Fan Huahao, Qiao Luhua, Kang Kyung-Don, Fan Junfen, Wei Wensheng, Luo Guangxiang
Department of Microbiology, Peking University School of Basic Medical Sciences, Beijing, China.
Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA.
J Virol. 2017 Jun 9;91(13). doi: 10.1128/JVI.00280-17. Print 2017 Jul 1.
Hepatitis C virus (HCV) requires multiple receptors for its attachment to and entry into cells. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules, such as CD81, Claudin-1 (CLDN1), Occludin (OCLN), SR-BI, and low-density lipoprotein receptor (LDLR), function mainly at postattachment steps and are considered postattachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockouts of SDC-1 and SDC-2 resulted in remarkable reductions of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockouts did not affect HCV infection. Defective HCV attachment to SDC-1 and/or SDC-2 knockout cells was completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of the attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the postattachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread, but very-low-density lipoprotein (VLDL)-containing mouse serum did not affect HCV cell-to-cell transmission, although it inhibited cell-free infection. These findings demonstrate that attachment receptors are essential for initial HCV binding and that postattachment receptors are important for both HCV cell-free and cell-to-cell transmission. The importance and underlying molecular mechanisms of cell surface receptors in HCV cell-free and cell-to-cell transmission are poorly understood. The role of some of the HCV attachment and postattachment receptors in HCV infection and cell-to-cell spread remains controversial. Using CRISPR-Cas9-mediated knockouts of specific cellular genes, we demonstrate that both SDC-1 and SDC-2, but not SDC-3 or SDC-4, are bona fide HCV attachment receptors. We also used a newly developed luciferase-based reporter system to quantitatively determine the importance of attachment and postattachment receptors in HCV cell-to-cell transmission. SDC-1, SDC-2, TIM-1, and SR-BI were found to modestly promote HCV cell-to-cell spread. CD81, CLDN1, OCLN, and LDLR play more important roles in HCV cell-to-cell transmission. Likewise, apolipoprotein E (apoE) is critically important for HCV cell-to-cell spread, unlike VLDL-containing mouse serum, which did not affect HCV cell-to-cell spread. These findings suggest that the mechanism(s) of HCV cell-to-cell spread differs from that of cell-free infection.
丙型肝炎病毒（HCV）需要多种受体来附着并进入细胞。我们之前的研究发现，人类多配体蛋白聚糖-1（SDC-1）、SDC-2以及含T细胞免疫球蛋白和粘蛋白结构域蛋白1（TIM-1）是HCV附着受体。其他细胞表面分子，如CD81、紧密连接蛋白-1（CLDN1）、闭合蛋白（OCLN）、清道夫受体BI（SR-BI）和低密度脂蛋白受体（LDLR），主要在附着后步骤发挥作用，被认为是附着后受体。不同受体在HCV非细胞和细胞间传播中的潜在分子机制仍不清楚。在本研究中，我们使用了成簇规律间隔短回文重复序列（CRISPR）-Cas9技术、基因特异性小干扰RNA以及新开发的基于荧光素酶的报告系统，以定量确定各个受体在HCV非细胞和细胞间传播中的重要性。SDC-1和SDC-2的敲除导致HCV感染和细胞附着显著减少，而SDC-3和SDC-4的敲除不影响HCV感染。SDC-1和/或SDC-2敲除细胞上有缺陷的HCV附着通过SDC-1和SDC-2而非SDC-4的表达完全恢复。附着受体SDC-1、SDC-2和TIM-1的敲除也适度降低了HCV细胞间传播。相反，附着后受体CD81、CLDN1、OCLN、SR-BI和LDLR的沉默和敲除极大地损害了HCV非细胞和细胞间传播。此外，发现载脂蛋白E对HCV细胞间传播很重要，但含极低密度脂蛋白（VLDL）的小鼠血清虽然抑制非细胞感染，但不影响HCV细胞间传播。这些发现表明，附着受体对HCV的初始结合至关重要，而附着后受体对HCV非细胞和细胞间传播都很重要。细胞表面受体在HCV非细胞和细胞间传播中的重要性及潜在分子机制了解甚少。一些HCV附着和附着后受体在HCV感染和细胞间传播中的作用仍存在争议。使用CRISPR-Cas9介导的特定细胞基因敲除，我们证明SDC-1和SDC-2是真正的HCV附着受体，而SDC-3和SDC-4不是。我们还使用新开发的基于荧光素酶的报告系统定量确定附着和附着后受体在HCV细胞间传播中的重要性。发现SDC-1、SDC-2、TIM-1和SR-BI适度促进HCV细胞间传播。CD81、CLDN1、OCLN和LDLR在HCV细胞间传播中发挥更重要的作用。同样，载脂蛋白E（apoE）对HCV细胞间传播至关重要，与含VLDL的小鼠血清不同，后者不影响HCV细胞间传播。这些发现表明，HCV细胞间传播的机制与非细胞感染的机制不同。
