Isolation of the envelope of vesicular stomatitis virus

S E Taube, L I Rothfield
1978 Journal of Virology  
Vesicular stomatitis virus was disrupted by a combination of freezing and thawing, osmotic shock, and sonic treatment. Subviral components were separated by isopycnic centrifugation. The low-density, lipid-rich fractions were pooled and shown to contain primarily viral glycoprotein. Further purification of this material resulted in the isolation of a preparation of vesicles which contained only the G protein and the same phospholipids as in the intact virions and exhibited spikelike structures
more » ... ikelike structures similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes. A series of events describing the maturation Virus. Our original stock of VSV Indiana (3Bof vesicular stomatitis virus (VSV) and other Glasgow) was a gift from Robert Wagner. A heatenveloped RNA viruses has been postulated resistant variant was selected for the ability to survive based on in vivo studies (5, 7, 15). The molecular heating for 10 min at 50°C and was cloned by three mechanisms involved in assembly have not been rounds of plaque isolation. This isolate was used to investigated directly, since the various stages prepare a large stock of virus in monolayers of BHKianvestigteddiearely, sin the varivo s. tes 21 cells and was stored in portions at -80°C. All cannvrot beclearlyutisolatedin theinvivo system.ha experiments were performed with virus grown directly In vitro reconstitution systems have not been from this seed stock. available, in part due to the inability to obtain Virus was prepared by infecting BHK-21 cell monointact viral envelopes. layers in Costar 150-cm2 plastic flasks with 3 ml of VSV has a nucleocapsid core which contains inoculum at a multiplicity of 0.1 to 1.0 plaque-forming RNA and the major nucleocapsid protein, N (6, units (PFU)/cell. After 30 min at 37°C, 20 ml of fresh 16). Two minor proteins, L and NS, are also MEM-C was added, and the infection was allowed to associated with the nucleocapsid (4). The enveproceed for an additional 18 to 20 h at 37°C. The lope of the viion appears to be a lipid bi.layer culture fluid was collected and cleared of cell debris ler fo th evro st be a lipid m biaye by low-speed centrifugation (1,000 x g) for 30 min at derived irom the host cell plasma membrane 40C (clarified virus). (12). A single glycoprotein species, G, is associ-Virus purification. Virus was maintained at 4°C ated with the envelope and appears as spikes on throughout the purification. Clarified virus was centhe virion (12, 15). The M or matrix protein is trifuged for 1 h at 30,000 rpm in a Spinco 6OTi rotor. believed to lie between the nucleocapsid core Pellets were resuspended in TSE buffer (0.05 M and the envelope (2, 15). Nucleocapsid cores Tris-0-15 M NaCl-0.5 mM EDTA, pH 7.7) and pelwith the ability to transcribe can be isolated (3), leted again in the same way. These pellets were resusbut until now there have been no procedures pended to 2% of the starting volume in TSE. The virus available for the isolation of intact viral enve-was sonically treated with a titanium microprobe on lopes. a Branson Sonifier (model W185) for two bursts of 15 lopes. p s at 30 to 40 W separated by a 30-s interval and was In this paper we report the successful isolation then applied to 5 to 40% linear sucrose gradients of VSV envelopes without the use of detergents. (weight/weight solutions of sucrose prepared in TSE buffer with 5 mM MgCl replacing the EDTA; TSM). MATERIALS ANI) METHODS Gradients were spun for 90 min at 21,000 rpm in an SW27 rotor. The single visible band of virus was Cells. BHK-21 cells and mouse L cells (clone 929) collected in a syringe by puncturing the side of the were obtained from the American Type Culture Coltube. The virus was diluted with TSM and sedimented lection (ATCC). Monolayer cultures were maintained in an SW27 rotor at 25,000 rpm for 90 min. The final in complete medium (MEM-C), which consisted of pellet was resuspended in TSM, sonically treated as Eagle minimal essential medium (MEM) suppledescribed above, and stored at -800C. mented with 7% calf serum and antibiotics (100 U of Virus assay. Plaque assays were performed on Lpenicillin per ml and 100 itg of streptomycin per ml). cell monolayers prepared by adding 1% 1.8 M CaCl2 to The mouse L cells were adapted to grow in suspen-Spinner cultures and plating approximately 5 x 10" sion and be maintained as Spinner cultures in comcells per 60-mm plastic petri dish. Monolayers were plete MEM suspension medium supplemented with 2 ready for use within 2 h after plating. The plaque mM glutamine. overlay medium consisted of MEM containing 1% 730 on May 9, 2020 by guest
doi:10.1128/jvi.26.3.730-735.1978 fatcat:mft5vzcptrgchcjctjmwaebmxu