携带外源基因的复制型HBV载体的构建
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摘要
背景和目的：
将病毒载体设计成能够携带外源基因并按着亲代病毒的侵入路径到达细胞体内的病毒变异体是病毒载体研究的新思路。对一些病毒家族来说，携带报告基因的复制型载体能够在简化和量化检测复制和感染、识别抗病毒和病毒易感细胞等方面成为有力的工具。乙型肝炎病毒(hepatitis B virus, HBV)，是引起乙型病毒性肝炎的小包裹DNA病毒，原则上在这个仅有3.2kb的基因组内插入外源基因不可避免地会影响本身的复制元件。HBV复制子通过前基因组RNA(pregenomic RNA, pgRNA)逆转录而来，pgRNA作为双顺反子mRNA，在形成核心蛋白(C)和逆转录酶(Pol)方面也是必需的， C和Pol开放读码框架(open reading frames, ORF)有150bp的重叠区。Pol ORF的下游区翻译一般不涉及内部核糖体进入位点(internal ribosome entry site, IRES)。我们设想将C、P重叠区域拉开，插入两个IRES分别用于表达外源基因和P蛋白，产生一个有功能的且不影响包膜蛋白表达的三顺反子pgRNA。为了减少对基因组长度的影响，我们利用仅含22个nt的RNA结合基序蛋白3(RNA-binding motif protein3, Rbm3) IRES，构建了分别携带399bp的杀稻瘟菌素抗性基因(blasticidin resistance, BsdR)和720bp的人绿色荧光蛋白(humanized Renilla green fluorescent protein, hrGFP)的HBV载体，研究发现仍有复制能力，能产生同野生型HBV一样的病毒蛋白，所形成的的病毒颗粒可用于感染HepRG细胞。这种新型的携带外源基因的HBV载体将成为研究HBV的有力工具。
方法：本课题分三部分进行探讨。
第一部分：22-nt Rbm3IRES在HBV前基因组RNA上的表达活性研究
1、构建携带CMV启动子和增强型绿色荧光蛋白(Enhanced Green FluorescentProtein, EGFP)的四种质粒： I： pCH-EMCV IRES-EGFP(包含脑炎心肌炎病毒(Encephalomyocarditis virus, EMCV) IRES及EGFP)；II：pCH-22nt IRES-EGFP(包含Rbm3IRES及EGFP)；III：pCH-BsdR-22nt IRES-EGFP(为三顺反子载体，依次包含Rbm3IRES，BsdR，Rbm3IRES及EGFP)；IV：pCH-pATG-EGFP(包含HBV C基因N端部分与EGFP)。ELISA检测I、II、III载体转染HepG2或Huh7细胞后72h的上清液中HBeAg的表达，并观察四种载体转染后EGFP的表达。
2、构建携带HBV C基因启动子和荧光素酶基因(Renilla luciferase, RLuc)的四种质粒：I：pHBV-EMCV IRES-RLuc(包含EMCV IRES及RLuc)；II：pHBV-22ntIRES-RLuc(包含Rbm3IRES及RLuc)；III：pHBV-BsdR-22nt IRES-RLuc(为三顺反子载体，依次包含Rbm3IRES，BsdR，Rbm3IRES及RLuc)；IV：pHBV-pATG-RLuc(包含HBV C基因N端部分与RLuc)。四种质粒分别同对照质粒pGL3-Control共转染HepG2或Huh7细胞，通过双荧光素酶检测试剂盒测定荧光素酶值。
第二部分：复制型HBV载体的构建及其表达与复制能力研究
构建两种复制型HBV载体：pCH-BsdR和pCH-hrGFP。两个质粒和携带野生型HBV的质粒pCH-3093分别转染肝癌细胞系HepG2和Huh7细胞。通过荧光显微镜观察外源基因hrGFP的表达，Bsd筛选细胞克隆。Northern blot检测质粒转染细胞后RNA的表达。Western blot、Native western blot分别用于检测HBV包膜、核心蛋白和组装的核心颗粒。ELISA测定细胞上清液中HBsAg和HBeAg的水平。内源性聚合酶反应(endogenous polymerase reaction, EPR)检测功能性P蛋白表达。Southern blot检测HBV复制中间体。荧光定量PCR法分析细胞上清液中HBV DNA含量。CsCl密度梯度离心法分离细胞上清液中的病毒颗粒。
第三部分：复制型HBV载体的感染特性研究
野生型HBV质粒pCH-3093、pCH-BsdR和pCH-hrGFP分别转染HepG2细胞，通过聚乙二醇8000沉淀上清中的重组病毒颗粒，用于感染HepRG细胞，感染8天后，经Northern blot检测HBV RNA的水平，ELISA测定细胞上清液中的HBsAg和HBeAg。病毒颗粒与高效价HBV免疫球蛋白（hepatitis B immuno-globulin, HBIG）预孵育1h后再感染HepRG细胞以验证重组HBV颗粒是否能被抗体阻断。
结果：
第一部分：22-nt Rbm3IRES在HBV前基因组RNA上的表达活性研究1、I、II、III号载体转染HepG2和Huh7后，HBeAg表达均无显著差异。从第一组质粒转染HepG2后观察荧光强度分析，携带Rbm3IRES双顺反子载体(II)中Rbm3IRES的翻译起始效率明显强于EMCV IRES(I)；相对于双顺反子载体(II)，在三顺反子载体上(III)串联的第二个Rbm3IRES引导的EGFP表达强度下降较多；但是仍明显高于HBV自身翻译起始序列引导的EGFP表达水平(IV)。2、从第二组质粒分别转染HepG2和Huh7细胞后有相似的荧光素酶结果，Rbm3IRES产生的荧光素酶活性(II)为EMCV IRES产生荧光素酶活性(I)的两倍有余（II VS I，P＜0.01）。三顺反子载体上Rbm3IRES产生的荧光素酶活性较双顺反子明显下降(III VSII，P＜0.01)，但是仍明显高于HBV自身翻译起始序列产生的荧光素酶活性(III VS IV，P＜0.05)。
第二部分：复制型HBV载体的构建及其表达与复制能力研究
成功构建HBV载体pCH-BsdR和pCH-hrGFP。转染细胞后能观察到高水平绿色荧光蛋白表达，经Bsd筛选可形成稳定的Bsd抗性细胞克隆。均可产生携带外源基因的pgRNA，有同野生型HBV相似的核心和包膜蛋白，载体之间HBsAg和HBeAg的分泌量无明显差异。BsdR载体转染细胞后能翻译出有功能的P蛋白。BsdR载体的复制能力为野生型HBV的40%，hrGFP载体复制能力明显减弱。然而，这两个载体都能够形成有包膜的病毒。
第三部分：复制型HBV载体的感染特性研究
利用pCH-BsdR和pCH-hrGFP能够制备重组病毒颗粒，能够感染HepRG细胞。感染8天后，Northern blot可以检测到前基因组和亚基因病毒RNA，ELISA测定出同野生型HBV一样的HBsAg和HBeAg的分泌趋势，HBV-hrGFP病毒感染HepRG后可以检测到hrGFP的表达。通过抗体阻断实验证实重组HBV通过与野生型HBV相同的方式(通过HBV外膜蛋白)感染HepaRG细胞。
结论：
1、不管是用CMV启动子还是HBV C基因启动子驱动，Rbm3IRES都比EMCVIRES有较高的翻译起始效率。在HBV C基因和P基因之间插入了外源基因的三顺反子载体中，串联的两个Rbm3IRES均有能力引导翻译起始过程。
2、把C、P蛋白分开表达，利用2个仅22-nt的强效IRES分别表达外源基因和P蛋白，保持了各结构蛋白的完整性又尽量少扩充基因组容量，实现载体功能并保持复制能力。
3、利用构建的HBV载体制备的重组HBV颗粒感染HepRG细胞，证明其能够像野生型HBV那样具有感染能力。
4、由于大量的报告基因和效应基因的长度一般小于500bp，这种新型的HBV载体有望成为研究和战胜HBV的有力工具。
Background and objective
Viral vectors are engineered virus variants able to deliver nonviral genetic informationinto cells, usually by the same routes as the parental viruses. For several virus families,replication-competent vectors carrying reporter genes have become invaluable tools foreasy and quantitative monitoring of replication and infection, and thus also for identifyingantivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNAvirus causing B-type hepatitis, such vectors are not available because insertions into its tiny3.2kb genome almost inevitably affect essential replication elements. HBV replicates byreverse transcription of the pregenomic (pg) RNA which is also required as bicistronicmRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open readingframes (ORFs) overlap by some150basepairs. Translation of the downstream Pol ORFdoes not involve a conventional internal ribosome entry site (IRES). We reasoned thatduplicating the overlap region and providing artificial IRES control for translation of bothPol and an in-between inserted transgene might yield a functional tricistronic pgRNA,without interfering with envelope protein expression. As IRESs we used a22nucleotideelement termed Rbm3IRES to minimize genome size increase. Analogous plasmids forcomplete HBV genomes carrying399bp and720bp transgenes for blasticidin resistance(BsdR) and humanized Renilla green fluorescent protein (hrGFP) were constructed. Theyboth produced core and envelope proteins like wild-type HBV and remained replication.Both vectors, however, formed enveloped virions which were infectious forHBV-susceptible HepaRG cells. The new HBV vectors should become highly useful toolsto better understand, and combat this important pathogen.
Method
The study was followed as three parts.
Part one: The activity of22-nt Rbm3IRES on the HBV pgRNA
1. The first series of model plasmids were driven by CMV promoter and encodedEGFP as marker for visual inspection. I: pCH-EMCV IRES-EGFP; II: pCH-22ntIRES-EGFP; III: pCH-BsdR-22nt IRES-EGFP; IV: pCH-pATG-EGFP. The EGFPexpression was observed after HepG2or Huh7cells were transfected by the four plasmidsfor72h. HBeAg secretion was measured by ELISA in I, II and III group.
2. The second series of model plasmids were driven by endogeous HBV core promoterand encoded Renilla luciferase (RLuc) for expression quantification. I: pHBV-EMCVIRES-RLuc; II: pHBV-22nt IRES-RLuc; III: pHBV-BsdR-22nt IRES-RLuc; IV:pHBV-pATG-RLuc. The Rluc constructs were cotransfected with the firely luciferasevector pGL3-Control and relative RLuc activity was monitored using the dual luciferaseluminometric assay.
Part two: Construciton, Expression and Replication competence of Replication-competentHBV vector
pCH-BsdR and pCH-hrGFP were constructed. HepG2and Huh7cells were transfectedby the two plasmids and wild type plasmid pCH3093separately. Expression of hrGFP wasobserved by fluorescence microscope. Expression of functional BsdR was subsequentlydemonstrated by formation of stable Bsd resistant cell clones. HBV RNA was determinedby Northern blot. Envelope protein, core protein and assembled capsids were shown byWestern blot and Native western blot respectively. HBsAg and HBeAg secrection weredetermined by ELISA. Functional Pol molecule was detected by endogenous polymerasereaction (EPR). Replicative DNA intermediates were analyzed by Southern blot, and theamounts of viral genomes in the culture supernatants were determined by quantitative PCR.Viral particles were separated by CsCl density gradient centrifugation.
Part three: Infection of Replication-competent HBV vector
HepG2cells were transfected by pCH-3093, pCH-BsdR and pCH-hrGFP respectively.Viral particles were collected from the culture supernatants by PEG8000precipitation.HepRG cells were infected by the enveloped virions for8days. HBV RNA wasdeterminded by Northern blot. HBsAg and HBeAg secrection were measured by ELISA.For blocking infection, the viral particles were preincubated with HBIG for1h beforeinfection.
Results
Part one: The activity of22-nt Rbm3IRES on the HBV pgRNA
1. From the result of HBeAg ELISA, there were no significant difference amongconstruct I, II and III. EGFP fluorescence from the Rbm3IRES was stronger in HepG2cells than that from the corresponding EMCV IRES construct (II vs. I). EGFP fluorescencefrom the tricistronic construct (III) was substantially reduced yet remained easily detectable,clearly exceeding the signals from construct IV.
2. Both HepG2and Huh7cells gave very similar results. Relative RLuc activity fromthe Rbm3IRES was more than2-fold higher than from the EMCV IRES (II vs. I, P＜0.01).RLuc activity from the tricistronic construct was reduced significantly compared withthe bicistronic EMCV IRES construct (III vs. II, P＜0.01), and it clearly exceeded that fromthe construct having the RLuc gene fused to the authentic Pol start (III vs. IV, P＜0.05).
Part two: Construciton, Expression and Replication competence of Replication-competentHBV vector
pCH-BsdR and pCH-hrGFP were constructed. After transfected into HepG2or Huh7cells, high level hrGFP expression could be seen. Stable Bsd resistant cell clones could beformed after screened by Bsd. The recombinant pgRNA carrying transgenes could bemeasured by Northern blot. The two vectors produced core and envelope proteins likewild-type HBV. There were no significant differences in HBsAg and HBeAg secretionbetween groups. The data demonstrated that functional Pol was translated from the BsdRvector. While the hrGFP vector replicated poorly, the BsdR vector generated around40%asmuch replicative DNA as wild-type HBV. Both vectors formed enveloped virions.
Part three: Infection of Replication-competent HBV vector
Enveloped virions formed by both vectors were infectious for HBV-susceptibleHepaRG cells. All inocula led to detectable presence of genomic and subgenomic viralRNAs8days post-inoculation. Infection by and active viral replication of the BsdR vectorparticles were supported by a wild-type HBV-like net increase of secreted HBsAg andHBeAg. GFP expression in hrGFP-HBV vector inoculated cells indicates successfulinfection. The test of preincubation with neutralizing hepatitis B immuno-globulin (HBIG)demonstrated that the infection was indeed mediated by the viral envelope proteins, as inwild-type HBV infection.
Conclusion
1. Wether driven by CMV or the authentic HBV core promoter, the Rbm3IRESdirected higher levels of reporter gene expression in human hepatoma cells than the EMCVIRES from bicistronic constructs. Importantly, substantial expression of the mostdownstream cistron was also achieved from the tricistronic constructs mimicking pgRNAwith a transgene inserted between core and Pol ORF.
2. The C and P protein were expressed separately. Two22-nt IRES were used toexpress transgenes and P protein separately, which could maintain integrity and replicationcompetence, decrease the size of gene expansion, and realize the vector function.
3. Both vectors formed enveloped virions which were infectious for HBV-susceptibleHepaRG cells. It demonstrated that the replication-competent HBV vector was infectious asthe wild-type HBV.
4. Because numerous reporter and effector genes with sizes of around500bp or lessare available, the new HBV vectors should become highly useful tools to better understand,and combat this important pathogen.
将病毒载体设计成能够携带外源基因并按着亲代病毒的侵入路径到达细胞体内的病毒变异体是病毒载体研究的新思路。对一些病毒家族来说，携带报告基因的复制型载体能够在简化和量化检测复制和感染、识别抗病毒和病毒易感细胞等方面成为有力的工具。乙型肝炎病毒(hepatitis B virus, HBV)，是引起乙型病毒性肝炎的小包裹DNA病毒，原则上在这个仅有3.2kb的基因组内插入外源基因不可避免地会影响本身的复制元件。HBV复制子通过前基因组RNA(pregenomic RNA, pgRNA)逆转录而来，pgRNA作为双顺反子mRNA，在形成核心蛋白(C)和逆转录酶(Pol)方面也是必需的， C和Pol开放读码框架(open reading frames, ORF)有150bp的重叠区。Pol ORF的下游区翻译一般不涉及内部核糖体进入位点(internal ribosome entry site, IRES)。我们设想将C、P重叠区域拉开，插入两个IRES分别用于表达外源基因和P蛋白，产生一个有功能的且不影响包膜蛋白表达的三顺反子pgRNA。为了减少对基因组长度的影响，我们利用仅含22个nt的RNA结合基序蛋白3(RNA-binding motif protein3, Rbm3) IRES，构建了分别携带399bp的杀稻瘟菌素抗性基因(blasticidin resistance, BsdR)和720bp的人绿色荧光蛋白(humanized Renilla green fluorescent protein, hrGFP)的HBV载体，研究发现仍有复制能力，能产生同野生型HBV一样的病毒蛋白，所形成的的病毒颗粒可用于感染HepRG细胞。这种新型的携带外源基因的HBV载体将成为研究HBV的有力工具。
方法：本课题分三部分进行探讨。
第一部分：22-nt Rbm3IRES在HBV前基因组RNA上的表达活性研究
1、构建携带CMV启动子和增强型绿色荧光蛋白(Enhanced Green FluorescentProtein, EGFP)的四种质粒： I： pCH-EMCV IRES-EGFP(包含脑炎心肌炎病毒(Encephalomyocarditis virus, EMCV) IRES及EGFP)；II：pCH-22nt IRES-EGFP(包含Rbm3IRES及EGFP)；III：pCH-BsdR-22nt IRES-EGFP(为三顺反子载体，依次包含Rbm3IRES，BsdR，Rbm3IRES及EGFP)；IV：pCH-pATG-EGFP(包含HBV C基因N端部分与EGFP)。ELISA检测I、II、III载体转染HepG2或Huh7细胞后72h的上清液中HBeAg的表达，并观察四种载体转染后EGFP的表达。
2、构建携带HBV C基因启动子和荧光素酶基因(Renilla luciferase, RLuc)的四种质粒：I：pHBV-EMCV IRES-RLuc(包含EMCV IRES及RLuc)；II：pHBV-22ntIRES-RLuc(包含Rbm3IRES及RLuc)；III：pHBV-BsdR-22nt IRES-RLuc(为三顺反子载体，依次包含Rbm3IRES，BsdR，Rbm3IRES及RLuc)；IV：pHBV-pATG-RLuc(包含HBV C基因N端部分与RLuc)。四种质粒分别同对照质粒pGL3-Control共转染HepG2或Huh7细胞，通过双荧光素酶检测试剂盒测定荧光素酶值。
第二部分：复制型HBV载体的构建及其表达与复制能力研究
构建两种复制型HBV载体：pCH-BsdR和pCH-hrGFP。两个质粒和携带野生型HBV的质粒pCH-3093分别转染肝癌细胞系HepG2和Huh7细胞。通过荧光显微镜观察外源基因hrGFP的表达，Bsd筛选细胞克隆。Northern blot检测质粒转染细胞后RNA的表达。Western blot、Native western blot分别用于检测HBV包膜、核心蛋白和组装的核心颗粒。ELISA测定细胞上清液中HBsAg和HBeAg的水平。内源性聚合酶反应(endogenous polymerase reaction, EPR)检测功能性P蛋白表达。Southern blot检测HBV复制中间体。荧光定量PCR法分析细胞上清液中HBV DNA含量。CsCl密度梯度离心法分离细胞上清液中的病毒颗粒。
第三部分：复制型HBV载体的感染特性研究
野生型HBV质粒pCH-3093、pCH-BsdR和pCH-hrGFP分别转染HepG2细胞，通过聚乙二醇8000沉淀上清中的重组病毒颗粒，用于感染HepRG细胞，感染8天后，经Northern blot检测HBV RNA的水平，ELISA测定细胞上清液中的HBsAg和HBeAg。病毒颗粒与高效价HBV免疫球蛋白（hepatitis B immuno-globulin, HBIG）预孵育1h后再感染HepRG细胞以验证重组HBV颗粒是否能被抗体阻断。
结果：
第一部分：22-nt Rbm3IRES在HBV前基因组RNA上的表达活性研究1、I、II、III号载体转染HepG2和Huh7后，HBeAg表达均无显著差异。从第一组质粒转染HepG2后观察荧光强度分析，携带Rbm3IRES双顺反子载体(II)中Rbm3IRES的翻译起始效率明显强于EMCV IRES(I)；相对于双顺反子载体(II)，在三顺反子载体上(III)串联的第二个Rbm3IRES引导的EGFP表达强度下降较多；但是仍明显高于HBV自身翻译起始序列引导的EGFP表达水平(IV)。2、从第二组质粒分别转染HepG2和Huh7细胞后有相似的荧光素酶结果，Rbm3IRES产生的荧光素酶活性(II)为EMCV IRES产生荧光素酶活性(I)的两倍有余（II VS I，P＜0.01）。三顺反子载体上Rbm3IRES产生的荧光素酶活性较双顺反子明显下降(III VSII，P＜0.01)，但是仍明显高于HBV自身翻译起始序列产生的荧光素酶活性(III VS IV，P＜0.05)。
第二部分：复制型HBV载体的构建及其表达与复制能力研究
成功构建HBV载体pCH-BsdR和pCH-hrGFP。转染细胞后能观察到高水平绿色荧光蛋白表达，经Bsd筛选可形成稳定的Bsd抗性细胞克隆。均可产生携带外源基因的pgRNA，有同野生型HBV相似的核心和包膜蛋白，载体之间HBsAg和HBeAg的分泌量无明显差异。BsdR载体转染细胞后能翻译出有功能的P蛋白。BsdR载体的复制能力为野生型HBV的40%，hrGFP载体复制能力明显减弱。然而，这两个载体都能够形成有包膜的病毒。
第三部分：复制型HBV载体的感染特性研究
利用pCH-BsdR和pCH-hrGFP能够制备重组病毒颗粒，能够感染HepRG细胞。感染8天后，Northern blot可以检测到前基因组和亚基因病毒RNA，ELISA测定出同野生型HBV一样的HBsAg和HBeAg的分泌趋势，HBV-hrGFP病毒感染HepRG后可以检测到hrGFP的表达。通过抗体阻断实验证实重组HBV通过与野生型HBV相同的方式(通过HBV外膜蛋白)感染HepaRG细胞。
结论：
1、不管是用CMV启动子还是HBV C基因启动子驱动，Rbm3IRES都比EMCVIRES有较高的翻译起始效率。在HBV C基因和P基因之间插入了外源基因的三顺反子载体中，串联的两个Rbm3IRES均有能力引导翻译起始过程。
2、把C、P蛋白分开表达，利用2个仅22-nt的强效IRES分别表达外源基因和P蛋白，保持了各结构蛋白的完整性又尽量少扩充基因组容量，实现载体功能并保持复制能力。
3、利用构建的HBV载体制备的重组HBV颗粒感染HepRG细胞，证明其能够像野生型HBV那样具有感染能力。
4、由于大量的报告基因和效应基因的长度一般小于500bp，这种新型的HBV载体有望成为研究和战胜HBV的有力工具。
Background and objective
Viral vectors are engineered virus variants able to deliver nonviral genetic informationinto cells, usually by the same routes as the parental viruses. For several virus families,replication-competent vectors carrying reporter genes have become invaluable tools foreasy and quantitative monitoring of replication and infection, and thus also for identifyingantivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNAvirus causing B-type hepatitis, such vectors are not available because insertions into its tiny3.2kb genome almost inevitably affect essential replication elements. HBV replicates byreverse transcription of the pregenomic (pg) RNA which is also required as bicistronicmRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open readingframes (ORFs) overlap by some150basepairs. Translation of the downstream Pol ORFdoes not involve a conventional internal ribosome entry site (IRES). We reasoned thatduplicating the overlap region and providing artificial IRES control for translation of bothPol and an in-between inserted transgene might yield a functional tricistronic pgRNA,without interfering with envelope protein expression. As IRESs we used a22nucleotideelement termed Rbm3IRES to minimize genome size increase. Analogous plasmids forcomplete HBV genomes carrying399bp and720bp transgenes for blasticidin resistance(BsdR) and humanized Renilla green fluorescent protein (hrGFP) were constructed. Theyboth produced core and envelope proteins like wild-type HBV and remained replication.Both vectors, however, formed enveloped virions which were infectious forHBV-susceptible HepaRG cells. The new HBV vectors should become highly useful toolsto better understand, and combat this important pathogen.
Method
The study was followed as three parts.
Part one: The activity of22-nt Rbm3IRES on the HBV pgRNA
1. The first series of model plasmids were driven by CMV promoter and encodedEGFP as marker for visual inspection. I: pCH-EMCV IRES-EGFP; II: pCH-22ntIRES-EGFP; III: pCH-BsdR-22nt IRES-EGFP; IV: pCH-pATG-EGFP. The EGFPexpression was observed after HepG2or Huh7cells were transfected by the four plasmidsfor72h. HBeAg secretion was measured by ELISA in I, II and III group.
2. The second series of model plasmids were driven by endogeous HBV core promoterand encoded Renilla luciferase (RLuc) for expression quantification. I: pHBV-EMCVIRES-RLuc; II: pHBV-22nt IRES-RLuc; III: pHBV-BsdR-22nt IRES-RLuc; IV:pHBV-pATG-RLuc. The Rluc constructs were cotransfected with the firely luciferasevector pGL3-Control and relative RLuc activity was monitored using the dual luciferaseluminometric assay.
Part two: Construciton, Expression and Replication competence of Replication-competentHBV vector
pCH-BsdR and pCH-hrGFP were constructed. HepG2and Huh7cells were transfectedby the two plasmids and wild type plasmid pCH3093separately. Expression of hrGFP wasobserved by fluorescence microscope. Expression of functional BsdR was subsequentlydemonstrated by formation of stable Bsd resistant cell clones. HBV RNA was determinedby Northern blot. Envelope protein, core protein and assembled capsids were shown byWestern blot and Native western blot respectively. HBsAg and HBeAg secrection weredetermined by ELISA. Functional Pol molecule was detected by endogenous polymerasereaction (EPR). Replicative DNA intermediates were analyzed by Southern blot, and theamounts of viral genomes in the culture supernatants were determined by quantitative PCR.Viral particles were separated by CsCl density gradient centrifugation.
Part three: Infection of Replication-competent HBV vector
HepG2cells were transfected by pCH-3093, pCH-BsdR and pCH-hrGFP respectively.Viral particles were collected from the culture supernatants by PEG8000precipitation.HepRG cells were infected by the enveloped virions for8days. HBV RNA wasdeterminded by Northern blot. HBsAg and HBeAg secrection were measured by ELISA.For blocking infection, the viral particles were preincubated with HBIG for1h beforeinfection.
Results
Part one: The activity of22-nt Rbm3IRES on the HBV pgRNA
1. From the result of HBeAg ELISA, there were no significant difference amongconstruct I, II and III. EGFP fluorescence from the Rbm3IRES was stronger in HepG2cells than that from the corresponding EMCV IRES construct (II vs. I). EGFP fluorescencefrom the tricistronic construct (III) was substantially reduced yet remained easily detectable,clearly exceeding the signals from construct IV.
2. Both HepG2and Huh7cells gave very similar results. Relative RLuc activity fromthe Rbm3IRES was more than2-fold higher than from the EMCV IRES (II vs. I, P＜0.01).RLuc activity from the tricistronic construct was reduced significantly compared withthe bicistronic EMCV IRES construct (III vs. II, P＜0.01), and it clearly exceeded that fromthe construct having the RLuc gene fused to the authentic Pol start (III vs. IV, P＜0.05).
Part two: Construciton, Expression and Replication competence of Replication-competentHBV vector
pCH-BsdR and pCH-hrGFP were constructed. After transfected into HepG2or Huh7cells, high level hrGFP expression could be seen. Stable Bsd resistant cell clones could beformed after screened by Bsd. The recombinant pgRNA carrying transgenes could bemeasured by Northern blot. The two vectors produced core and envelope proteins likewild-type HBV. There were no significant differences in HBsAg and HBeAg secretionbetween groups. The data demonstrated that functional Pol was translated from the BsdRvector. While the hrGFP vector replicated poorly, the BsdR vector generated around40%asmuch replicative DNA as wild-type HBV. Both vectors formed enveloped virions.
Part three: Infection of Replication-competent HBV vector
Enveloped virions formed by both vectors were infectious for HBV-susceptibleHepaRG cells. All inocula led to detectable presence of genomic and subgenomic viralRNAs8days post-inoculation. Infection by and active viral replication of the BsdR vectorparticles were supported by a wild-type HBV-like net increase of secreted HBsAg andHBeAg. GFP expression in hrGFP-HBV vector inoculated cells indicates successfulinfection. The test of preincubation with neutralizing hepatitis B immuno-globulin (HBIG)demonstrated that the infection was indeed mediated by the viral envelope proteins, as inwild-type HBV infection.
Conclusion
1. Wether driven by CMV or the authentic HBV core promoter, the Rbm3IRESdirected higher levels of reporter gene expression in human hepatoma cells than the EMCVIRES from bicistronic constructs. Importantly, substantial expression of the mostdownstream cistron was also achieved from the tricistronic constructs mimicking pgRNAwith a transgene inserted between core and Pol ORF.
2. The C and P protein were expressed separately. Two22-nt IRES were used toexpress transgenes and P protein separately, which could maintain integrity and replicationcompetence, decrease the size of gene expansion, and realize the vector function.
3. Both vectors formed enveloped virions which were infectious for HBV-susceptibleHepaRG cells. It demonstrated that the replication-competent HBV vector was infectious asthe wild-type HBV.
4. Because numerous reporter and effector genes with sizes of around500bp or lessare available, the new HBV vectors should become highly useful tools to better understand,and combat this important pathogen.
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