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.

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