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

定义黄病毒基因组包装和病毒组装所需的分子相互作用

• 批准号: 10750591
• 负责人: Joyce Jose
• 金额: $ 38.59万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750591
• 关键词: Address; Affect; Affinity; Affinity Chromatography; Amino Acids; Antiviral Agents; Arboviruses; Architecture; Binding; Biological Assay; Candidate Disease Gene; Capsid; Capsid Proteins; Cell Line; Cells; Cellular Membrane; Cellular Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Complex; Congenital Abnormality; Coupled; Data; Dengue; Dengue Virus; Disease; Encephalitis; Endoplasmic Reticulum; Experimental Designs; Fetus; Flavivirus; Flavivirus Infections; Gene Proteins; Genes; Genome; Genomics; Golgi Apparatus; Guide RNA; Human; Image; Imaging Techniques; Infection Control; Integration Host Factors; Intervention; Kinetics; Knowledge; Label; Libraries; Licensing; Mango - dietary; Mass Spectrum Analysis; Maternal-Fetal Exchange; Mediating; Membrane; Methodology; Microcephaly; Microtubules; Mission; Modeling; Modification; Molecular Virology; Mothers; National Institute of Allergy and Infectious Disease; Nonstructural Protein; Nuclear; Nuclear Structure; Organelles; Pathway interactions; Peptide Hydrolases; Persons; Powassan virus; Process; Production; Proteins; Protocols documentation; Public Health; RNA; RNA Binding; RNA replication; RNA-Protein Interaction; Replication-Associated Process; Reporting; Role; Site; Small Interfering RNA; Structural Protein; Structure; Testing; Therapeutic Intervention; Transmission Electron Microscopy; Vaccines; Vector-transmitted infectious disease; Vesicle; Viral; Viral Genome; Viral Hemorrhagic Fevers; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Visualization; West Nile; Work; Yellow Fever; ZIKA; ZIKV infection; Zika Virus; aptamer; arthropod-borne; candidate selection; combat; confocal imaging; design; electron tomography; genome-wide; genomic RNA; knock-down; molecular imaging; mutant; mutation screening; novel; prevent; protein biomarkers; recruit; retrograde transport; single molecule; small hairpin RNA; spatiotemporal; targeted treatment; tool; trafficking; transmission process; viral RNA; viral transmission; virus host interaction; virus identification

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.

人类的节肢动物传播黄病毒，例如寨卡，登革热，西尼罗河和Powassan病毒会导致人类的出血热，先天性疾病和致命性脑炎。寨卡病毒（ZIKV）具有从母亲到胎儿以及通过性接触水平的人类到人向人类传播的独特能力。在当前的建议中，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，旨在表征形成的基本机制和修饰的膜结构的细胞内运输，以促进成熟和释放。我们介绍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capsID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架动物相互作用的机制。通过创建用于实时成像的突变ZIKV的库，我们确定了cap蛋白和NS2A配位病毒组装中的关键氨基酸。使用标记的NS2A ZIKV，我们介绍了来自感染细胞的NS2A的第一个病毒和宿主相互作用，这些病毒和宿主的质谱方法避免了质谱法，并鉴定了与小头畸形，RNA运输和ER修饰相关的NS2A相互作用的宿主蛋白。使用共聚焦和透射电子显微镜，我们发现ZIKV NS4B修饰了规范的分泌途径，并介导了源自质生出口位点的囊泡的同型融合，这些囊泡来自Golgi Apparatus附近的大型核周结构。以我们的初步数据为基础，并使用迄今为止开发的独特ZIKV感染克隆和工具；我们提出了一组严格的实验集，旨在测试我们的假设，即黄素病毒修改ER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，我们将使用带有共聚焦成像的标记的病毒RNA和蛋白质构建库来确认病毒RNA的命运，确定可能参与病毒萌芽并与病毒和宿主蛋白相互作用的C，NS2A和RNA的特定区域，并评估RNA是否会促进RNA相互作用的蛋白（是否会促进组装组装）（目标1）。其次，使用高级亲和力纯化 - 质谱法和siRNA和CRISPR敲低方法，我们将确定ER中的基因组途径，这些途径是病毒生产和细胞膜修饰的关键（AIM 2）。第三，我们将研究组装病毒的ER出口，NS4B介导的大囊泡形成以及通过微管的囊泡逆行转运，并确定其对ZIKV成熟动力学的影响（AIM 3）。我们的发现将建立一个以前未描述的ZIKV组装的基本细胞机制，并确定干预ZIKV感染和其他相关病毒的关键目标，以满足全球公共健康需求，并为NIAID促进NIAID的使命，以理解，治疗，治疗和预防感染性疾病。这些知识可以产生新的与ER相关的靶标，用于治疗干预，以减轻母体界面处的病毒传播。