Cellular Components Involved in Japanese Encephalitis Virus Entry

描述（由申请人提供）：该实验室的长期目标是了解日本脑炎病毒（JEV）如何进入人体细胞内部，这是感染过程的第一步，涉及病毒与其靶细胞之间的多重高度协调的相互作用。这方面的病毒研究是特别相关的，因为病毒进入是一个共同的特点，是必不可少的启动，传播和维持生产性感染的所有人类和动物病毒。虽然参与JEV进入的病毒衍生因子是众所周知的（特别是病毒糖蛋白E，其连接JEV进入的细胞），但是， 病毒体到细胞表面，将它们递送到内体，并催化病毒-内体膜融合），但是参与该多步骤过程的宿主因子仍然难以捉摸。特别是，鉴定JEV进入的细胞表面受体一直是一个重大挑战 在JEV生物学中，这主要是由于缺乏受体筛选所需非易感细胞。我们现在已经鉴定了一种非敏感的非人细胞系，其具有JEV进入的阻断，但完全支持随后的进入后步骤;该细胞系仍然对另外两种包膜RNA病毒的感染高度敏感，这两种包膜RNA病毒与JEV一样，通过网格蛋白依赖性内吞途径进入细胞。该细胞系为我们提供了一个独特的机会，以确定和验证参与JEV进入的宿主因素。乙脑病毒是一种蚊媒黄病毒 它与西尼罗河病毒、圣路易斯脑炎病毒和墨累谷脑炎病毒密切相关，所有这些病毒都会导致人类致命的神经系统疾病。本文提出的工作涉及使用两种互补的、技术先进的基因组规模的基因筛选，用于获得和丧失JEV进入的功能（分别为目的1和2），以鉴定人类宿主中对病毒进入至关重要的细胞因子和机制，并剖析由特定宿主因子调节的离散进入步骤。将进行两个独立但互补的目标：在目标1中，将使用迭代cDNA文库筛选方法来鉴定高度JEV敏感的非神经元和神经元人类细胞中的一种或多种细胞类型特异性细胞基因，其赋予新开发的JEV非敏感的非人类细胞系对JEV感染的敏感性。在目标2中，将采用多重RNAi筛选方法来鉴定一种或多种细胞类型特异性细胞基因，所述细胞基因在JEV进入两种高度JEV易感的人类非神经元细胞和神经元细胞中起重要作用。候选宿主因子将进行功能表征，以评估其在促进JEV进入中的潜在作用，使用各种创新的分子生物学，细胞生物学和生物化学技术。这项研究的结果将（i）提供一个独特的机会，以获得一个完整的了解如何JEV宿主细胞相互作用发生在JEV进入的水平，（ii）阐明了新的细胞/组织嗜性和发病机制的JEV，和可能的其他密切相关的脑炎黄病毒，和（iii）为开发能够抑制JEV感染早期阶段的新型抗病毒干预提供新的靶点。

DESCRIPTION (provided by applicant): The long-term goal of this laboratory is to understand how Japanese encephalitis virus (JEV) gains access to the interior of human cells, the first step in an infection process that involves a cascade of multiple, highly coordinated interactions between the virus and its target cells. This aspect of virus research is of particular relevance because virus entry is a common feature that is essential to the initiation, dissemination, and maintenance of productive infection by all human and animal viruses. While the virus-derived factors involved in JEV entry are well known (particularly viral glycoprotein E, which attaches the virions to the cell surface, delivers them to endosomes, and catalyzes the viral-endosome membrane fusion), the host factors that participate in this multistep process remain elusive. In particular, identification of the cell-surface receptor(s) for JEV entry has been a major challenge in JEV biology, primarily due to a lack of availability of non-susceptible cells, which are require for receptor screening. We have now identified a non-susceptible non-human cell line that has a block in JEV entry but fully supports the subsequent post-entry steps; this cell line remains highly susceptible to infection by two other enveloped RNA viruses which, like JEV, enter the cells through clathrin-dependent endocytic pathway. This cell line offers us a unique opportunity to identify and validate the host factors involved in JEV entry. JEV is a mosquito-borne flavivirus that is closely related to West Nile virus, St. Louis encephalitis virus, and Murray Valley encephalitis virus, all of which cause fatal neurological disease in humans. The work proposed here involves the use of two complementary, technologically advanced genome- scale genetic screens for gain- and loss-of-function of JEV entry (Aims 1 and 2, respectively) to identify the cellular factors and mechanisms in the human host that are critical for viral entry and to dissect the discrete entry steps that are regulated by specific host factors. Two independent but complementary aims will be carried out: In Aim 1, an iterative cDNA library screening approach will be used to identify one or more cell type-specific cellular genes in highly JEV-susceptible non-neuronal and neuronal human cells that confer susceptibility to JEV infection on a newly developed, JEV-non-susceptible non-human cell line. In Aim 2, a multiplexed RNAi screening approach will be employed to identify one or more cell type-specific cellular genes that play an important role in JEV entry into two highly JEV-susceptible human non-neuronal and neuronal cells. Candidate host factors will be functionally characterized to assess their potential role in promoting JEV entry, using a variety of innovative molecular biology, cell biology, and biochemical techniques. The outcomes of this research will (i) provide a unique opportunity to gain a complete understanding of how JEV-host cell interactions occur at the level of JEV entry, (ii) shed new light on the cell/tissue tropism and pathogenesis of JEV, and possibly other closely related encephalitic flaviviruses, and (iii) provide new targets for the development of novel antiviral interventions capable of inhibiting the early steps of JEV infection.