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

丙型肝炎病毒和疟原虫肝细胞进入中 CD81 受体相互作用物的表征。

基本信息

批准号：
246964086
负责人：
Professorin Dr. Gisa Gerold
金额：
--
依托单位：
Institut für Biochemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/246964086?language=en
关键词：
Characterization CD81 receptor interactors hepatitis

项目摘要

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.

CD 81是一种跨膜蛋白，是两种人类病原体的受体，即丙型肝炎病毒（HCV）和疟原虫（疟疾的病原体）。尽管它们的分子组成、传播和病理学有很大差异，但这两种病原体都需要CD 81进入肝细胞并在其中复制。此外，HCV和人类病原体恶性疟原虫（Plasmodium falciparum）均通过天然地仅感染人类而显示狭窄的宿主向性，并且CD 81是几种宿主向性决定因素之一。由于CD 81缺乏信号结构域，我们假设它通过蛋白质-蛋白质相互作用（PPI）协调HCV和疟原虫的摄取。我们先前的工作在人肝癌细胞中鉴定了68种CD 81 PPI，并且可以表明至少10种CD 81相互作用物是HCV和疟原虫感染所必需的。目前，HCV和疟原虫CD 81依赖性进入所需的全套宿主因子仍然难以捉摸。此外，我们缺乏关于蛋白质如何引导病原体进入的知识。在此，我们提出了基于我们先前的CD 81受体相互作用组学来确认HCV和疟原虫进入宿主因子，并深入表征宿主因子的分子功能，具体而言，本工作的目的是（1）确认CD 81相互作用因子与HCV和疟原虫感染的相关性，（2）评估所发现的宿主因子对不同包膜病毒、HCV基因型和疟原虫种属的特异性，（3）评估宿主因子对HCV和疟原虫感染的特异性。（3）机械地表征所选择的宿主因子;（4）确定宿主因子对HCV和恶性疟原虫的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CD 81相互作用因子的敲低表型，并评估细胞对HCV和疟原虫的易感性。在这种RNA干扰试验中得分的基因将被CRISPR/Cas9敲除，这是一种我们最近成功应用于肝癌细胞的技术。为了评估宿主因素的特异性，我们将用三种包膜病毒（水泡性口炎病毒、冠状病毒229 E、呼吸道合胞病毒）、七种HCV基因型和三种疟原虫感染敲除细胞。广义HCV和疟原虫宿主因子将根据其支持的特定进入步骤、其亚细胞定位、病原体摄取期间的酶活性（如适用）和蛋白质中的关键结构域进行机械分析。在最后的项目阶段，宿主因子的组织表达分析将揭示HCV和疟原虫子孢子的肝嗜性的可能贡献。最后，敲除细胞与宿主因子的小鼠和猕猴直系同源物的互补将突出HCV和疟原虫的可能物种限制。总之，这项工作将阐明HCV和疟原虫进入肝细胞的过程，并揭示细胞膜运输和信号传导的重要方面。