Objective： The combination of interferon （IFN） and ribavirin （RBV） is the standard therapy for hepatitis C virus （HCV） infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet...Objective： The combination of interferon （IFN） and ribavirin （RBV） is the standard therapy for hepatitis C virus （HCV） infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet fimited. This study aimed to investigate the mechanism of the poor response in a case ofHCV genotype 2a infection. Methods： We analyzed dynamic change of HCV RNA from a patient, infected with HCV genotype 2a, showing a poor virological response to 1FN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing. Then we constructed subgenomic Japanese fulminant hepatitis-1 （JFH1） replicon and different chimeric replicons with humanized Gaussia luciferase gene. The chimeric replicons were derived from subgenomic JFH1 replicon, in which the NS5A region was replaced by the patient＇s sequence from the pre/post- treatment, and the chimeric replicons＇ susceptibility to IFN were evaluated by relative Gausia Luciferase activity. Results： The pretreatment HCV sequences appeared almost uniform, and the quasispecies variation was further more simplified after 12 weeks of therapy. Besides, the quasispecies variation seemed to be more diversified in the NS5A, relatively, a region crucial for IFN response, and each of chimeric replicons exhibited distinct response to IFN. Conclusions： During the course of the chronic infection, HCV population seems to be adapted to the patient＇s immunological system, and further to be selected by combination of 1FN/RBV therapy, indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN. In addition, each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region （ISDR） of NSSA during chronic infection and IFN/RBV therapy.展开更多
目的在原核与真核细胞中表达丙型肝炎病毒(hepatitis C virus,HCV)2a型JFH1(japanese fulminanthepatitis 1)株NS5A蛋白,分析JFH1NS5A蛋白与其它基因型NS5A蛋白间的差异,为进一步研究NS5A蛋白在HCV病毒复制中的作用奠定基础。方法PCR特...目的在原核与真核细胞中表达丙型肝炎病毒(hepatitis C virus,HCV)2a型JFH1(japanese fulminanthepatitis 1)株NS5A蛋白,分析JFH1NS5A蛋白与其它基因型NS5A蛋白间的差异,为进一步研究NS5A蛋白在HCV病毒复制中的作用奠定基础。方法PCR特异扩增JFH1NS5A基因,克隆入pET-32a和pEGFP-N1载体中,构建JFH1NS5A的原核和真核重组表达质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。将pET-JFH1NS5A转化大肠杆菌BL21(DE3),pEGFP-JFH1NS5A转染HEK293T细胞,在原核与真核系统中进行表达,并以SDS-PAGE、荧光显微术和Westernblot方法检测。利用PubMedBLAST软件对JFH1与HCV1a、1b、2b和2c型NS5A蛋白间的同源性进行了分析。结果酶切鉴定及测序结果表明成功构建了重组质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。SDS-PAGE电泳检测到融合蛋白在原核细胞中的表达,荧光显微镜观察和Westernblot检测到了GFP-JFH1NS5A融合蛋白在HEK293T细胞中表达。BLAST分析显示,JFH1和H77、HC-J4、HCV-J8和BEBE1病毒株NS5A蛋白的同源性分别为57%、60%、68%和74%。结论JFH1NS5A蛋白在原核与真核细胞中表达成功,为研究HCVNS5A在JFH1株病毒高效复制中的作用提供了材料。展开更多
AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations...AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5 A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.展开更多
目的构建丙型肝炎病毒JFH1全长c DNA克隆,获取突变位点信息。方法应用长片段RT长片段RT-PCR技术对不同时期传代的JFH1病毒株进行分段扩增。通过共同酶切位点连接成9.7Kb从DNA片段,与p Blue Script2ks(+)形成克隆载体,进一步测序分析。...目的构建丙型肝炎病毒JFH1全长c DNA克隆,获取突变位点信息。方法应用长片段RT长片段RT-PCR技术对不同时期传代的JFH1病毒株进行分段扩增。通过共同酶切位点连接成9.7Kb从DNA片段,与p Blue Script2ks(+)形成克隆载体,进一步测序分析。结果获取丙型肝炎病毒JFH1系列适应性突变株全长c DNA模板,共发现23个单核苷酸突变。结论丙型肝炎病毒JFH1系列存在单核苷酸突变位点,为进一步了解HCV JFH1适应性突变位点功能奠定基础。展开更多
文摘Objective： The combination of interferon （IFN） and ribavirin （RBV） is the standard therapy for hepatitis C virus （HCV） infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet fimited. This study aimed to investigate the mechanism of the poor response in a case ofHCV genotype 2a infection. Methods： We analyzed dynamic change of HCV RNA from a patient, infected with HCV genotype 2a, showing a poor virological response to 1FN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing. Then we constructed subgenomic Japanese fulminant hepatitis-1 （JFH1） replicon and different chimeric replicons with humanized Gaussia luciferase gene. The chimeric replicons were derived from subgenomic JFH1 replicon, in which the NS5A region was replaced by the patient＇s sequence from the pre/post- treatment, and the chimeric replicons＇ susceptibility to IFN were evaluated by relative Gausia Luciferase activity. Results： The pretreatment HCV sequences appeared almost uniform, and the quasispecies variation was further more simplified after 12 weeks of therapy. Besides, the quasispecies variation seemed to be more diversified in the NS5A, relatively, a region crucial for IFN response, and each of chimeric replicons exhibited distinct response to IFN. Conclusions： During the course of the chronic infection, HCV population seems to be adapted to the patient＇s immunological system, and further to be selected by combination of 1FN/RBV therapy, indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN. In addition, each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region （ISDR） of NSSA during chronic infection and IFN/RBV therapy.
文摘目的在原核与真核细胞中表达丙型肝炎病毒(hepatitis C virus,HCV)2a型JFH1(japanese fulminanthepatitis 1)株NS5A蛋白,分析JFH1NS5A蛋白与其它基因型NS5A蛋白间的差异,为进一步研究NS5A蛋白在HCV病毒复制中的作用奠定基础。方法PCR特异扩增JFH1NS5A基因,克隆入pET-32a和pEGFP-N1载体中,构建JFH1NS5A的原核和真核重组表达质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。将pET-JFH1NS5A转化大肠杆菌BL21(DE3),pEGFP-JFH1NS5A转染HEK293T细胞,在原核与真核系统中进行表达,并以SDS-PAGE、荧光显微术和Westernblot方法检测。利用PubMedBLAST软件对JFH1与HCV1a、1b、2b和2c型NS5A蛋白间的同源性进行了分析。结果酶切鉴定及测序结果表明成功构建了重组质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。SDS-PAGE电泳检测到融合蛋白在原核细胞中的表达,荧光显微镜观察和Westernblot检测到了GFP-JFH1NS5A融合蛋白在HEK293T细胞中表达。BLAST分析显示,JFH1和H77、HC-J4、HCV-J8和BEBE1病毒株NS5A蛋白的同源性分别为57%、60%、68%和74%。结论JFH1NS5A蛋白在原核与真核细胞中表达成功,为研究HCVNS5A在JFH1株病毒高效复制中的作用提供了材料。
基金Beijing Natural Science Foundation,No.7161006Beijing Municipal Administration of Hospitals’ Youth Program,No.QML20161801 and No.QML20171801
文摘AIM To explore hepatitis C virus(HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms.METHODS A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5 A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and celllysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets(LDs) with NS5 A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5 A was analyzed further.RESULTS The plasmids named JFH1-m E2, JFH1-mp7, JFH1-m NS4 B, JFH1-m NS5 A, JFH1-m E2/NS5 A, JFH1-mp7/NS5 A, JFH1-m NS4 B/NS5 A, JFH1-m E2/p7/NS5 A, and m JFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units(FFUs)/m L. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5 A(C2274 R, I2340 T, and V2440 L) and p7(H781 Y) were critical adaptive mutations. The effect of NS5 A(C2274 R, I2340 T, and V2440 L), p7(H781 Y), and NS4 B(N1931 S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5 A proteins or the phosphorylation of NS5 A.CONCLUSION In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/m L, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.
