Visualization of Influenza Viral RNA Assembly

流感病毒 RNA 组装的可视化

基本信息

批准号：
10681630
负责人：
Seema S. Lakdawala
金额：
$ 38.16万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-11 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10681630
关键词：
Address Binding Proteins Biochemical Biochemistry Biology Biotin Biotinylation Birds Cell Culture System Cell Culture Techniques Cell Nucleus Cell membrane Cell model Cells Cessation of life Color Custom Cytoskeletal Proteins Data Endosomes Epidemic Epithelial Cells Event Family suidae Fluorescent in Situ Hybridization Future Genetic Genetic Materials Genetic Processes Genomic approach Goals Hospitalization Human Imaging Techniques Infection Influenza Influenza A Virus, H1N1 Subtype Influenza A virus Integration Host Factors Intracellular Transport Knowledge Light Location Methods Microscope Microscopy Microtubules Modeling Molecular Virology Movement Nature Nuclear Export Organelles Pathway interactions Pattern Physiological Plasma Play Population Primary Cell Cultures Process Proteins Public Health Publishing RNA RNA Transport Reassortant Viruses Recycling Research Research Personnel Role Route Study models Techniques Viral Viral Genome Viral Proteins Virion Virus Virus Assembly Virus Diseases Virus Replication Visualization Work Zoonoses airway epithelium base cell transformation cell type co-infection imaging approach influenza infection influenzavirus insight live cell imaging new therapeutic target novel nucleocytoplasmic transport pandemic disease pandemic influenza respiratory swine flu therapeutic target tool trafficking viral RNA virology

项目摘要

Influenza A viruses (IAV) pose a major public health threat through both seasonal epidemics and sporadic pandemics. The segmented nature of the viral genome promotes reassortment, a process where the genetic material between viruses is exchanged in a co-infected cell. In nature, reassortment leads to increased viral diversity and emergence of pandemic influenza viruses. For example, the 2009 influenza H1N1 (‘swine flu’) pandemic virus, emerged from reassortment of two circulating swine viruses. Prediction of future pandemic influenza viruses from circulating zoonotic virus populations is difficult because very little is known about the mechanism of reassortment within a single co-infected cell. To accurately define the process of reassortment, we must first understand the dynamics of intracellular viral RNA (vRNA) assembly. Influenza vRNA replicates in the nucleus and is transported to the plasma membrane for packaging, which requires one copy of all eight segments to assemble within a single virion to produce a fully infectious virus. In this proposal, we will build upon our previous data on influenza assembly and define 1) the assembly dynamics in physiologically relevant human and swine cell types, 2) the cellular proteins modulating vRNA transport, and 3) the location of reassortment within a co-infected cell. Our central hypothesis is that vRNA assembly occurs in a cell-type specific manner that correlates with IAV reassortment in different host species. The Specific Aims of this application will use a variety of sophisticated microscopy tools, including live cell imaging with a custom light-sheet microscope, to determine the assembly mechanism in various cell culture models. Aim 1 will utilize multicolor fluorescent in situ hybridization and live cell imaging techniques to explore the dynamics of influenza vRNA assembly in human and swine differentiated airway epithelial cells. Aim 2 will uncover the identity and roles of cellular cytoskeletal proteins and membranous organelles utilized during influenza vRNA assembly using biochemical approaches like proximity-dependent biotinylation. Aim 3 will combine imaging and genomic approaches to characterize the cellular location of vRNA intermingling during co-infection with two heterologous viruses in differentiated airway epithelial cells. The proposed work will address many outstanding questions in influenza biology regarding reassortment that have remained unanswered due to a lack of tools to track vRNA movement in live cells during a productive infection. In addition, these studies will identify novel host factors involved in vRNA packaging that can be pursued as potential therapeutic targets.

流感病毒（IAV）通过季节性流行病和零星大流行病构成了重大的公共卫生威胁。病毒基因组的分段性质促进了恢复，这一过程是在共同感染的细胞中交换病毒之间的遗传物质。在自然界中，恢复会导致病毒多样性的增加和大流行影响病毒的出现。例如，2009年的影响力H1N1（“猪流感”）大流行病毒，是由两种循环猪病毒的重新分类出现的。对未来大流行的预测影响了循环的人畜共患病毒种群的病毒，因为对单个共感染细胞内的重新分类机制知之甚少。为了准确定义重新分类的过程，我们必须首先了解细胞内病毒RNA（VRNA）组装的动力学。流感VRNA在细胞核中复制，并将其转运到质膜以进行包装，这需要在单个病毒中组装所有八个段的一个副本才能产生完全感染性的病毒。在此提案中，我们将基于对影响力组装的先前数据，并定义1）在物理相关的人和猪细胞类型中的组装动力学，2）调节VRNA转运的细胞蛋白，以及3）在共感染的细胞中浮雕的位置。我们的中心假设是，VRNA组装以细胞类型的特定方式发生，目的1将利用多色荧光原位杂交和活细胞成像技术来探索人和猪分化的气道上皮细胞中人和猪的影响力的动力学。 AIM 2将使用生物化学方法（如接近依赖性生物素化）揭示在影响力VRNA组装过程中使用的细胞细胞骨架蛋白和膜细胞器的身份和作用。 AIM 3将结合成像和基因组方法，以表征VRNA在分化气道上皮细胞中与两个异源病毒共同感染过程中VRNA相互交织的细胞位置。拟议的工作将解决影响力生物学的许多出色问题，这些问题由于缺乏在生产性感染过程中跟踪活细胞中VRNA运动的工具，因此尚未得到答复。此外，这些研究将确定可作为潜在治疗靶标的VRNA包装涉及的新型宿主因素。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

mSphere of Influence: Redefining an Influenza Virus-How Different Are Influenza Viruses and Extracellular Vesicles?

影响范围：重新定义流感病毒——流感病毒和细胞外囊泡有何不同？

DOI：
10.1128/msphere.00563-19
发表时间：
2019
期刊：
mSphere
影响因子：
4.8
作者：
Lakdawala,SeemaS
通讯作者：
Lakdawala,SeemaS

Learning the sequence of influenza A genome assembly during viral replication using point process models and fluorescence in situ hybridization.

使用点过程模型和荧光原位杂交了解病毒复制过程中甲型流感基因组组装的序列。

DOI：
10.1371/journal.pcbi.1006199
发表时间：
2019
期刊：
PLoS computational biology
影响因子：
4.3
作者：
Majarian,TimothyD;Murphy,RobertF;Lakdawala,SeemaS
通讯作者：
Lakdawala,SeemaS

Cell-Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Airborne Transmission in Ferret Model.

DOI：
10.3390/v13050719
发表时间：
2021-04-21
期刊：
Viruses
影响因子：
0
作者：
Le Sage V;Kormuth KA;Nturibi E;Lee JM;Frizzell SA;Myerburg MM;Bloom JD;Lakdawala SS
通讯作者：
Lakdawala SS

Teaching an Old Virus New Tricks: A Review on New Approaches to Study Age-Old Questions in Influenza Biology.