Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.
Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.
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表皮生长因子受体，claudin-1和occludin在丙型肝炎病毒多步入二极化肝癌球体中的作用。
DOI：
10.1371/journal.ppat.1011887
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发表时间：
2023-12
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
丙型肝炎病毒(HCV)进入的多步骤过程受到多种宿主因素的促进，包括表皮生长因子受体(EGFR)和紧密连接蛋白claudin-1(CLDN1)和occludin(OCLN)，它们被认为在丙型肝炎病毒进入过程的后期发挥作用。利用极化肝癌球体感染丙型肝炎病毒的单颗粒成像，我们观察到EGFR在丙型肝炎病毒进入过程中发挥多种功能，既有磷酸化依赖的，也有非磷酸化的。我们之前观察到，并且在这项研究中证实，EGFR不是丙型肝炎病毒迁移到紧密连接所必需的。EGFR是以一种不依赖于磷酸化的方式向丙型肝炎病毒募集网状蛋白所必需的。在网状蛋白募集后的一个阶段，病毒粒子内化需要EGFR的磷酸化。丙型肝炎病毒的进入激活了EGFR磷酸化下游的RAF-MEK-ERK信号通路。这一信号通路调节内化的丙型肝炎病毒分选和成熟为APPL1和EEA1相关的早期内小体，形成病毒粒子脱壳的部位。紧密连接蛋白CLDN1和OCLN在丙型肝炎病毒进入的两个不同阶段发挥作用。尽管CLDN1被认为是丙型肝炎病毒进入体内的“晚期受体”，但它是在紧密连接处有效积聚病毒粒子所必需的。缺乏CLDN1的HH-7.5细胞主要在最初的基底外侧表面积聚丙型肝炎病毒粒子。OCLN是病毒粒子内化后期所必需的。这项研究对异常复杂的丙型肝炎病毒内吞过程有了进一步的了解。丙型肝炎病毒是一种嗜肝的核糖核酸病毒。据估计，2019年有5800万人慢性感染丙型肝炎病毒，而与丙型肝炎病毒相关的肝细胞癌和肝硬变导致29万人死亡。丙型肝炎病毒进入肝细胞是高度复杂的，具有不同的阶段和多种宿主辅助因素。干扰丙型肝炎病毒与进入宿主因子的相互作用是治疗和基于疫苗的抗丙型肝炎病毒方法的潜在策略。然而，大多数宿主因子在丙型肝炎病毒进入中的确切作用还没有得到充分的认识。此外，使用生理上与体内肝细胞相似的极化细胞培养进入体内的研究有限。在本文中，我们评估了三个进入因子在极化的肝癌球体中的作用：表皮生长因子受体(EGFR)和紧密连接蛋白claudin-1(CLDN1)和occludin(OCLN)。我们观察到，EGFR在调节丙型肝炎病毒内化和转运到内体方面具有多种功能。该过程涉及EGFR磷酸化依赖和非依赖性作用以及RAF-MEK-ERK信号通路下游。我们还表明，尽管CLDN1和OCLN都位于紧密连接处，但它们在丙型肝炎病毒进入的两个不同阶段发挥作用。CLDN1是丙型肝炎病毒在基底膜的紧密连接处聚集所必需的，而OCLN则调节丙型肝炎病毒的内化。这些发现提供了对丙型肝炎病毒进入机制的洞察，并强调了潜在的干预策略。
The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a “late receptor” in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process. Hepatitis C virus (HCV) is a hepatotropic RNA virus. An estimated 58 million people are chronically infected with HCV, while HCV-associated hepatocellular carcinoma and cirrhosis accounted for 290, 000 deaths in 2019. HCV entry into hepatocytes is highly complex with distinct stages and multiple host cofactors. Perturbing the interaction of HCV with entry host factors is potential strategy for therapeutic and vaccine-based anti-HCV approaches. However, the precise functions of most of the host factors in HCV entry are not fully appreciated. Moreover, studies of entry using polarized cell culture that physiologically resembles hepatocytes in vivo are limited. In this paper, we evaluate the roles of three entry factors in polarized hepatoma spheroids: epidermal growth factor receptor (EGFR) and tight junction proteins claudin-1 (CLDN1) and occludin (OCLN). We observe that EGFR performs multiple functions to regulate HCV internalization and trafficking to endosomes. The process involves EGFR phosphorylation-dependent and -independent roles and downstream RAF-MEK-ERK signaling pathway. We also show that CLDN1 and OCLN, despite both localizing at the tight junction, function at two distinct stages of HCV entry. CLDN1 is required for the accumulation of HCV virions at the tight junction from the basolateral membrane, while OCLN regulates HCV internalization. These findings provide insights into the mechanism of HCV entry and highlight potential interventional strategies.
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