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 该提案概述了一项计划，该计划利用一种新颖且独特的方法来描述 HIV-1 衣壳组装的早期步骤。 最近可用的单分子技术。最早的步骤包括与肉豆蔻酰化 Gag (myr- Gag）与质膜的结合，Gag 的寡聚化，以及二聚体病毒基因组 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于研究 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 光度测定 (MP) 用于研究模型脂质膜系统中的 Gag-Gag 和 Gag-RNA 相互作用，即 支持脂质双层（SLB）。质量光度测定法是一种相对较新的表征物质分布的方法。 使用散射光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”观察非特异性吸附到表面的分子，或作为“动态测定”， 观察 SLB 上扩散的分子。在着陆测定中，分子的 MW 分布 测量表面的缔合/解离，提供有关低聚物分布的信息 状态和复合体。在动态测定中，复合物的位置和 MW 均记录为 时间的函数。后一种检测提供了前所未有的观察 Gag 和 Gag:RNA 的机会 以无标记方式在 SLB 中形成复合物。