Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Abstract

The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCERetroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.T he retroviral structural protein Gag provides the primary driving force for virus assembly at the inner leaflet of the plasma membrane (PM), and Gag alone is sufficient to assemble into virus-like particles (VLPs) in cells (1) and also in vitro (2). Gag is synthesized in the cytosol and then is targeted to the PM by the N-terminal domain, MA. How Gag interacts with the inner leaflet of the PM and how the biophysical properties of the PM...

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

“…In particular, we prepared (i) a "DSPS sample" (DSPS/DSPC/ DOPC/cholesterol at a 15:30:45:10 molar ratio), in which PS was concentrated in small ordered domains, and (ii) a "DOPS sample" (DOPS/DSPC/DOPC/cholesterol at a 15:30: 45:10 molar ratio), in which PS was distributed all over the liquid disordered matrix. A rough estimate based on the phase diagram published by Wen et al (43) suggested that the DOPS concentration in the disordered matrix of the DOPS sample was circa 10 times higher than that in the ordered phase. Similarly, the DSPS concentration in the small ordered domains of the DSPS sample was circa 5 times higher than that in the disordered phase.…”

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties

“…Protein was purified as previously described (8) and stored at -80 C until use. Preparation of 100 nm extruded liposomes and binding reactions were performed as previously described (25). Briefly, all binding reactions were performed with 15 mg (4.7 mM) protein and 50 mg (328À431 mM) lipid in 200 mL at 20 mM Tris-HCl pH 8, and adjusted with buffer to the stated NaCl concentration.…”

“…Although the effects of Chol on membrane thickness and lipid packing are well known (see (54)), the calculations of the previous sections demonstrate that bilayer structural perturbations induced by Chol constitute a general mechanism by which the molecule can indirectly mediate electrostatic interactions with proteins. To further explore retroviral MA-membrane association in the context of these interactions, we used liposome pelleting (25) to measure binding of RSV MA to LUVs with varying POPS and Chol concentrations, and at varying salt concentration. The data were analyzed in terms of calculated electrostatic potential above the membrane surface (Figs.…”

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

“…However, coarse grained dynamics studies [11] and new NMR experiments using full length acyl chain PIP 2 [40] showed the opposite. Moreover, recent experiments using either a multimerizable matrix domain of HIV-1 Gag [34] or RSV Gag [54] also exhibited contradictory results regarding their partitioning in lipid domains of giant unilamellar vesicles (GUVs). Therefore, the ability of the Gag/PIP 2 complex to partition preferentially into "raft" domains or more generally into PM pre-existing domains to enhance virus assembly is still a matter of controversy [36].…”