Abstract
The cholesterol and phospholipid composition of the membrane of vesicular stomatitis (VS) virus was altered by growth in a sterol auxotroph Chinese hamster ovary (CHO MI) host cell and by infection of CHO MI and baby hamster kidney (BHK)-21 cells supplemented with fatty acids and dimethylethanolamine. VS virus released from infected CHO MI sterol auxotroph cells grown in delipidated serum had a 50% lower ratio of cholesterol to phospholipid and an 80% drop in infectivity measured by plaque formation on L-929 cells compared with VS virus released from infected CHO MI cells grown in fetal calf serum. When VS virus was harvested from infected BHK-21 cells fed the choline analogue dimethylethanolamine, 29% of the membrane phospholipids were phosphatidyldimethylethanolamine (PDME); 87% of the PDME was located in the external monolayer of the virus membrane as determined by phospholipase C hydrolysis. Exogenous fatty acids added to the medium of cells infected with VS virus comprised up to 30% of the fatty acyl chains of the viral glycerophospholipids. The presence of PDME or unusual fatty acyl chains in the viral membrane had no effect on viral infectivity. These data indicate that the lipid composition of the VS virus membrane is determined primarily by the lipids available in the host cell and that only cholesterol content affects the biological activity of the virus membrane.
Highlights
Ovary (CHO MI) host cell and by infection of CHO MI vesicular stomatitis (VS) virus obtains its membrane by budding from the plasma and baby hamsterkidney (BHK)-%lcells supplemented membrane of infected cells (6)
VS virus pholipids are distributed asymmetrically with the majority of released from infected CHO MI sterol auxotroph cells the aminophospholipids in the internal monolayer and the grown in delipidated serum had a 50% lower ratio of cholesterol to phospholipidand an 80%dropin infectivity measured by plaque formation on L-929 cells compared with VS virus released from infected CHO MI cells grown in fetal calf serum.When VS virus was harvested from infected BHK-21 cells fed the choline analogue dimethylethanolamine,29%of the membrane bulk of the choline-containing phospholipids in the external monolayer ( 7 ) .The membrane is enriched in phosphatidylethanolamine and sphingomyelin and has a higher cholesterol/ phospholipid ratio than does the plasma membrane of the host BHK-21 cells (7)
(8).In such studies thleoss of infectivity could not be ascribed solely to thecholesterol depletion; this experiment was complicated by the loss of infectivity in the nondepleted control virus due to residual adherentvesicles (8).The in uiuo alteration of VS viral cholesterol content by the use of the CHO MI cell sterol auxotroph provided a more biological means of measuring the effecotf cholesterol content onviral infectivity stomatitis virus grown in BHK-PI, CHO wild type, and CHO M I cells with altered lipid compositions
Summary
Cell Culture Media-F-12 medium and Dulhecco's modified Eagle medium were obtained from GIBCO (Grand Island, NY) and fetal calf S ~ ~ IfrIoIm Flow Laboratories (McLean,VA). For the fatty acid effect this would have on the characteristic lipid compositon supplementation experiments, the CHO MI cells and the BHK-21 cells were passed directly into media containing 10%salt-fractionated serum and 20 pg/ml of either oleic, linoleic, or linolenic acid (Applied Science) and the cells allowed to grow to confluence before VS virus infection. The virus was harvested from the tissue culture media 18to 21 h postinfection and purified by differential, rate zonal, and equilibrium from infected BHK-21 cells grown in medium supplemented with 5 mM dimethylethanolamine (Table I). The phospholipids of the VS virus grown on the base-altered BHK21 cells were separated by thin layer chromatography on 250-pm Silica Gel G plates (Analtech,Newark, DE) using a one-dimensional, one-solvent system of chloroform/methanol/30% ammonia (65:25:5; v/v/v). Aliquots of the VS virus pholipids extracted and separated by thin layer chromatography as described under “Experimental Procedures.” The accessibility of the phospholipids of the base-supplemented VS virus to phospholipase C was determined after incubation of the virus with 0.75 unit/& of enzyme for h at 37°C (see “Experimental Procedures” and Fia. 1)
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