Morikawa Kenichi, Zhao Zijiang, Date Tomoko, Miyamoto Michiko, Murayama Asako, Akazawa Daisuke, Tanabe Junichi, Sone Saburo, Wakita Takaji
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan.
J Med Virol. 2007 Jun;79(6):714-23. doi: 10.1002/jmv.20842.
Because appropriate cell-culture systems or small-animal models have been lacking, the early steps in the HCV life cycle have been difficult to study. A cell culture system was developed recently that allows production of infectious HCV. In this study, infectious HCV particles produced in cultured cells were used. To clarify the role of CD81 in HCV attachment and entry, the effect of anti-CD81 antibody was examined. The antibody blocked HCV virion entry but not particle attachment. Only the fraction bound to a heparin affinity column and eluted with 0.3 M NaCl productively infected Huh7 cells, indicating that infectious HCV particles bind to heparin. Both heparin treatment of the virus particles and heparinase treatment of the Huh7 cells reduced virus-cell binding without substantially inhibiting HCV infectivity. Finally, to confirm the role of both heparin sulfate proteoglycan (HSPG) and CD81 in HCV entry, the effects of heparinase I and anti-CD81 antibody were analyzed. No productive RNA replication was detected in the Huh7 cells in the presence of both heparinase I and anti-CD81 antibody. In conclusion, these data suggested that both HSPG and CD81 are important for HCV entry. HSPG may play a role in the initial cell surface binding of infectious HCV particles and CD81 is conceivably correlated with HCV entry after viral attachment.
由于缺乏合适的细胞培养系统或小动物模型，丙型肝炎病毒（HCV）生命周期的早期步骤一直难以研究。最近开发了一种细胞培养系统，可用于生产具有传染性的HCV。在本研究中，使用了在培养细胞中产生的具有传染性的HCV颗粒。为了阐明CD81在HCV附着和进入中的作用，研究了抗CD81抗体的作用。该抗体阻断了HCV病毒体的进入，但未阻断颗粒的附着。只有与肝素亲和柱结合并用0.3M NaCl洗脱的部分能有效感染Huh7细胞，这表明具有传染性的HCV颗粒与肝素结合。对病毒颗粒进行肝素处理以及对Huh7细胞进行肝素酶处理均可降低病毒与细胞的结合，而不会显著抑制HCV的感染性。最后，为了证实硫酸乙酰肝素蛋白聚糖（HSPG）和CD81在HCV进入中的作用，分析了肝素酶I和抗CD81抗体的作用。在同时存在肝素酶I和抗CD81抗体的情况下，未在Huh7细胞中检测到有效的RNA复制。总之，这些数据表明HSPG和CD81对HCV进入都很重要。HSPG可能在具有传染性的HCV颗粒与细胞表面的初始结合中起作用，并且可以想象CD81与病毒附着后HCV的进入相关。
