Site-specific Treated with Maturation of Enveloped Cytochalasin B Viruses in L Cells

Treatment of infected L cells with 10 /~g/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites. The cytochalasins have a profound effect on the surface morphology of a wide variety of cultured mammalian cells. Within minutes after sensitive cells are treated with effective doses ofcytochalasin B (CB) ~ or cytochalasin D, the cytoplasm contracts and blebs (also called blisters, zeiotic knobs, or knobby protuberances) appear on the cell surface (l, 6, 8, 1416, 22, 23). Over the next 30-120 min the blebs aggregate to form clusters (also called rosettes, crowns, or bouquets) on the upper surface of substrate-adherent cells or at one pole in the case of suspension cells (14, 17). Adherent cells may react further by acquiring an "arborized" (20, 21, 26) or macrovacuolated (2, 3) morphology after 12-48 h in the presence of the drug. The cellular response to the cytochalasins is temperature-dependent (14), and it requires an input of metabolic energy (16, 22). Colchicine does not affect the cytoplasmic contraction or bleb formation promoted by the cytochalasins, but it blocks aggregation of blebs into clusters (17, 27). All morphologic effects of the cytochalasins are reversible when the drug is withdrawn from the cell growth medium (22, 26). Sundquist and his colleagues (27, 28) have demonstrated that CB-induced changes in cell surface morphology are accompanied by an equally dramatic relocalization of certain cell surface proteins. The case of surface ~2-microglobulin in Abbreviations used in this paper: CB, cytochalasin B; HA, hemagglutinin; MDCK, Madin-Darby canine kidney; VSV, vesicular stomatitis virus. Lu 106 (transformed human lung) cells is illustrative. Whereas B2-microglobulin is uniformly distributed on the surface of untreated cells, it is relocalized into a "cap" at the site of clustered blebs in cells treated with 10 #g/ml CB. The CBinduced redistribution of/32-microglobulin is reversible and it is inhibited by colchicine. CB is found to promote a similar relocalization of H-2 antigens in transformed 3T3 cells (28), /32-microglobulin in HeLa cells, and measles virus hemaglutinin in persistently infected Lu 106 cells (27). The studies described below have focused on the effect of CB on the surface distribution of viral glycoproteins in infected cells. The project was undertaken to test the idea that if CB were to restrict the distribution of viral glycoproteins to particular areas of the infected cell membrane (as it does in the case of the measles virus hemagglutinin), then virus particles should mature only from those glycoprotein-containing sites. Viruses should bud preferentially from membrane regions containing their specific envelope glycoproteins. Our experimental tests involved infecting L-929 cells with vesicular stomatitis virus (VSV) or with influenza virus and treating the infected cells with CB. Cells were then examined for the surface distribution of virus-specific glycoproteins and for the sites of virus maturation. MATERIALS AND METHODS Cell and Virus Growth: L-929 (mouse) cells were propagated in suspension at 37°C in Eagle's minimal essential medium modified for suspenTHE JOURNAL OF CELL BIOLOGY • VOLUME 100 FEBRUARY 1985 357-363 © The Rockefeller University Press • 0021-9525/85/02/357107 $1.00 35 7 on N ovem er 6, 2017 jcb.rress. org D ow nladed fom sion cultures (Flow Laboratories, Inc., Rockville, MD) containing 3% fetal calf serum and antibiotics as previously described (12). Experiments involving adherent cells were carried out with subconfluent monolayer cultures prepared by allowing suspension cells to attach overnight to glass or plastic surfaces in Dulbecco's modified minimal essential medium or in Eagle's basal medium containing 3% fetal calf serum and antibiotics. The cell reaction to CB was comparable on glass and on plastic surfaces, and it did not depend on the identity of the medium employed. CB (Sigma Chemical Co., St. Louis, MO) was added to cell cultures from a 1 mg/ml stock solution prepared in ethanol. Solvent in the absence of drug did not affect cell morphology or glycoprotein localization. Stock cultures of VSV-lndiana (Mudd-Summers strain) were grown on monolayer cultures of baby hamster kidney-21 cells as previously described (11). L cells were infected at a multiplicity of infection of 2 in cell growth medium (containing 1.5% rather than 3% fetal calf serum). The virus was allowed to attach to cells for 1 h at room temperature, and infection was continued for a total of 6-10 h at 37*. Cultures showing marked cytopathologic effects (such as cell rounding or detachment from the substrate) were not employed for cytochalasin experiments. The WSN strain of influenza A (the gift of Dr. W. Young and Dr. G. Nichols, University of Virginia) was grown on Madin-Darby canine kidney (MDCK) cells and employed to infect L cells as described above for VSV, Light and Electron Microscopy: L-cell monolayerswere prepared for phase-contrast microscopy by growing them in 25 cm 2 plastic tissue culture flasks. Flasks of cells were drug-treated as described in the text and photographed at various intervals, in a Leitz Diavert inverted, phase-contrast tissue culture microscope (E. Leitz, Inc., Rockleigh, N J), which was maintained in a warm (37*) environment. Scanning electron microscopy was carried out with L-cell monolayers prepared and drug-treated on round (2.2 cm in diameter) glass coverslips. Cells were fixed with 2% glutaraldehyde, critical point-dried, and coated with gold as described previously (4). Photographs were taken on a JEOL 70T scanning electron microscope (JEOL USA, Electron Optics Div., Peabody, MA) operated at 25 kV. Transmission electron microscopy was carried out with monolayer cultures of L cells prepared in glass petri dishes of l0 cm diam. After experimental operations (such as virus infection and drug treatment) were complete, cells were fixed immediately for l0 min at room temperature in PBS (Dulbecco's phosphate-buffered saline) containing 2% glutaraldehyde and then removed from the dish by scraping. Thereafter, cells were centrifuged (at 1,000 g for l rain) into a hard pellet, fixed overnight at 4* in 2% glutaraldehyde-PBS, postfixed in 1% OsO4-PBS for 1-2 h at room temperature and stained en bloc for l h with 1% uranyl acetate. Samples were then dehydrated, embedded in plastic (Poly/Bed 812: Polysciences, Inc., Warrington, PA), sectioned, examined in a JEOL 100cx electron microscope, and photographed at a magnification of