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Envelope and Host Cell Plasma Membranes (H9 Plasma Membranes) ROLAND C Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5181-5185, June 1993 Medical Sciences Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes (H9 plasma membranes) ROLAND C. ALOIA*tt§, HuIROu TIANt, AND FRED C. JENSEN1 *Anesthesia Service, J. L. Pettis Veterans Administration Hospital, Loma Linda, CA 92357; Departments of tAnesthesiology and tBiochemistry, Loma Linda University School of Medicine, Loma Linda, CA 92350; and lImmuneResponse Corporation, Carlsbad, CA 92008 Communicated by Robert C. Gallo, January 4, 1993 (receivedfor review August 11, 1992) ABSTRACT Previous studies have indicated that human IIIRF, Sph is 11-13 mol % (6). It is presently unclear what immunodeficiency virus (HIV) is enclosed with a lipid envelope physical mechanism(s) account for these lipid variations. similar in composition to cell plasma membranes and to other They could reflect variations in cell lines and growth condi- viruses. Further, the fluidity, as measured by spin resonance tions or genetic differences in HIV isolates, in which enve- spectroscopy, is low and the viral envelope is among the most lope or other proteins select different lipids from the bulk highly ordered membranes analyzed. However, the relation- lipid pool within the cell surface membrane during the ship between viral envelope lipids and those of the host cell is budding process (5). Lipid envelopes ofother viruses possess not known. Here we demonstrate that the phospholipids within a lipid profile different from their host surface membranes, as the envelopes ofHIV-1RF and HIV-2-L are similar to each other has been shown for Semliki Forest virus (12), simian virus 5 but significantly different from their respective host cell surface (13, 14), respiratory syncytial virus (15), Sindbis virus (16), membranes. Further, we demonstrate that the cholesterol-to- and vesicular stomatitis virus (17). Viral budding through phospholipid molar ratio ofthe viral envelope is approximately specific regions of the cell membrane has been suggested for 2.5 times that of the host cell surface membranes. Consistent some of these enveloped viruses (see refs. 9, 18, and 44 for with the elevated cholesterol-to-phospholipid molar ratio, the reviews). viral envelopes of HIV-1RF and HIV-2-L were shown to be Here we present evidence that HIV types 1 and 2 (HIV-1RF 7.5% and 10.5% more ordered than the plasma membranes of and HIV-2-L) exhibit a phospholipid profile and fluidity their respective host cells. These data demonstrate that HIV-1 significantly different from those ofthe plasma membranes of and HIV-2-L select specific lipid domains within the surface the host cells in which they are grown. Such differences are membrane of their host cells through which to emerge during strongly suggestive of a selective sequestration of lipids viral maturation. occurring during the budding process, in which the viral proteins select specific domains within the host cell mem- Electron microscopic studies (1-3) have demonstrated that brane through which to emerge during maturation. Further, the human immunodeficiency virus (HIV) is surrounded by a we provide data on the C/P ratios of six additional laboratory lipid envelope derived from the host cell plasma membrane. and clinical HIV and simian immunodeficiency virus (SIV) The initial report of the lipid composition of the HIV dem- isolates. onstrated that the envelope phospholipids were similar to those of other enveloped viruses and the erythrocyte plasma membrane (4, 5). The demonstration of high levels of phos- MATERIALS AND METHODS pholipids normally found in cell surface membranes [sphin- Materials. Spin probes were purchased from Molecular gomyelin (Sph), phosphatidylethanolamine (PE), phosphati- Probes and Aldrich and checked with two-dimensional chro- dylcholine (PC), and phosphatidylserine (PS)] supported the matography for purity (19). All solvents were HPLC grade electron microscopic observations depicting the enclosure of from Fisher. the viral capsid with a lipid envelope derived from the host HIV Isolates. HIV-1RF was purchased from John Dahlberg cell plasma membrane during the budding process (4, 5). (formerly at Universal Biotechnology; Rockville, MD) and Further, these studies (4, 5) and others (6) indicated that HIV HIV-2-L was from James Whitman, Jr. (Advanced Biotech- had a low lipid-to-protein (weight) ratio and a high choles- nologies; Columbia, MD), along with H9 cells, from which terol-to-phospholipid molar ratio (C/P ratio). the viral isolates were grown. Essentially 15 liters of infected Electron spin resonance (ESR) analyses of HIV have H9 cells (1 x 106 per ml) were clarified with a JCF-Z indicated that the flexibility of an incorporated spin-labeled (Beckman) rotor in continuous flow centrifugation. The cell probe molecule (a 5-nitroxide derivative of stearic acid; pack was washed and banded between 1.12 and 1.18 g/ml on 5-NS) is restricted and that the viral envelope is among the a 5-45% sucrose density gradient, washed again with phos- most "ordered" membranes analyzed (5, 7, 8). Order pa- phate-buffered saline (PBS), and resuspended in 1.5 ml for rameters, which were calculated to be >0.7 (when spectral analysis. Purity was checked by viral particle count, PAGE/ recordings were made at 37°C), were comparable to those of Western blot analysis, and p24 and protein concentration, other enveloped viral systems exhibiting high order param- and it was determined to be greater than 92.5%. Viral isolates eters (>0.7) and C/P ratios >1.0 (5, 9-11). were stored frozen at -70°C and shipped on dry ice. Infected Although all HIV isolates examined to date exhibit high C/P ratios and elevated order parameters, differences in Abbreviations: HIV, human immunodeficiency virus; SIV, simian phospholipid classes have been reported. For example, in immunodeficiency virus; 5-NS, the N-oxy-4',4'-dimethyloxazolidine HIV isolates LK013, F7529, and 4105 (5) and HZ321 (9) Sph derivative of 5-ketostearate; C/P ratio, cholesterol-to-phospholipid is 25-28 mol %, whereas in isolates HTLV-IIIB and HTLV- molar ratio; Sph, sphingomyelin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidyl- inositol; PA, phosphatidic acid. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at Anesthesia payment. This article must therefore be hereby marked "advertisement" Service, 112A, J.L. Pettis Veterans Hospital, 11201 Benton Street, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Loma Linda, CA 92357. 5181 Downloaded by guest on September 24, 2021 5182 Medical Sciences: Aloia et al. Proc. Natl. Acad. Sci. USA 90 (1993) and uninfected H9 cells were grown under identical condi- mixer (South Shore Scientific, Santa Ana, CA), in a com- tions and washed three times in PBS before being frozen as pletely enclosed glass vessel sealed with a Teflon washer and a pellet and shipped on dry ice. a stainless steel cap. The extracting solvent was chloroform/ Plasma Membrane Isolation. All manipulations of cells methanol (2:1, vol/vol), 5% (vol/vol) freshly prepared satu- during plasma membrane isolation were performed within a rated (28% wt/wt) ammonia, and 0.05% butylated hydroxy- sterile hood of a biosafety level 3 (P3) facility. H9 cells were toluene (5). A rotor blade projecting through the cap of the homogenized by using a Kirkland Emulsiflex device (Aves- Omni-mixer homogenized the samples without aerosol dis- tin, Ottawa), which is completely enclosed to permit the persion in an atmosphere flushed with argon gas. The lipid isolation of membrane fractions with a minimum of aerosol extracts were removed from the P3 facility, concentrated by formation. Twenty milliliters of a homogenizing buffer (10 low-pressure, low-temperature, rotary evaporation, and pu- mM Hepes/5 mM MgC92/1 mM EDTA/0.1 mM phenylmeth- rified of nonlipid contaminants on a Sephadex G-25 column ylsufonyl fluoride/i mM dithiothreitol/10 mM KCl, pH 7.6) in a solvent ofchloroform/methanol (19:1, vol/vol) saturated was added to a packed pellet of 2-5 x 109 frozen cells and the with water (5, 26, 27). The column eluate was concentrated mixture was allowed to thaw briefly at 37°C. The cells were and chromatographed, and lipid spots were identified from disrupted by the piston action (5-cm stroke distance; 1 cycle standards run under identical conditions (see Fig. 1 for per sec) of the Emulsiflex device, forcing the suspension solvent composition). Lipid phosphorus was quantified as through a 5-mm orifice apposed by a plunger of slightly described previously (5, 27, 28). Cholesterol was assayed smaller diameter. with the Boehringer Mannheim Diagnostics High Perfor- After cell disruption, plasma membranes were isolated by mance-K kit (catalog no. 692905) (27). Protein was deter- a modified sucrose gradient procedure (20-23). The cell mined by the Bradford assay (29) according to Bio-Rad. homogenate was transferred from the syringe receptacle of HIV Spin-Labeling and Spectral Recording. HIV-1RF and the Emulsiflex to a 50-ml tube, placed in a JA-20 rotor within HIV-2-L (7-9 mg/ml) and plasma membranes (14-16 mg/ml) a sterile hood ofthe P3 facility, and spun at 400 x g (Beckman were spin-labeled as described (5, 7, 8) within the P3 facility. JA21 centrifuge). The supernatant was decanted into a 28-ml Basically, 20 ,ul of sample was added to the 5-NS spin label tube, which was bottom loaded with 22.5%, 32%, 37%, and (N-oxy-4',4'-dimethyloxazolidine derivative of 5-ketostear- 45% sucrose and centrifuged for 3 hr at 100,000 x g with a ate) previously dried at the bottom of a microcentrifuge tube, Beckman L4 ultracentrifuge (within the P3 facility). The and after mixing, the sample was drawn into a 50-,ul capillary plasma membrane fraction (22.5%/32% interface) was di- and the ends were heat sealed.
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