Characterization of CD81 receptor interactors in hepatitis C virus and Plasmodium liver cell entry.

CD81是一种跨膜蛋白，是两种人类病原体的受体，丙型肝炎病毒(HCV)和疟疾的病原体疟原虫。尽管它们的分子组成、传播和病理都有很大的不同，但这两种病原体都需要CD81进入肝细胞并在其中复制。此外，丙型肝炎病毒和人类病原体恶性疟原虫都表现出狭隘的宿主趋向性，只自然感染人类，而CD81是几个宿主趋向性决定因素之一。由于CD81缺乏信号域，我们假设它通过蛋白质-蛋白质-相互作用(PPI)来协调丙型肝炎病毒和疟疾的摄取。我们之前的工作在人肝癌细胞中发现了68个CD81 PPI，并可能表明至少有10个CD81相互作用是丙型肝炎病毒和疟疾感染所必需的。目前，依赖CD81的丙型肝炎病毒和疟原虫进入所需的全套宿主因素仍然难以捉摸。此外，我们缺乏关于这些蛋白质如何引导病原体进入的知识。在这里，我们建议在我们以前的CD81受体相互作用的基础上确认进入丙型肝炎病毒和疟原虫的宿主因子，并深入地表征宿主因子的分子功能。具体地说，这项工作旨在(1)确认CD81相互作用因子与丙型肝炎病毒和疟原虫感染的相关性；(2)评估已发现的不同包膜病毒、丙型肝炎病毒基因型和疟原虫物种的宿主因子的特异性；(3)对选定的宿主因子进行机械表征；(4)确定宿主因子对丙型肝炎病毒和恶性疟原虫狭窄组织和宿主嗜性的贡献。在项目的第一阶段，我们将确认肝癌细胞中所有68个CD81相互作用物的敲除表型，并评估细胞对丙型肝炎病毒和疟疾的敏感性。在这种RNA干扰实验中得分的基因将被我们最近成功应用于肝癌细胞的CRISPR/Cas9技术敲除。为了评价宿主因子的特异性，我们用三种囊膜病毒(水泡性口炎病毒、冠状病毒229E、呼吸道合胞病毒)、七种丙型肝炎病毒和三种疟原虫感染敲除细胞。广泛的丙型肝炎病毒和疟原虫宿主因子将根据它们支持的特定进入步骤、它们的亚细胞定位、如果适用的话、它们在病原体摄取期间的酶活性以及蛋白质中的关键区域来进行机械分析。在最后一个项目阶段，宿主因子的组织表达分析将揭示可能对丙型肝炎病毒和疟原虫子孢子的肝脏趋向性的贡献。最后，用宿主因子的小鼠和猕猴同源基因来补充敲除细胞将突出丙型肝炎病毒和疟原虫可能的物种限制。总之，这项工作将阐明丙型肝炎病毒和疟原虫进入肝细胞的过程，并揭示细胞膜运输和信号转导的重要方面。

CD81 is a transmembrane protein and the receptor for two human pathogens, hepatitis C virus (HCV) and Plasmodium, the causative agent of malaria. Despite substantial differences in their molecular makeup, their transmission and pathology, both pathogens require CD81 to enter liver cells and replicate in these. Also, HCV and the human pathogen Plasmodium falciparum both display a narrow host tropism by naturally only infecting humans and CD81 is one of several host tropism determinants. Since CD81 lacks signaling domains, we had hypothesized that it coordinates HCV and Plasmodium uptake through protein-protein-interactions (PPI). Our previous work identified 68 CD81 PPIs in human hepatoma cells and could show that at least 10 of the CD81 interactors are required for HCV and Plasmodium infection. Currently, the full set of host factors required for CD81-dependent entry of HCV and Plasmodium remains elusive. Moreover, we lack knowledge on how the proteins guide pathogen entry. Here, we propose to confirm HCV and Plasmodium entry host factors based on our previous CD81 receptor interactomics and to characterize the host factors molecular function in depth.Specifically, this work aims at (1) confirming the relevance of CD81 interactors for HCV and Plasmodium infection; (2) evaluating the specificity of discovered host factors for diverse enveloped viruses, HCV genotypes and Plasmodium species; (3) mechanistically characterizing selected host factors; (4) determining the contribution of the host factors to the narrow tissue and host tropism of HCV and Plasmodium falciparum. In the first phase of the project we will confirm knockdown phenotypes of all 68 CD81 interactors in hepatoma cells and evaluate the cells susceptibility to HCV and Plasmodium. Genes, which score in this RNA interference assay, will be knocked out by CRISPR/Cas9, a technique, which we successfully applied to hepatoma cells recently. To evaluate the specificity of the host factors, we will infect knockout cells with three enveloped viruses (vesicular stomatitis virus, coronavirus 229E, respiratory syncytial virus), seven HCV genotypes and three Plasmodium species. Broad HCV and Plasmodium host factors will be mechanistically analyzed in terms of the specific entry step they support, their subcellular localization, if applicable their enzymatic activity during pathogen uptake and critical domains in the protein. In the last project phase tissue expression analysis of host factors will reveal possible contributions to liver tropism of HCV and Plasmodium sporozoites. Lastly, complementation of knockout cells with mouse and macaque orthologues of the host factors will highlight possible species restrictions for HCV and Plasmodium. In sum, this work will shed light on the entry of HCV and Plasmodium into liver cells and reveal important aspects of cellular membrane trafficking and signaling.