Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses
Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses
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DOI：
10.1128/jvi.02460-05
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Bartenschlager, Ralf
中科院分区：
文献类型：
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作者：
Koutsoudakis, George;Kaul, Artur;Bartenschlager, Ralf
缺乏一个有效的系统来生产丙型肝炎病毒（HCV）颗粒阻碍了HCV生命周期的分析。最近，我们沿着与其他人证明，Huh 7肝癌细胞转染一种新的HCV分离株（JFH 1）产生感染性病毒。为了促进HCV复制的研究，我们生成了基于JFH 1的双顺反子荧光素酶报告病毒基因组。我们发现，报告构建体的RNA复制仅轻微减弱，并且产生的病毒滴度仅比亲本病毒低三到五倍，使得这些报告病毒成为定量分析HCV感染的理想工具。为了扩大系统的范围，我们创建了两个嵌合的JFH 1荧光素酶报告病毒与Con1（基因型1b）和J6CF（基因型2a）菌株的结构蛋白。使用这些和真实的JFH 1报告病毒，我们分析了HCV生命周期的早期步骤。我们的数据表明，这些分离株之间的病毒进入模式是保守的，并涉及CD81作为pH依赖性病毒进入的关键受体。竞争研究和时间过程实验表明，HCV与细胞表面驻留的糖胺聚糖的相互作用有助于Huh7细胞的有效感染，并且CD81在后附着步骤中起作用。这里描述的报告病毒应该有助于研究病毒的生命周期和HCV抑制剂的开发。
The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.