Initiation of HIV Capsid Assembly Monitored by Mass Photometry

摘要 这项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。最早的步骤包括肉豆蔻酰化GAG的结合(MYR- GAG)与质膜结合，GAG的寡聚，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在研究上。 涉及三种必需成分中的两种：蛋白质、核糖核酸和脂类。在这里，我们建议使用质量 用光度法(MP)研究模型脂膜系统中Gag-Gag和Gag-RNA的相互作用，即 支持类脂双分子层(SLB)。质量光度法是一种相对较新的表征污染物分布的方法。 使用散射光干涉法测量样品中的相对分子质量。数据可以记录为 “着陆试验”观察分子非特异性地吸附在表面上，或作为“动态试验”， 观察分子在SLB上的扩散。在着陆实验中，分子的相对分子质量分布 测量与表面的缔合/解离，提供关于齐聚物分布的信息 状态和情结。在动态测定中，络合物的位置和相对分子质量都被记录为 时间的作用。后一种分析方法提供了一个前所未有的机会来观察Gag和Gag：RNA SLB中的复合体以无标记的方式。

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.