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A novel minimal in vitro system for analyzing HIV-1 Gag-mediated budding

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Abstract

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. 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.

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Acknowledgments

The authors acknowledge grant support from UCLABs Fund (D.G., S.G., J.X., U.M.), from the National Science Foundation through Grant No. DMR-13-10687 (RZ) and the UCR Chancellor's Strategic Fund and Collaborative Research Seed Grant (D.G., U.M., R.Z., S.G, I-C.H, A.L.N. R).

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Authors and Affiliations

  1. Department of Physics & Astronomy, University of California, Riverside, CA, 92521, USA

    Dong Gui, Sharad Gupta, Jun Xu, Roya Zandi & Umar Mohideen

  2. Department of Cell Biology & Neuroscience, University of California, Riverside, CA, USA

    Sarjeet Gill & I-Chueh Huang

  3. Department of Plant Pathology & Microbiology, University of California, Riverside, CA, USA

    A.L.N. Rao

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  1. Dong Gui
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Correspondence to Umar Mohideen.

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Gui, D., Gupta, S., Xu, J. et al. A novel minimal in vitro system for analyzing HIV-1 Gag-mediated budding. J Biol Phys 41, 135–149 (2015). https://doi.org/10.1007/s10867-014-9370-z

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