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、西尼罗河病毒和鲍瓦桑病毒，会引起人类的出血热、先天性疾病和致命性脑炎。寨卡病毒(ZIKV)具有独特的人传人能力，可通过母婴垂直传播和性接触水平传播。在目前的提案中，我们确定了一个先前未确定的ZIKV在活胎盘细胞中组装的细胞机制，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转运的潜在机制。我们提供了支持我们的新假设的初步数据，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-宿主相互作用的机制。通过建立一个用于活体成像的突变型ZIKV文库，我们确定了衣壳蛋白和NS2A协调病毒组装的关键氨基酸。使用标记的NS2AZIKV，我们展示了第一个来自感染细胞的NS2A病毒和宿主相互作用组，到目前为止，这些感染细胞避开了质谱学方法，并鉴定了与小头畸形、RNA运输和内质网修饰相关的NS2A型相互作用宿主蛋白。利用共聚焦显微镜和透射电子显微镜，我们发现ZIKV NS4B改变了典型的分泌途径，并介导了源自内质网出口部位的小泡的同型融合，形成了靠近高尔基体的大型核周结构。在我们初步数据的基础上，并使用我们迄今开发的独特的ZIKV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，即黄病毒修改ER和宿主分泌途径，形成大型泡状结构，促进组装和细胞内运输。首先，使用带有共聚焦成像的标记病毒RNA和蛋白质构造物的文库，我们将确认病毒RNA的命运，识别可能参与病毒萌发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相互作用蛋白质是否促进组装(目标1)。其次，使用先进的亲和纯化-质谱法以及siRNA和CRISPR敲除方法，我们将确定内质网中对病毒生产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退出，NS4B介导的大小泡的形成，以及小泡通过微管的逆行运输，并确定其对ZIKV成熟动力学的影响(目标3)。我们的发现将为ZIKV的组装建立一个以前未描述的必要的细胞机制，并确定干预ZIKV感染和其他相关病毒的关键靶点，满足重大的全球公共卫生需求，并为NIAID了解、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相关目标，用于治疗干预，以减少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.