Initiation of HIV Capsid Assembly Monitored by Mass Photometry

通过质谱光度法监测 HIV 衣壳组装的启动

• 批准号: 10649361
• 负责人: James R Williamson
• 金额: $ 22.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-13 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10649361
• 关键词: Adsorption; Binding; Binding Proteins; Biochemical; Biological Assay; Biology; Body Weight Changes; Capsid; Cell Nucleus; Cell membrane; Cells; Complex; Coupled; Data; Diffusion; Dissociation; Equilibrium; Guide RNA; HIV; HIV-1; In Vitro; Interferometry; Label; Lipid Bilayers; Lipids; Literature; Measurement; Measures; Membrane; Membrane Lipids; Methods; Modeling; Molecular Weight; Monitor; Pathway interactions; Phase; Photometry; Positioning Attribute; Process; Proteins; RNA; RNA Binding; Research; Sampling; Scheme; Set protein; Surface; System; Techniques; Thermodynamics; Time; Virus Assembly; dimer; equilibrium model; expectation; experimental study; gag Gene Products; genomic RNA; innovation; light scattering; monomer; myristoylation; novel; preference; recruit; single molecule; viral genomics

SUMMARY This proposal outlines a plan to characterize the early steps of HIV-1 capsid assembly using a novel and recently available single molecule technique. The earliest steps include association of myristoylated Gag (myr- Gag) with the plasma membrane, oligomerization of Gag, and recruitment of dimeric viral genomic RNA. There is a large literature on the early steps, but the in vitro biochemical data has primarily focused on studies involving two of the three required components: protein, RNA, and lipid. Here, we propose to use mass photometry (MP) to study Gag-Gag and Gag-RNA interactions in a model lipid membrane system, namely supported lipid bilayers (SLBs). Mass photometry is a relatively new method to characterize the distribution of molecular weights (MW) in a sample using interferometry of scattered light. Data can be recorded either as a “landing assay” observing molecules adsorbing to a surface non-specifically, or as a “dynamic assay”, observing molecules diffusing on an SLB. In the landing assay, the distribution of MW for molecules associating/dissociating from the surface is measured, providing information on the distribution of oligomeric states and complexes. In the dynamic assay, both the position and MW of the complexes are recorded as a function of time. The latter assay provides an unprecedented opportunity to observe Gag and Gag:RNA complexes in an SLB in a label-free manner.

概括 该建议概述了使用小说和 最近可用的单分子技术。最早的步骤包括Myristoymet的插科打术（Myr- GAG）具有质膜，GAG的低聚和二聚体病毒基因组RNA的募集。那里 是关于早期步骤的大型文献，但是体外生化数据主要集中在研究上 涉及三个所需组件中的两个：蛋白质，RNA和脂质。在这里，我们建议使用质量 在模型脂质膜系统中研究GAG-GAG和GAG-RNA相互作用的光度法（MP），即 支持的脂质双层（SLB）。质量光度法是一种表征分布的相对新方法 样品中使用散射光的干扰中的分子量（MW）。数据可以记录为 “着陆测定”观察分子吸附到表面非特异性的，或作为“动态测定”， 观察分子在SLB上扩散。在着陆测定中，分子的MW分布 测量与表面的关联/解离，提供有关低聚的分布的信息 国家和复合物。在动态测定中，复合物的位置和MW都记录为 时间的功能。后来的测定提供了一个前所未有的机会来观察堵嘴和堵嘴：RNA 以不含标签的方式以SLB为单位的复合物。