Blanchard Emmanuelle, Belouzard Sandrine, Goueslain Lucie, Wakita Takaji, Dubuisson Jean, Wychowski Czeslaw, Rouillé Yves
Equipe Hépatite C, CNRS-UMR8161, Institut de Biologie de Lille, 1 rue du Professeur Calmette, BP447, 59021 Lille cedex, France.
J Virol. 2006 Jul;80(14):6964-72. doi: 10.1128/JVI.00024-06.
Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.
由于丙型肝炎病毒（HCV）在细胞培养增殖方面存在困难，其进入细胞的机制尚不清楚。在此，我们使用HCV包膜糖蛋白假型化的逆转录病毒颗粒（HCVpp）以及在细胞培养中增殖的HCV克隆JFH-1（HCVcc），研究了HCV进入细胞的结合后细胞机制。与靶细胞接触3小时后，通过定量实时PCR检测HCVpp的进入情况，并通过免疫印迹分析和对感染细胞中表达的HCV蛋白进行免疫荧光检测来定量HCVcc感染。通过小干扰RNA介导的网格蛋白重链耗竭以及使用氯丙嗪（一种质膜上网格蛋白包被小窝形成的抑制剂）来评估网格蛋白介导的内吞作用在HCV进入细胞中的功能作用。在这两种情况下，HCVpp的进入和HCVcc的感染均受到抑制。用巴弗洛霉素A1或氯喹预处理靶细胞也可抑制HCVcc感染，这两种药物已知会干扰内体酸化。这些数据表明，HCV通过网格蛋白介导的内吞作用进入靶细胞，随后在酸性内体区室中发生融合步骤。
