green fluorescent protein into the herpes simplexvirus type1capsid. J Virol,1998;72:7563-7568.
69. McNamee EE, Taylor TJ, Knipe DM. A dominant-negative herpesvirus protein inhibitsintranuclear targeting of viral proteins: effects on DNA replication and late geneexpression. J Virol,2000;74:10122-10131.
70. Posada R, Pettoello-Mantovani M, Sieweke M, et al. Suppression of HIV type1replication by a dominant-negative Ets-1mutant. AIDS Res Hum Retroviruses,2000;16:1981-1989.
71. Szilvay AM, B e SO, Kalland KH. Co-expression of a trans-dominant negative mutant ofthe human immunodeficiency virus type1(HIV-1) Rev protein affects theRev-dependent splicing pattern and expression of HIV-1RNAs. J Gen Virol,1999;80:1965-1974.
72. Scaglioni P, Melegari M, Takahashi M. Use of dominant negative mutants of thehepadnaviral core protein as antiviral agents. Hepatology,1996;24:1010-1017.
73. Zhang W, Zhang X, Tian C, et al. Cytidine deaminase APOBEC3B interacts withheterogeneous nuclear ribonucleoprotein K and suppresses hepatitis B virus expression.Cell Microbiol,2008;10:112-121.
1. Ganem D, Prince AM. Hepatitis B virus infection natural history and clinicalconsequences. N Engl J Med,2004;350:1118-1129.
2. Guidotti LG, Rochford R, Chung J, et al. Viral clearance without destruction of infectedcells during acute HBV infection. Science,1999;284:825-828.
3. Thimme R, Wieland S, Steiger C, et al. CD8+T cells mediate viral clearance and diseasepathogenesis during acute hepatitis B virus infection. J Virol,2003;77:68-76.
4. Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of alpha/beta interferoneliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. J Virol,2000;74:4165-4173.
5. Navaratnam N, Fujino T, Bayliss J, et al. Escherichia coli cytidine deaminase provides amolecular model for ApoB RNA editing and a mechanism for RNA substraterecognition.J Mol Biol,1998;275:695-714.
6. Betts L, Xiang S, Short SA, et al. Cytidine deaminase. The2.3A crystal structure of anenzyme: transition-state analog complex. J Mol Biol,1994;235:635-656.
7. Johansson E, Mejlhede N, Neuhard J, et al. Crystal structure of the tetrameric cytidinedeaminase from Bacillus subtilis at2.0A resolution. Biochemistry,2002;41:2563-2570.
8. Xie K, Sowden MP, Dance GS, et al. The structure of a yeast RNA-editing deaminaseprovides insight into the fold and function of activation-induced deaminase andAPOBEC-1. Proc Natl Acad Sci U S A,2004;101:8114-8119.
9. Chung SJ, Fromme JC, Verdine GL. Structure of human cytidine deaminase bound to apotent inhibitor. J Med Chem,2005;48:658-660.
10.吴小霞，马义才. APOBEC家族的防御机制.现代预防医学,2006;33:41-43.
11. Smith HC, Gott JM, Hanson MR. A guide to RNA editing. RNA,1997;3:1105-1123.
12. Powell LM, Wallis SC, Pease RJ, et al. A novel form of tissue specifi cRNA processingproduces apolipoprotein-B48in intestine. Cell,1987;50:831-840.
13. Chen SH, Habib G, Yang CY, et al. Apolipoprotein B-48is the product of a messengerRNA with an organ-specific in frame stop codon. Science,1987;238:363-366.
14. Teng B, Burant CF, Davidson NO. Molecular cloning of an apolipoprotein BmessengerRNA editing protein. Science,1993;260:1816-1819.
15. Anant S, Mac Ginnitie AJ, Davidson NO. APOBEC-1, the catalytic subunit of themammalian apolipoprotein B mRNA editing enzyme, is a novel RNA-binding protein. JBiol Chem,1995;270:14762-14767.
16.李卫中，洪敏，李琦涵. APOBEC家族：介导天然抗病毒免疫的新型宿主细胞因子.病毒学报,2006;22:74-77.
17. Navaratnam N, Bhattacharya S, Fujino T, et al. Evolutionary origins of apoB mRNAediting: catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif atits active site. Cell,1995;81:187-195.
18. Mehta A, Kinter MT, Sherman NE, et al. Molecular cloning of apobec-1complementation factor, a novel RNA-binding protein involved in the editing ofapolipoprotein B mRNA. Mol Cell Biol,2000;20:1846-1854.
19. Blanc V, Henderson JO, Kennedy S, et al. Mutagenesis of apobec-1complementationfactor reveals distinct domains that modulate RNA binding, protein-protein interactionwith apobec-1, and complementation of C to U RNA-editing activity. J Biol Chem,2001;276:46386-46393.
20. Lau PP, Zhu HJ, Baldini HA, et al. Dimeric structure of a human apo B mRNA editingprotein and cloning and chromosomal localization of its gene. Proc Natl Acad Sci USA,1994;91:8522-8526.
21. Galloway CA, Kumar A, Krucinska J, et al. APOBEC-1complementation factor (ACF)forms RNA-dependent multimers. Biochem Biophys Res Commun,2010;398:38-43.
22. Sowden MP, Ballatori N, Jensen KL, et al. The editosome for cytidine to uridine mRNAediting has a native complexity of27S: identification of intracellular domainscontaining active and inactive editing factors. J Cell Sci,2002;115:1027-1039.
23. Navaratnam N, Patel D, Shah RR, et al. An additional editing site is present inapolipoprotein B mRNA. Nucleic Acids Re,1991;19:1741-1744.
24. Backus JW, Schock D, Smith HC. Only cytidines5' of the apolipoprotein B mRNAmooring sequence are edited. Biochim Biophys Acta,1994;1219:1-14.
25. Skuse GR, Cappione AJ, Sowden M, et al. The neurofibromatosis type I messenger RNAundergoes base modification RNA editing. Nucleic Acids Res,1996;24:478-485.
26. Sowden M, Hamm JK, pinelli S, et al. Determinants involved in regulating theproportion of edited apolipoprotein B RNAs. RNA,1996;2:274-288.
27. Rosenberg BR, Hamilton CE, Mwangi MM, et al. Transcriptome-wide sequencingreveals numerous APOBEC1mRNA-editing targets in transcript3' UTRs. NatStruct Mol Biol,2011;18:230-236.
28. Lehmann DM, Galloway CA, Macelrevey C, et al. Functional characterization ofAPOBEC-1complementation factor phosphorylation sites. Biochim Biophys Acta,2007;1773:408-418.
29. Lehmann DM, Galloway CA, Sowden MP, et al. Metabolic regulation of apoB mRNAediting is associated with phosphorylation of APOBEC-1comple-mentation factor.Nucleic Acids Res,2006;34:3299-3308.
30. Mukhopadhyay D, Anant S, Lee RM, et al. C→U editing of neurofibromatosis1mRNAoccurs in tumors that express both the type II transcript and apobec-1, the catalytic subunitof the apolipoprotein B mRNA-editing enzyme. Am J Hum Genet,2002;70:38-50.
31. Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequentlylocated at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U SA,2004;101:2999-3004.
32. Smith HC, Mark P, Kefang X, et al. In: Grosjean H, editor. Structure and Function ofMammalian Cytidine Deaminases that Mediate Expressed Sequence Diversification. NY:Springer-Verlag,2005; p:1610-2145.
33. Hirano K, Young SG, Farese Jr RV, et al. Targeted disruption of the mouse apobec-1gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48. J BiolChem,1996;271:9887-9890.
34. Nakamuta M, Chang BHJ, Zsigmond E, et al. Complete phenotypic characterization ofthe apobec-1knockout mice with a wild-type genetic background and a humanapolipoprotein B transgenic background, and restoration of apolipoprotein B mRNAediting by somatic gen transfer of Apobec-1. J Biol Chem,1996;271:25981-25988.
35. Yamanaka S, Poksay KS, Driscoll DM, et al. Hyperediting of multiple cytidines ofapolipoprotein B mRNA by APOBEC-1requires auxiliary protein(s) but not a mooringsequence motif. J Biol Chem,1996;271:11506-11510.
36. Yamanaka S, Poksay KS, Arnold KS, et al. A novel translational repressor mRNA isedited extensively in livers containing tumors caused by the transgene expression of theapoB mRNA-editing enzyme. Genes Dev,1997;11:321-333.
37. Yamanaka S, Balestra M, Ferrell L, et al. Apolipoprotein B mRNA editing proteininduces hepatocellular carcinoma and dysplasia in transgenic animals. Proc Natl Acad SciUSA,1995;92:8483-8487.
38. Harris RS, Petersen-Mahrt SK, Neuberger MS. RNA editing enzyme APOBEC1andsome of its homologs can act as DNA mutators. Mol Cell,2002;10:1247-1253.
39. Petersen-Mahrt SK, Neuberger MS. In vitro deamination of cytosine to uracil insingle-stranded DNA by apolipoprotein B editing complex catalytic subunit1(APOBEC1). J Biol Chem,2003;278:19583-19586.
40. Gee P, Ando Y, Kitayama H, et al. APOBEC1-mediated editing and attenuation ofHSV-1DNA implicates an antiviral role in neurons during encephalitis. J Virol,2011;85:9726-9736.
41. Bishop KN, Holmes RK, Sheehy AM, et al. APOBEC-mediated editing of viral RNA.Science,2004;305:645.
42. Kracker S, Durandy A. Insights into the B cell specific process of immunoglobulin classswitch recombination. Immunol Lett,2011;138:97-103.
43. Di Noia JMNM. Molecular mechanisms of antibody somatic hypermutation. Annu RevBiochem,2007;76:1-22.
44. Muramatsu M, Anant SV, Sugai S, et al.Specific expression of activation-inducedcytidine deaminase (AID), a novel member of the RNA-editing deaminase family ingerminal center B cells. J Biol Chem,1999;274:18470-18476.
45. Martin ASM. Antibody alterations. Nature,2001;412:870-871.
46. Papavasiliou FNSD. Somatic hypermutation of immunoglobulin genes: mergingmechanisms for genetic diversity. Cell,2002;109:S35-44.
47. Odegard Vh SD. Targeting of omatic hypermutation.Nat Rev Immunol,2006;6:573-583.
48. Arakawa HHJ, Buerstedde M. Requirement of the activation-induced deaminase (AID)gene for immunoglobulin gene conversion. Science,2001;295:1301.
49. Fugmann SD, Schatz DG. Immunology. One AID to unite them all. Science,2002;295:1244-1245.
50. Petersen-Mahrt SK, Harris RS, Neuberger MS. AID mutates E. coli suggesting a DNAdeamination mechanism for antibody diversification. Nature,2002;418:99-103.
51. Revy PMT, Levy Y, Geissmann F, et al. Activation-induced cytidine deaminase (AID)deficiency causes the autosomal recessive form of the hyper-IgM syndrome (HIGM2).Cell,2000;102:565-575.
52. Muramatsu M, Fagarasan KK, Yamada S, et al. Class switch recombination andhypermutation require activation-induced cytidine deaminase (AID), a potential RNAediting enzyme. Cell,2000;102:553-563.
53. Okazaki IM, Honjo KAT. Role of AID in tumorigenesis. Adv Immunol,2007;94:245-273.
54. McCarthy HWW, Barron LL, Cromwell CC, et al. High expression of activation-induced cytidine deaminase (AID) and splice variants is a distinctive feature ofpoor-prognosis chronic lymphocytic leukemia. Blood,2003;101:4903-4908.
55. Marantidou F, Stalika DA, Korkolopoulou E, et al. Activation-induced cytidinedeaminase splicing patterns in chronic lympho-cytic leukemia. Blood Cells MolDis,2010;44:262-267.
56. Albesiano E, Damle MB, Allen RN, et al. Activation-induced cytidine deaminase inchronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic,variably sized fraction of the clone. Blood,2003;102:3333-3339.
57. Wu XD, Chang SK, Nowakowski GS, et al. Alternative splicing regulatesactivation-induced cytidine deaminase (AID): implications for suppression of AIDmutagenic activity in normal and malignant B cells. Blood,2008;112:4675-4682.
58. van Maldegem FJR, van Dijk R, Bende RJ, et al. Activation-induced cytidine deaminasesplice variants are defective because of the lack of struc-tural support for the catalyticsite. J Immunol,2010;184:2487-2491.
59. Endo Y, Marusawa H, Kinoshita K, et al.Expression of activation-induced cytidinedeaminase in human hepatocytes via NF-kappaB signaling. Oncogene,2007;26:5587-5595.
60. Sayegh CEQM, Agata Y, Murre C. E-proteins directly regulate expression ofactivation-induced deaminase in mature B cells. Nat Immunol,2003;4:586-593.
61. Jannek Hauser NS, Wallenius A, Baradaran S, et al. B-cell receptor activation inhibitsAID expression through calmodulin inhibition of E-proteins. Proc Natl Acad Sci USA,2008;105:1267-1272.
62. Ta VT, Catalan NH, Durandy N, et al. AID mutant anal-yses indicate requirement forclass-switch-specific cofactors. Nat Immunol,2003;4:843-848.
63. Okuyama S, Marusawa H, Matsumoto T, et al. Excessive activity of apolipoprotein BmRNA editing enzyme catalytic polypeptide2(APOBEC2) contributes to liver and lungtumorigenesis. Int J Cancer,2012;130:1294-1301.
64. Prochnow C, Bransteitter R, Klein MG, et al. The APOBEC-2crystal structure andfunctional implications for the deaminase AID. Nature,2007;445:447-451.
65. Lada AG, Krick CF, Kozmin SG, et al. Mutator effects and mutation signatures of editingdeaminases produced in bacteria and yeast. Biochemistry (Mosc),2011;76:131-146.
66. Sato Y, Probst HC, Tatsumi R, et al. Deficiency in APOBEC2leads to a shift in musclefiber type, diminished body mass, and myopathy. J Biol Chem,2010;285:7111-7118.
67. OhAinle M, Kerns JA, Malik HS, et al. Adaptive evolution and antiviral activity of theconserved mammalian cytidine deaminase APOBEC3H. J Virol,2006;80:3853-3862.
68. Jarmuz A, Chester A, Bayliss J, et al. An anthropoid-specific locus of orphan C to URNA-editing enzymes on chromosome22. Genomics,2002;79:285-296.
69. Hersberger M, Patarroyo-White S, Qian X, et al. Regulatable liver expression of therabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide1(APOBEC-1) inmice lack ing endogenous APOBEC-1leads to aberrant hyper editing. Biochem J,2003;369:255-262.
70. Yu Q, Konig R, Pillai S, et al. Single-strand specificity of APOBEC3G accounts forminus-strand deamination of the HIV genome. Nat St ruct Mol Biol,2004;11:435-442.
71. Sheehy AM, Gaddis NC, Choi JD, et al. Isolation of a human gene that inhibits HIV-1infection and is suppressed by the viral Vif protein. Nature,2002;418:646-650.
72. Smith HC. APOBEC3G: a double agent in defense. Trends Biochem Sci,2011;36:239-244.
73. Mangeat B, Turelli P, Caron G, et al. Broad antiretroviral defence by human APOBEC3Gthrough lethal editing of nascent reverse transcripts. Nature,2003;424:99-103.
74. Zhang H, Yang B, Pomerantz RJ, et al. The cytidine deaminase CEM15induceshypermutation in newly synthesized HIV-1DNA. Nature,2003;424:94-98.
75. Hultquist JF, Lengyel JA, Refsland EW, et al. Human and Rhesus APOBEC3D,APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity torestrict Vif-deficient HIV-1. J Virol,2011;85:11220-11234.
76. Zielonka J, Bravo IG, Marino D, et al.Restriction of equine infectious anemia virus byequine APOBEC3cytidine deaminases. J Virol,2009;83:7547-7559.
77. Dang Y, Wang X, Esselman WJ, et al. Identification of APOBEC3DE as anotherantiretroviral factor from the human APOBEC family. J Virol,2006;80:10522-10533.
78. Delebecque F, Suspene R, Calattini S, et al. Restriction of foamy viruses by APOBECcytidine deaminases. J Virol,2006;80:605-614.
79. Henry M, Guetard D, Suspene R, et al. Genetic editing of HBV DNA by monodomainhuman APOBEC3cytidine deam-inases and the recombinant nature of APOBEC3G.PLoS One,2009;4:e4277.
80. Liddament MT, Brown WL, Schumacher AJ, et al. APOBEC3F properties andhypermutation preferences indicate activity against HIV-1in vivo.Curr Biol,2004;14:1385-1391.
81. Patenaude AMOA, Hu Y, Campo VH, et al. Active nuclear import and cytoplasmicretention of activation-induced deaminase. Nat Struct Mol Biol,2009;16:517-527.
82. Thielen BK, McNevin JP, McElrath MJ, et al. Innate immune signaling induces highlevels of TC-specific deaminase activity in primary monocyte-derived cells throughexpression of APOBEC3isoforms. J Biol Chem,2010;285:27753-27766.
83. Bulliard Y, Narvaiza I, Bertero A, et al. Structure-function analyses point to apolynucleotide-accommodating groove essential for APOBEC3A restriction activities. JVirol,2011;85:1765-1776.
84. Bonvin M, Achermann F, Greeve I, et al. Interferon-inducible expression of APOBEC3editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.Hepatology,2006;43:1364-1374.
85. Xu R, Zhang X, Zhang W, et al. Association of human APOBEC3cytidine deaminaseswiththe generation of hepatitis virus B x antigen mutants and hepatocellular carcinoma.Hepatology,2007;46:1810-1820.
86. Chiu YL, Witkowska HE, Hall SC, et al. High-molecular-mass APOBEC3G complexesrestrict Alu retrotransposition. Proc Natl Acad Sci USA,2006;103:15588-15593.
87. Schumann GG. APOBEC3proteins: major players in intracellular defence againstLINE-1-mediated retrotransposition.Biochem Soc Trans,2007;35:637-642.
88. Esnault C, Millet J, Schwartz O, et al. Dual inhibitory effects of APOBEC familyproteins on retrotransposition of mammalian endogenous retroviruses. Nucleic Acids Res,2006;34:1522-1531.
89. Schumacher AJ, Nissley DV, Harris RS. APOBEC3G hypermutates genomic DNA andinhibits Ty1retrotransposition in yeast. Proc Natl Acad Sci USA,2005;102:9854-9859.
90. Hulme AE, Bogerd HP, Cullen BR, et al. Selective inhibition of Alu retro-transpositionby APOBEC3G. Gene,2007;390:199-205.
91. Bogerd HP, Wiegand HL, Doehle BP, et al. APOBEC3A and APOBEC3B are potentinhibitors of LTR-retrotransposon function in human cells. Nucleic Acids Res,2006;34:89-95.
92. Chen H, Lilley CE, Yu Q, et al. APOBEC3A is a potent inhibitor of adeno-associatedvirus and retrotransposons. Curr Biol,2006;16:480-485.
93. Muckenfuss H, Hamdorf M, Held U, et al. APOBEC3proteins inhibit human LINE-1retrotransposition. J Biol Chem,2006;281:22161-22172.
94. Stenglein MD, Harris RS. APOBEC3B and APOBEC3F inhibit L1retrotrans-position bya DNA deamination-independent mechanism. J Biol Chem,2006;281:16837-16841.
95. Kinomoto M, Kanno T, Shimura M, et al. All APOBEC3family proteins differentiallyinhibit LINE-1retrotransposition. Nucleic Acids Res,2007;35:2955-2964.
96. Tan L, Sarkis PT, Wang T, et al. Sole copy of Z2-type human cytidine deaminaseAPOBEC3H has inhibitory activity against retrotransposons and HIV-1. FASEB J,2009;23:279-287.
97. Wichroski MJ, Robb GB, Rana TM. Human retroviral host restriction factors APOBEC3Gand APOBEC3F localize to mRNA processing bodies. PLoS Pathog,2006;2:e41.
98. Landry S, Narvaiza I, Linfesty DC, et al. APOBEC3A can activate the DNAdamageresponse and cause cell-cycle arrest. EMBO Rep,2011;12:444-450.
99. Rogozin IB, Basu MK, Jordan IK, et al. APOBEC4, a new member of theAID/APOBEC family of polynucleotide (deoxy) cytidine deaminases predicted bycomputational analysis. Cell Cycle,2005;4:1281-1285.
100.Gunter S, Sommer G, Plikat U, et al. Naturally occurring hepatitis B virus genomesbearing the hallmarks of retroviral G to A hypermutation. Virology,1997;18:104-108.
101.Turelli P, Mangeat B, Jost S, et al. Inhibition of hepatitis B virus replication byAPOBEC3G. Science,2004;303:1829.
102.R sler C, K ck J, Malim MH, et al. Comment on ‘Inhibition of hepatitis B virusreplication’ by APOBEC3G. Science,2004;305:1403a.
103.R sler C, K ck J, Kann M, et al. APOBEC-mediated interference with hepadnavirusproduction. Hepatology,2005;42:301-309.
104.Nguyen DH, Gummuluru S, Hu J. Deamination-independent inhibition of hepatitis Bvirus reverse transcription by APOBEC3G. J Virol,2007;81:4465-4472.
105.Noguchi C, Hiraga N, Mori N, et al. Dual effects of APOBEC3G on hepatitis B virus. JGen Virol,2007;88:432-440.
106.Bonvin M, Greeve J. Effects of point mutations in the cytidine deaminase domains ofAPOBEC3B on replication and hypermutation of hepatitis B virus in vitro. J Gen Virol,2007;88:3270-3274.
107.Suspene R, Henry M, Guillot S, et al. Recovery of APOBEC3G-edited humanimmunodeficiency virus G to A hypermutants by differential DNA denaturation PCR. JGen Virol,2005;86:125-129.
108.Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNAstrands by APOBEC3cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A,2005;102:8321-8326.
109.Baumert TF, R sler C, Malim MH, et al. Hepatitis B virus DNA is subject to extensiveediting by the human deaminase APOBEC3C. Hepatology,2007;46:682-689.
110.Zhang W, Zhang X, Tian C, et al. Cytidine deaminase APOBEC3B interacts withheterogeneous nuclear ribonucleoprotein K and suppresses hepatitis B virus expression.Cell Microbiol,2008;10:112-121.
111.Murakami Y, Saigo K, Takashima H, et al. Large scaled analysis of hepatitis B virus(HBV) DNA integration in HBV related hepatocellular carcinomas. Gut,2005;54:1162-1168.
112.Iavarone M, Trabuet JB, Delpuech O, et al. Characterization of hepatitis B virus Xprotein mutants in tumour and nontumour liver cells using laser capture microdissection.J Hepatol,2003;39:253-261.
113.Jost S, Turelli P, Mangeat B, et al. Induction of antiviral cytidine deaminases does notexplain the inhibition of hepatitis B virus replication by interferons. J Virol,2007;81:10588-10596.