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

节肢动物传播的黄病毒，如寨卡病毒、登革热病毒、西尼罗河病毒和波瓦桑病毒，会导致人类出现出血热、先天性疾病和致命的脑炎。寨卡病毒（ZIKV）具有独特的人传人能力，可从母体向胎儿垂直传播，并通过性接触水平传播。在目前的提案中，我们确定了ZIKV在活胎盘细胞中组装的先前未定义的细胞机制，并旨在表征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。我们提供的初步数据支持我们的新假设，即基因组RNA的适体标记和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-宿主相互作用的机制。通过建立一个用于实时成像的突变ZIKV文库，我们鉴定了衣壳蛋白和NS2A协调病毒组装的关键氨基酸。利用标记的NS2A ZIKV，我们首次从感染细胞中发现了NS2A的病毒与宿主相互作用组，这些细胞迄今为止躲过了质谱分析方法，并鉴定了与小头畸形、RNA运输和内质网修饰相关的NS2A相互作用宿主蛋白。通过共聚焦和透射电镜，我们发现ZIKV NS4B修饰了典型的分泌途径，并介导源自内质网出口位点的囊泡的同型融合，形成靠近高尔基体的大核周结构。以我们的初步数据为基础，利用我们迄今为止开发的独特的寨卡病毒感染克隆和工具；我们提出了一套严格的实验，旨在验证我们的假设，即黄病毒修饰内质网和宿主分泌途径，形成促进组装和细胞内运输的大泡状结构。首先，利用共聚焦成像的标记病毒RNA和蛋白质构建物文库，我们将确认病毒RNA的命运，鉴定C、NS2A和RNA中可能参与病毒出芽和与病毒和宿主蛋白相互作用的特定区域，并评估RNA相互作用蛋白是否促进组装（Aim 1）。其次，使用先进的亲和纯化-质谱法和siRNA和CRISPR敲低方法，我们将确定内质网中对病毒产生和细胞膜修饰至关重要的基因组途径（目的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（目的3）。我们的研究结果将为ZIKV组装建立一个先前未描述的基本细胞机制，并确定干预ZIKV感染和其他相关病毒的关键目标，解决重大的全球公共卫生需求，并为NIAID理解、治疗和预防传染病的使命做出贡献。这一知识可以产生新的er相关的治疗干预靶点，以减轻病毒在母胎界面的传播。

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.