Institut National de la Santé et de la Recherche Médicale, U748, Strasbourg, France.
Hepatology. 2010 Apr;51(4):1144-57. doi: 10.1002/hep.23445.
The tight junction protein claudin-1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry-the first step of viral infection. Due to the lack of neutralizing anti-CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface-expressed CLDN1 specifically inhibit HCV infection in a dose-dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti-CLDN1 and anti-CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti-CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1-E2 interaction. Using fluorescent-labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti-CLDN1 antibodies inhibit CD81-CLDN1 association. In contrast, CLDN1-CLDN1 and CD81-CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81-CLDN1 interactions.
These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2-CD81-CLDN1 interactions.
已证实紧密连接蛋白 Claudin-1（CLDN1）对于丙型肝炎病毒（HCV）进入——病毒感染的第一步是必不可少的。由于缺乏中和抗 CLDN1 抗体，CLDN1 在病毒进入过程中的作用知之甚少。在这项研究中，我们通过遗传免疫产生了针对人 CLDN1 细胞外环的抗体，并使用这些抗体在感染性 HCV 细胞培养系统和人肝细胞中研究 CLDN1 在 HCV 进入过程中的机制作用。针对细胞表面表达的 CLDN1 的抗体特异性地以剂量依赖的方式抑制 HCV 感染。针对 CLDN1、清道夫受体 B1 和 CD81 的抗体与单独使用任一试剂相比具有附加的中和能力。针对 CLDN1 和抗 CD81 抗体的动力学研究表明，HCV 与这两种进入因子的相互作用在内化过程中发生在相似的时间。在没有检测到 CLDN1-E2 相互作用的情况下，抗 CLDN1 抗体抑制包膜糖蛋白 E2 与 HCV 许可细胞系的结合。使用荧光标记的进入因子和荧光共振能量转移方法，我们证明抗 CLDN1 抗体抑制 CD81-CLDN1 缔合。相比之下，CLDN1-CLDN1 和 CD81-CD81 缔合未被调节。总之，我们的结果表明，针对 CLDN1 的抗体通过减少 E2 与细胞表面的结合并破坏 CD81-CLDN1 相互作用来中和 HCV 感染性。
这些结果进一步定义了 CLDN1 在 HCV 进入过程中的功能，并强调了针对 E2-CD81-CLDN1 相互作用的新抗病毒策略。
