HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes

Abstract

Membrane binding of the HIV-1 group-specific antigen (Gag) structural protein, a critical step in viral assembly at the plasma membrane, is mediated by the myristoylated, highly basic matrix (MA) domain, which interacts with negatively charged lipids in the inner leaflet. According to a popular model, virus particles bud from membrane rafts, microdomains enriched in cholesterol and high-melting phospholipids with higher order than found outside rafts. How Gag might recognize membrane rafts, if they exist in the inner leaflet, is unknown. Using a liposome flotation assay with proteins translated in vitro, we investigated whether Gag can sense the composition of the hydrophobic part of the bilayer, by fixing lipid head group composition and varying hydrophobic properties. In liposomes composed solely of phosphatidylserine and phosphatidylcholine, and with the same overall membrane negative charge, Gag strongly preferred lipids with both acyl chains unsaturated over those with only one chain unsaturated. Adding cholesterol increased Gag binding and led to closer packing of phospholipids. However, higher membrane order, as measured by electron spin resonance, was not correlated with increased Gag binding. Gag proteins from two other retroviruses gave similar results. These liposome binding preferences were qualitatively recapitulated by purified myristoylated HIV-1 MA. Phosphatidylinositol 4,5-bisphosphate and cholesterol enhanced binding in an additive manner. Taken together, these results show that Gag is sensitive both to the acyl chains of phosphatidylserine and to cholesterol concentration and other details of the membrane environment. These observations may help explain how retroviruses acquire a raft-like lipid composition.

Fig. 1.

Acyl chain saturation influences PS-driven HIV-1 Gag liposome binding. [³⁵S]methionine-labeled HIV-1 Gag synthesized in a reticulocyte extract was incubated with liposomes composed of one of the following three mixtures of two phospholipids: DOPC and DOPS; POPC and POPS; or egg PC and brain PS. [The oleoyl acyl chain has one double bond (18:1), whereas the palmitoyl acyl chain has no double bonds (16:0)]. The percentage of Gag that floated with the liposomes in a sucrose gradient, as determined by SDS/PAGE and fluorography, was plotted as a function of increasing concentration of the negatively charged lipid PS. Error bars indicate SD for replicas done at 40% and 80% PS for all three lipid compositions and at 60% and 70% PS for the natural phospholipids.

Fig. 2.

Cholesterol concentration influences HIV-1 Gag binding to liposomes. Liposomes were made with a fixed 30% PS and varying ratios of cholesterol and DOPC or POPC (and in one case also containing 5% DSPC, 18:0, 18:0). HIV-1 Gag synthesis and flotation analyses were as described in Fig. 1. (A) Examples of lipid ratios for each of the four lipid compositions used and the corresponding fluorograms. MB, membrane bound (floated liposomes); NMB, nonmembrane bound (one-fourth of the sample loaded compared with MB). (B) Quantification of flotation reactions. The symbols are the same as those in A. Error bars represent SD for the three, three-component mixtures repeated in triplicate at 9% and 36% cholesterol, for DOPC/DOPS/0% cholesterol, and for egg PC/brain PS/45% cholesterol. (C) ESR measurements for each of the four liposome compositions from 0–63% cholesterol. S, order parameter.

Fig. 3.

Membrane order has complex influence on HIV-1 Gag–liposome binding at fixed PS and cholesterol concentrations. HIV-1 Gag and flotation analyses were as described in Fig. 1. (A and B) Phase diagrams situate the DOPC and POPC mixtures tested relative to the Ld + Lo phase coexistence region. (A) The leftmost triangle crossing the low-Tm lipid axis represents a composition of low-Tm lipids DOPC/DOPS/cholesterol (40%/20%/40%). Each triangle moving sequentially to the right has an additional 5.7% DOPC replaced with 5.7% of the high-Tm lipid DSPC. The rightmost X symbol represents a composition of high-Tm lipids DSPC/DPPS/cholesterol (40%/20%/40%). Each X moving sequentially to the left has 5.7% DSPC replaced by 5.7% of the low-Tm DOPC. Note that the boundaries of the four-component phase diagrams containing PS have not been determined, but they are shown here based on the published three-component phase diagrams (19) to provide a guide to the expected phase behavior of the four-component PS-containing mixtures. (B) As in A, except with POPC lipids instead of DOPC lipids. The left starting point for the low-Tm lipids POPC/POPS/cholesterol (38%/30%/32%) is marked by a circle, with each sequential point having 5.4% POPC replaced by 5.4% of the high-Tm lipid DSPC. The right starting point for the high-Tm lipids DSPC/DPPS/cholesterol (38%/30%/32%) is marked by a dash, with each sequential point having 5.4% DSPC replaced with 5.4% of the low-Tm lipid POPC. All compositions yield a single phase (i.e., not coexistence of the two phases, Ld + Lo). Lipid order increases from the left to right (Ld to Lo) as the percentage of high-Tm lipid increases. (C and E) ESR and Gag binding measurements for the DOPC-containing liposomes. (D and F) ESR and Gag binding measurements for the POPC-containing liposomes. The symbols in C and D are the same as those in A and B. Error bars represent SD for at least three replicas for each data point.

Fig. 4.

Acyl chain type and presence of cholesterol influence liposome binding of other retroviral Gag proteins. The three Gag proteins were expressed in a reticulocyte lysate and analyzed by flotation as in Fig. 1. RSV, Rous sarcoma virus (alpharetrovirus genus); MLV, murine leukemia virus (gamma retrovirus genus). The lipid compositions were as follows: 0% = 70% egg PC/30% brain PS; 36% = 34% egg PC/30% brain PS/36% cholesterol (white bars); Ld = 10% DSPC/30% POPS/50% POPC/10% cholesterol; Lo = 22% DSPC/30% DPPS/23% POPC/25% cholesterol. The histograms represent at least three replicas; error bars show SD. Order parameter (S), as determined via ESR, is 0.156 for the Ld composition and 0.332 for the Lo composition.

Fig. 5.

Acyl chain type and presence of cholesterol influence liposome binding of purified HIV-1 MA. Myristoylated MA was purified from an E. coli expression system. Fifteen micrograms of protein was mixed with 50 μg of liposomes containing either 0% cholesterol or 36% cholesterol, or with an Ld or Lo composition, both as defined in Fig. 4. Binding was measured after flotation by SDS/PAGE and Coomassie blue staining and densitometry. (A) Representative flotation result showing the stained MA band. (B) Quantitation. For all these lipid compositions, the concentration of PS was constant (30%). Bars represent the average of no fewer than three replicas. Error bars represent SD.

Fig. 6.

Cholesterol enhancement of HIV-1 Gag binding to membranes containing PI(4,5)P2. Gag protein was synthesized and analyzed by flotation as in Fig. 1. White bars, binding to liposomes containing 0% or 36% cholesterol (data taken from Fig. 4); gray bars, binding to liposomes with 2% PC being substituted with 2% PI(4,5)P2. The data represent the average of no fewer than three flotations, with error bars representing the SDs.