Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.HIV-1³ is an enveloped retrovirus, which acquires its lipid envelope by budding from the plasma membrane of the infected host cell. Several reports have shown that the viral membrane is enriched in sphingomyelin (SM), including the unusual sphingolipid dihydrosphingomyelin (DHSM) and collectively referred to as sphingomyelins (SMs), glycosphingolipids, cholesterol (CHOL), saturated phosphoglycerolipids and phosphoinositides (1–4). Moreover the CHOL/phospholipid and protein/lipid ratios of the HIV-1 membrane are high, corresponding to a highly ordered membrane, and are presumed to be different from the overall host cell plasma membrane. Accordingly, the HIV-1 envelope has been considered to be a large raft-like membrane microdomain (3). This is in line with previous reports describing enrichment of raft markers in the HIV-1 membrane and its sensitivity to CHOL-depleting agents (5–9). Furthermore, HIV-1 glycoproteins have been suggested to localize within membrane rafts due to palmitoylation of two cysteines (10), and the main structural Gag protein has been shown to rapidly relocalize to detergent-resistant membranes after initial membrane binding (6).Membrane microdomains are dynamic assemblies resulting from the lateral interaction of lipids and proteins. Two phases coexist in the plasma membrane: the liquid-ordered phase (l_o), mainly composed of CHOL and sphingolipids (SPLs), and the liquid disordered phase (l_d), mainly composed of glycerophospholipids (11–13). In the activated state, l_o microdomains can coalesce and serve as platforms for membrane trafficking, signaling, and virus budding (14, 15). The first method to biochemically enrich membrane rafts was the purification of detergent-resistant membranes, based on their resistance to extraction with non-ionic detergent at 4 °C (16). However, this and other methods based on antibody or cholera toxin binding may lead to artificial aggregation of membrane microdomains and thus do not necessarily represent their native state (17, 18). For these reasons and because the association and dissociation of membrane microdomains appears to occur on a rapid time-scale and the raft size is too small to be optically resolved, the raft concept remains controversial. However, the determination of the HIV-1 lipidome, a native membrane purified without any detergent, has provided strong evidence for the existence of these microdomains (3).Fluorescent lipid analogs that partition preferentially into a specialized lipid phase could be an attractive tool to study membrane microdomains. However, partitioning of such dyes mainly depends on the local chemical environment and not on the phase state of the membrane (19–21). In contrast, Laurdan (6-dodecanoyl-2-dimethylaminonapthalene) is a lipophilic dye that binds to membranes independent of their phase state but reports the phase state by a change in its fluorescence emission (20). Laurdan exhibits a blue shift in its emission spectrum with increasing membrane condensation. This is caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. Thus, excitation of membrane bound Laurdan leads to two emission maxima representing differences in membrane lateral structure. Quantification of membrane order is achieved by computing the Generalized Polarization (GP) value, which is defined as normalized intensity ratio of the two emission channels. GP values range from +1 (most condensed) to −1 (most fluid). They are not biased by probe concentration, membrane ruffles, and surface modifications, such as lipoprotein binding. Furthermore, there is no preferential interaction with a specific lipid, fatty acid, or head group (20, 21). GP value correspondence to different lipid phases was estimated using liposomes with a composition similar to that of cellular membranes (22, 23). Using an equimolar mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine, CHOL, and SM as an l_o membrane, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as an l_d and solid ordered (s_o) phase, GP values below +0.25 were shown to correspond to the l_d phase, GP values between +0.25 and +0.5 to the l_o phase, and GP values above +0.5 to the s_o phase (22, 23).Laurdan has been extensively used to characterize domain formation and lateral lipid segregation in model membranes composed of different phospholipid mixtures or lipids extracted from cellular membranes (19, 22–25). It has also been used to study the membrane structure in living cells. Gaus and coworkers observed l_o domains enriched on membrane protrusions (filopodia), adhesion points, and cell-cell contacts (26). They also used Laurdan to address the physical properties of the plasma membrane around the T-cell receptor in activated T cells, observing larger and more stably ordered membrane domains at sites of T-cell activation (27). Quantitative determination of cellular plasma membrane order by fluorescence spectroscopy is complicated due to the rapid internalization and redistribution of the probes to other cellular membranes, making it difficult to interpret the fluorescence measurements over the whole cell. This problem is not encountered in purified virus particles, because they contain only a single membrane. We therefore developed an assay to study viral membrane lateral structure by fluorescence spectroscopy. For this purpose, isogenic HIV-1 particles were produced in two different cell lines, and their GP profiles were determined. In parallel, the lipid constituents were quantified by mass spectrometry. The viral membrane displayed a l_o structure in both cases, but this was more prominent for the virus derived from the T-cell line MT-4 compared with virus derived from 293T cells. The validity of this result was supported by comparing the lipidome of the two viruses, which revealed a significantly higher SMs/phosphatidylcholine (PC) ratio for the MT-4-derived virus. Accordingly, treatment with SM-binding toxins inactivated MT-4-derived virus more efficiently than 293T-derived virus, whereas both viruses exhibited similar infectivities before treatment. The reported approach allows rapid determination of differences in viral membrane order, permitting screening for compounds that perturb l_o domains, which may act as antivirals of a novel type.