Thursday, April 25, 2024
4/25/2024
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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