Molecular mechanisms linking viral replication and neuropathogenesis

项目概要/摘要 感染发育中的大脑的病毒，包括寨卡病毒（ZIKV），风疹病毒和巨细胞病毒， 严重的出生缺陷小头畸形就是这样一种出生缺陷，即头部和大脑尺寸严重缩小， 并且常常伴有智力残疾。这种由病毒引起的神经系统疾病， 神经发病机制，可以由多种机制引起。最近发现的一种机制是ZIKV 非结构蛋白4A（NS 4A）通过破坏人ANKLE 2蛋白而导致小头畸形。有趣的是， 在编码ANKLE 2的基因中具有突变的个体患有小头畸形。ANKLE 2是保守的 从蠕虫到人类，它对大脑发育过程中协调细胞分裂至关重要。ANKLE 2不是一个 酶，而是通过介导蛋白质相互作用在细胞分裂和发育中获得这种功能。 NS 4A与ANKLE 2发生物理相互作用，并以ANKLE 2依赖性方式破坏大脑发育。 果蝇大脑发育模型。ANKLE 2还促进ZIKV复制。综合来看，这些研究 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过程。 通路因此，NS 4A-ANKLE 2蛋白相互作用代表了研究病毒的重要模型。 神经发病机制以及它如何在分子水平上与病毒复制和遗传性疾病联系在一起。 这项工作的长期目标是破译病毒-宿主蛋白质相互作用如何影响病毒复制， 这些发现将推动治疗靶点鉴定和药物开发。的 本提案的目的是剖析ZIKV NS 4A之间的蛋白质相互作用的机制， 和人ANKLE 2促进ZIKV复制并抑制脑发育。为了实现这一目标，我们 将检验ANKLE 2通过与NS 4A相互作用促进病毒复制的中心假设， 通过招募参与ZIKV复制的其他宿主因子到复制位点，这破坏了ZIKV的生理功能。 大脑发育所需的ANKLE 2蛋白相互作用。以下具体目标将检验这一假设： 目的1：剖析NS 4A-ANKLE 2蛋白相互作用在ZIKV复制和发病机制中的影响。 目的2：揭示ANKLE 2在ZIKV复制和发病机制中的分子功能。 当完成后，这项工作将描绘如何一个单一的病毒宿主蛋白质相互作用重新布线的发展， 在分子水平上促进病毒复制和造成神经疾病的途径。这将揭示 详细的生化洞察病毒与宿主的相互作用与氨基酸水平的分辨率，新的宿主因子， 在ZIKV复制中发挥作用，以及以前未知的对大脑发育至关重要的蛋白质。从长远来看， 这里建立的方法可以用来揭示其他疾病背后的分子机制， 病毒和遗传病因学。

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.