HIV-1 Gag Polyprotein Affinity to the Lipid Membrane Is Independent of Its Surface Charge

Abstract

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

Keywords: Gag polyprotein; bilayer lipid membrane; binding constant; boundary potential; cholesterol; human immunodeficiency virus (HIV); inner field compensation method; membrane charge; surface potential; ζ-potential.

Figure 1

Determination of the charge density on the surface of the BLMs. (A) Example of changes of the Δφ_b at the BLM of mixture 2 as a result of the increase of electrolyte ionic strength by 1 M KCl addition to the one side of the membrane. (B) The growth of the Δφ_b at the BLM as a function of the KCl concentration. In the legend, ratios are given in mol%. (C) The surface potential at the BLM as a function of the KCl concentration. The approximation curves were plotted using Equations (1)–(4). Each point in the plots was obtained by averaging the results of 3–5 independent experiments. Error bars in the plots represent the standard deviation. If the error bars are smaller than the size of the circle in the plot, the error bars are not shown.

Figure 2

The difference in boundary potentials measured after addition of Gag to the one side of the membrane. BLMs were formed from the mixtures 1–6 (in the legend ratios are given in mol%). Each point in the plots was obtained by averaging the results of 3–5 independent experiments. Error bars in the plots represent the standard deviation. If the error bars are smaller than the size of the circle in the plot, the error bars are not shown.

Figure 3

Isotherms of the Gag adsorption onto the membranes. In the legend, the ratios are given in mol% for mixtures 1–6. Fitting curves were plotted using Equation (5) for mixtures 1–5 and Equation (6) for mixture 6. Each point in the plots was obtained by averaging the results of 3–5 independent experiments. Error bars in the plots represent the standard deviation. If the error bars are smaller than the size of the circle in the plot, the error bars are not shown.