Defining the molecular interactions required for flavivirus genome packaging and virus assembly

节肢动物传播的黄病毒，例如寨卡病毒、登革热病毒、西尼罗河病毒和波瓦桑病毒，会引起人类出血热、先天性疾病和致命性脑炎。寨卡病毒（ZIKV）具有独特的人际传播能力，可以从母体垂直传播给胎儿，也可以通过性接触水平传播。在当前的提案中，我们确定了活胎盘细胞中 ZIKV 组装的先前未定义的细胞机制，旨在描述促进成熟和释放的修饰膜结构的形成和细胞内运输的潜在机制。我们提供了支持我们新假设的初步数据，即基因组 RNA 的适体标记并使用荧光蛋白标记的衣壳和非结构蛋白 2A (NS2A)，我们可以可视化和理解活细胞中病毒组装和病毒与宿主相互作用的机制。通过创建用于实时成像的突变型 ZIKV 文库，我们鉴定了衣壳蛋白和协调病毒组装的 NS2A 中的关键氨基酸。使用标记的 NS2A ZIKV，我们展示了来自感染细胞的 NS2A 的第一个病毒和宿主相互作用组，迄今为止，这些细胞已逃避质谱方法并鉴定出与小头畸形、RNA 运输和 ER 修饰相关的 NS2A 相互作用宿主蛋白。使用共聚焦和透射电子显微镜，我们发现 ZIKV NS4B 修改了经典分泌途径，并介导源自 ER 出口位点的囊泡的同型融合，在高尔基体附近形成大型核周结构。基于我们的初步数据并使用我们迄今为止开发的独特的 ZIKV 感染性克隆和工具；我们提出了一系列严格的实验，旨在检验我们的假设，即黄病毒修改内质网和宿主分泌途径，形成促进组装和细胞内运输的大囊泡结构。首先，利用共聚焦成像标记的病毒 RNA 和蛋白质构建体文库，我们将确认病毒 RNA 的命运，识别可能参与病毒出芽以及与病毒和宿主蛋白质相互作用的 C、NS2A 和 RNA 的特定区域，并评估 RNA 相互作用蛋白是否促进组装（目标 1）。其次，使用先进的亲和纯化质谱法以及 siRNA 和 CRISPR 敲低方法，我们将确定 ER 中对于病毒产生和细胞膜修饰至关重要的基因组途径（目标 2）。第三，我们将研究组装病毒的内质网出口、NS4B 介导的大囊泡形成以及通过微管的囊泡逆行运输，并确定其对 ZIKV 成熟动力学的影响（目标 3）。我们的研究结果将为 ZIKV 的组装建立一种以前未描述过的重要细胞机制，并确定干预 ZIKV 感染和其他相关病毒的关键目标，满足全球重大公共卫生需求，并为 NIAID 理解、治疗和预防传染病的使命做出贡献。这些知识可以产生新的 ER 相关治疗干预目标，以减轻母婴界面的病毒传播。

Arthropod-borne flaviviruses such as Zika, dengue, West Nile, and Powassan viruses cause hemorrhagic fever, congenital diseases, and fatal encephalitis in humans. Zika virus (ZIKV) has the unique ability of human-to- human transmission vertically from mother to fetus and horizontally through sexual contact. In the current proposal, we identify a previously undefined cellular mechanism of ZIKV assembly in live placental cells and aim to characterize the underlying mechanisms of formation and intracellular trafficking of modified membrane structures that facilitate maturation and release. We present preliminary data supporting our novel hypothesis that aptamer tagging of genomic RNA and using fluorescent-protein tagged capsid and nonstructural protein 2A (NS2A), we can visualize and understand mechanisms of virus assembly and virus-host interactions in live cells. By creating a library of mutant ZIKV for live imaging, we identified critical amino acids in capsid protein and NS2A coordinating virus assembly. Using tagged NS2A ZIKV, we present the first viral and host interactome of NS2A from infected cells that have thus far evaded mass spectrometry approaches and identified NS2A-interacting host proteins associated with microcephaly, RNA trafficking, and ER modifications. Using confocal and transmission electron microscopy, we found that ZIKV NS4B modifies the canonical secretory pathway and mediates homotypic fusion of vesicles originating from the ER exit sites forming large perinuclear structures near the Golgi apparatus. Building on our preliminary data and using unique ZIKV infectious clones and tools we have so far developed; we propose a rigorous set of experiments designed to test our hypothesis that flaviviruses modify the ER and host secretory pathways to form large vesicular structures that facilitate assembly and intracellular trafficking. First, using a library of labeled viral RNA and protein constructs with confocal imaging, we will confirm the fate of viral RNA, identify the specific regions of C, NS2A, and RNA that may participate in virus budding and interaction with viral and host proteins, and evaluate whether RNA interacting proteins facilitate assembly (Aim 1). Second, using an advanced affinity purification-mass spectrometry and siRNA and CRISPR knockdown approach, we will determine the genomic pathways in the ER that are key for virus production and cellular membrane modification (Aim 2). And third, we will investigate the ER exit of assembled viruses, NS4B mediated large vesicle formation, and vesicular retrograde transport via microtubules and determine its effect on the dynamics of ZIKV maturation (Aim 3). Our findings will establish a previously undescribed and essential cellular mechanism for the assembly of ZIKV and identify critical targets for intervention in ZIKV infections and other related viruses, addressing a significant global public health need and contributing to the NIAID mission to understand, treat, and prevent infectious diseases. This knowledge can generate new ER-associated targets for therapeutic intervention to mitigate viral transmission at the maternal-fetal interface.