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.

总结 该提案概述了一项计划，利用一种新的和 最近可用的单分子技术。最早的步骤包括豆蔻酰化的Gag（myr-100）与蛋白质的结合。 Gag）与质膜、Gag的寡聚化和二聚体病毒基因组RNA的募集。那里 是关于早期步骤的大量文献，但体外生化数据主要集中在研究 涉及三种必需成分中的两种：蛋白质，RNA和脂质。在这里，我们建议使用质量 光度法（MP）来研究模型脂质膜系统中的Gag-Gag和Gag-RNA相互作用，即 支持的脂质双层（SLB）。质量光度法是一种相对较新的表征物质分布的方法 使用散射光的干涉测量法测量样品中的分子量（MW）。数据可以记录为 “着陆测定”，非特异性地观察吸附到表面的分子，或作为“动态测定”， 观察分子在SLB上的扩散。在着陆测定中，分子的MW分布 测量从表面缔合/解离，提供关于低聚物的分布的信息。 状态和复合体。在动态测定中，复合物的位置和MW均记录为 时间的功能。后一种检测方法为观察Gag和Gag：RNA提供了前所未有的机会 以无标记的方式在SLB中形成复合物。