A biomimetic minimalist model membrane was used to study the mechanism and
kinetics of cell-free in vitro HIV-1 Gag budding from a giant unilamellar
vesicle (GUV). Real time interaction of Gag, RNA and lipid leading to the
formation of mini-vesicles was measured using confocal microscopy. Gag forms
resolution limited punctae on the GUV lipid membrane. Introduction of the Gag
and urea to a GUV solution containing RNA led to the budding of mini-vesicles
on the inside surface of the GUV. The GUV diameter showed a linear decrease in
time due to bud formation. Both bud formation and decrease in GUV size were
proportional to Gag concentration. In the absence of RNA, addition of urea to
GUVs incubated with Gag also resulted in subvesicle formation but exterior to
the surface. These observations suggest the possibility that clustering of GAG
proteins leads to membrane invagination even in the absence of host cell
proteins. The method presented here is promising, and allows for systematic
study of the dynamics of assembly of immature HIV and help classify the
hierarchy of factors that impact the Gag protein initiated assembly of
retroviruses such as HIV.Comment: 27 pages, 9 Figures and 0 Table
