Development of Tools to Study Retroviral Gag Assembly on Giant Unilamellar Vesicles(GUV)

Abstract

The retroviral Gag polyprotein provides the principal driving force for virus assembly and budding from the cellular plasma membrane. The binding of Gag to the plasma membrane (PM) is governed by several mechanisms, including electrostatics, hydrophobics, Gag multimierization, and recognition of specific lipid head groups. To better understand how Gag interacts with the PM, control proteins GFP-poly(K/R)n (n=4/8/12 residues), were purified and used to compare Gag membrane binding to an electrostatic membrane binding protein. Preliminary protein-liposome binding experiments suggest that the anionic lipids, PS and PI(4,5)P2, contribute to the recruitment of polycationic proteins as expected. Unlike previous results, which showed that HIV-1 Gag responded strongly to cholesterol, GFP-poly(K/R)n responded weakly to cholesterol. PI(4,5)P2 enhances more liposome binding for retroviral Gag than for the control protein GFP-poly(K/R)n. This study may shed light on how the retroviral Gag protein interacts electrostatically with membranes, and recognizes specific lipids such as PI(4,5)P2.Membrane binding is not a prerequisite for Gag multimerization, however, it might enhance the formation of Gag-Gag interactions. It is known that assembly of Gag occurs on the PM, but it remains unclear when and where Gag multimerization takes place, and if membrane lipid composition influences Gag assembly. To visualize by fluorescence microscopy the interaction of Gag with membranes and to detect Gag-Gag interactions, Rous Sarcoma Virus Gag is labeled with different Alexa-flurophores using Sfp synthase. Giant unilamellar vesicles (GUVs), composed of lipids similar to cellular inner leaflet lipid composition, were employed to study Gag assembly by measuring fluorescence resonance energy transfer (FRET). This study may provide a starting point for understanding where Gag multimerization happens as well as how membrane composition affects Gag assembly.