Cellular Components Involved in Japanese Encephalitis Virus Entry

描述(申请人提供)：该实验室的长期目标是了解乙型脑炎病毒(JEV)如何进入人类细胞内部，这是感染过程的第一步，涉及病毒与其目标细胞之间的一系列多个、高度协调的相互作用。病毒研究的这一方面具有特别重要的意义，因为病毒进入是启动、传播和维持所有人和动物病毒的生产性感染所必需的共同特征。虽然JEV进入过程中涉及的病毒衍生因子是众所周知的(特别是病毒糖蛋白E，它结合了 虽然病毒粒子到达细胞表面，将它们运送到内小体，并催化病毒-内吞体膜融合)，但参与这一多步骤过程的宿主因素仍然难以捉摸。特别是，识别乙脑病毒进入的细胞表面受体(S)一直是一个重大挑战 在乙型脑炎病毒生物学中，主要是由于缺乏非敏感细胞，这是受体筛选所需的。我们现在已经确定了一种不敏感的非人类细胞系，它阻止了乙脑病毒的进入，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病毒高度敏感，这两种病毒与乙脑病毒一样，通过依赖于笼蛋白的内吞途径进入细胞。该细胞系为我们提供了一个独特的机会来鉴定和验证JEV进入的宿主因素。乙脑病毒是一种由蚊子传播的黄病毒。 这与西尼罗河病毒、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疾病。这里提出的工作涉及使用两个互补的、技术先进的基因组规模的基因筛查，以检测乙脑病毒进入的获得和丧失功能(分别为目标1和2)，以确定人类宿主中对病毒进入至关重要的细胞因素和机制，并剖析由特定宿主因素调节的离散进入步骤。将实现两个独立但互补的目标：在目标1中，将使用迭代cDNA文库筛选方法在高度易感乙脑病毒的非神经细胞和神经细胞中鉴定一个或多个细胞类型特异的细胞基因，这些基因使新开发的不敏感乙脑病毒的非人类细胞系对乙脑病毒感染易感。在目标2中，将采用多重RNAi筛选方法来鉴定在乙脑病毒进入两个高度敏感的人类非神经细胞和神经细胞中起重要作用的一个或多个细胞类型特异的细胞基因。将利用各种创新的分子生物学、细胞生物学和生化技术，对候选宿主因子进行功能表征，以评估它们在促进乙脑病毒进入方面的潜在作用。这项研究的结果将(I)提供一个独特的机会，以便在JEV进入的水平上全面了解JEV与宿主细胞的相互作用，(Ii)揭示JEV的细胞/组织趋向性和发病机制，以及可能与之密切相关的其他脑炎性黄病毒，以及(Iii)为开发能够抑制JEV感染早期步骤的新型抗病毒干预措施提供新的靶点。 公共卫生相关性：日本脑炎病毒(JEV)感染可导致人类致命的神经系统疾病。目前还没有临床批准的抗病毒药物或治疗乙脑病毒感染的特殊疗法。由于其蚊媒的流行，目前对包括美国在内的北美地区存在严重的健康威胁。这项拟议的研究将阐明JEV进入的机制，并剖析受特定JEV进入因子调控的离散进入步骤，这可能会促进新抗病毒药物的开发，这些药物通过阻断JEV感染的这一关键步骤发挥作用。

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. PUBLIC HEALTH RELEVANCE: Japanese encephalitis virus (JEV) infection causes fatal neurological disease in humans. There are no clinically approved antiviral drugs or specific therapies for the treatment of JEV infection. Because of the prevalence of its mosquito vectors, there currently exists a serious health threat to na¿ve areas, including the United States. The proposed research will elucidate the mechanism of JEV entry and dissect the discrete entry steps that are regulated by specific JEV entry factors, which may foster development of new antivirals that act by blocking this key step in JEV infection.