Molecular mechanisms linking viral replication and neuropathogenesis

项目摘要/摘要 感染发育中大脑的病毒，包括寨卡病毒(ZIKV)、风疹病毒和巨细胞病毒，会导致 严重的出生缺陷。小头畸形就是这样一种出生缺陷，它的头部和大脑尺寸严重缩小， 而且常常伴随着智力残疾。这种由病毒引起的神经系统疾病，或称病毒 神经发病机制，可由多种机制引起。最近发现的一种机制是ZIKV 非结构蛋白4A(NS4A)通过破坏人类ANKLE2蛋白而导致小头畸形症。有趣的是， 编码ANKLE2基因突变的个体患有小头畸形症。ANKLE2是保守的 对人类来说是蠕虫，在大脑发育过程中对协调细胞分裂是必不可少的。ANKLE2不是 酶，而不是通过调节蛋白质相互作用而在细胞分裂和发育中获得这一功能。 NS4A与ANKLE2在物理上相互作用，并以ANKLE2依赖的方式扰乱大脑发育 脑发育的果蝇模型。ANKLE2还促进ZIKV复制。总而言之，这些研究 研究表明，在选择宿主蛋白进行复制的过程中，ZIKV失调了一个重要的发育 路径。因此，NS4A-ANKLE2蛋白的相互作用是研究病毒的一个重要模型 神经发病机制及其与病毒复制和遗传性疾病在分子水平上的联系。 这项工作的长期目标是破译病毒-宿主蛋白相互作用如何影响病毒复制和 发病机制，因为这些发现将推动治疗靶点的确定和药物开发。这个 本研究的目的是分析ZIKV NS4A蛋白相互作用的机制。 人类ANKLE2促进ZIKV复制，抑制大脑发育。为了实现这一目标，我们 将检验核心假设，即ANKLE2通过与NS4A和NS4A的相互作用促进病毒复制 通过招募参与ZIKV复制的其他宿主因素到复制站点，这扰乱了生理 ANKLE2蛋白相互作用是大脑发育所必需的。以下具体目标将检验这一假设： 目的1：分析NS4A-ANKLE2蛋白相互作用在ZIKV复制和致病中的作用。 目的2：揭示ANKLE2在ZIKV复制和致病中的分子功能。 当这项工作完成后，这项工作将描绘出单个病毒-宿主蛋白相互作用如何改变发育 促进病毒复制并在分子水平上造成神经疾病的途径。这将揭示 对病毒-宿主相互作用的详细生化洞察与氨基酸水平的解析，新的宿主因素 在ZIKV复制中发挥作用，以及以前未知的大脑发育关键蛋白。从长远来看， 建立的方法可以用来揭示其他疾病背后的分子机制 病毒性和遗传性病因。

PROJECT SUMMARY/ABSTRACT Viruses that infect the developing brain, including Zika virus (ZIKV), rubella virus, and cytomegalovirus, cause major birth defects. Microcephaly is one such birth defect, in which head and brain size are severely reduced, and is often accompanied by intellectual disability. This virally-inflicted neurological disease, or viral neuropathogenesis, can be caused by multiple mechanisms. One recently identified mechanism by which ZIKV non-structural protein 4A (NS4A) causes microcephaly is by disrupting the human ANKLE2 protein. Interestingly, individuals with mutations in the gene encoding ANKLE2 suffer from microcephaly. ANKLE2 is conserved from worms to humans, and is essential for coordinating cell division during brain development. ANKLE2 is not an enzyme, and instead derives this function in cell division and development by mediating protein interactions. NS4A physically interacts with ANKLE2 and disrupts brain development in an ANKLE2-dependent manner in a fruit fly model of brain development. ANKLE2 also promotes ZIKV replication. Taken together, these studies show that in the process of coopting a host protein for replication, ZIKV dysregulates an important developmental pathway. Thus, the NS4A-ANKLE2 protein interaction represents an important model to study viral neuropathogenesis and how it is connected to viral replication and hereditary disorders at the molecular level. The long-term goal of this work is to decipher how virus-host protein interactions impact virus replication and pathogenesis, as these discoveries will fuel therapeutic target identification and drug development. The objective of this proposal is to dissect the mechanisms by which the protein interaction between ZIKV NS4A and human ANKLE2 promote ZIKV replication and inhibit brain development. To accomplish this objective, we will test the central hypothesis that ANKLE2 promotes viral replication through its interaction with NS4A and by recruiting other host factors involved in ZIKV replication to sites of replication, and this disrupts physiological ANKLE2 protein interactions required for brain development. The following specific aims will test this hypothesis: Aim 1: Dissect the impact of the NS4A-ANKLE2 protein interaction in ZIKV replication and pathogenesis. Aim 2: Unravel the molecular function of ANKLE2 in ZIKV replication and pathogenesis. When completed, this work will delineate how a single virus-host protein interaction rewires a developmental pathway to facilitate virus replication and inflict neurological disease at the molecular level. This will reveal detailed biochemical insight into a virus-host interaction with amino acid-level resolution, new host factors that play a role in ZIKV replication, and previously unknown proteins key to brain development. In the long term, the methods established here could be employed to uncover the molecular mechanisms behind other diseases with viral and hereditary etiologies.