The structure of vesicular stomatitis virus membrane A phosphorus nuclear magnetic resonance approach

The proton decoupled 40.48 M Hz ³¹P NMR spectrum of intact and unperturbed membrane-enclosed vesicular stomatitis virus (serotype Indiana) exhibited two distinct maxima. These can be resolved into a narrow, symmetric line and a broad asymmetric line. The ³¹P NMR spectrum of a multilamellar (unsonicated) preparation of the extracted viral lipids exhibited a line shape similar to that of the intact virus. A sonicated vesicle preparation of the extracted viral lipids exhibited a narrow symmetric line. The narrow component in the intact virus spectrum may be attributed to small membrane fragments. Phospholipase C digestion of the intact virus resulted in substantial reduction in intensity of both components which suggests that much of the contribution to both peaks is due to phosphate in the phospholipid polar head groups.The phospholipid phosphates in both sonicated and unsonicated preparations of the extracted viral lipids exhibited substantially longer relaxation times than did those in the intact virus. The short relaxation time emanating from the intact virus preparation is caused by immobilization of the phospholipid head groups which could be due to lipid-protein interactions. Trypsin treatment of vesicular stomatitis virions, which results in complete removal of the exterior hydrophilic segment of the membrane glycoprotein, increased the ³¹P relaxation time to a value similar to that observed in the protein-free total lipid extracts; this finding provides supporting evidence for the role of virus glycoprotein in shortened relaxation times. A reversible temperature-dependent change in apparent line width and absence of an effect of cholesterol on the ³¹P phospholipid spectrum were also demonstrated.     

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.     

Isolation of the envelope of vesicular stomatitis virus.

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 similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes.     

Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid in the condensed form found in native virions.

Vesicular stomatitis virus was extracted with 60 mM octylglucoside in the absence of salts and in the presence of 0.5 M NaCl. The resulting extracted virus particles were examined by electron microscopy, and the proteins present were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extraction in the absence of salts yielded subviral structures which we cell "skeletons" as originally suggested by Cartwright et al. (J. Gen. Virol. 7:19-32, 1970). The skeletons contained the viral N, M, and L proteins, but they lacked the glycoprotein (G) entirely. Morphologically, the skeletons resembled intact vesicular stomatitis virus but they were slightly longer and smaller in diameter. Like native vesicular stomatitis virus, skeletons were found to have lateral striations spaced 5.0 to 6.0 nm apart along the length of the structure. In contrast to extraction in the absence of NaCl, extraction of vesicular stomatitis virus with 60 mM octylglucoside in the presence of 0.5 M NaCl yielded highly extended viral nucleocapsids in which N was the predominant protein; no M or G proteins could be detected. These results support the view that the M protein is involved in maintaining the nucleocapsid in the compact form found in native virions.     

Reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis.

The glycoprotein of vesicular stomatitis (VS) virus was selectively liberated from the virion membrane by the dialyzable nonionic detergent, beta-D-octylglucoside. The isolated viral glycoprotein could be rendered virtually free of phospholipid and detergent, under which conditions it formed tail-to-tail glycoprotein micelles in the form of rosettes. When mixtures of viral glycoprotein and egg lecithin were dialyzed free of octylglucoside, glycoprotein vesicles formed spontaneously with spikes protruding in the same external orientation as the VS virion membrane. The glycoprotein vesicles exhibited increased and uniform buoyant density, indicating relative homogeneity in the proportion of glycoprotein and phosphatidylcholine in each glycoprotein liposome. Evidence for similar insertion and orientation of VS viral glycoprotein in both phosphatidylcholine vesicles and virion membrane was substantiated by the finding that proteolytic digestion with thermolysin gave rise to hydrophobic glycoprotein tail fragments in vesicle or virion membranes that migrated identically in polyacrylamide gels.     

Permeability properties of the membrane of vesicular stomatitis virions.

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus.     

Mosquito cells infected with vesicular stomatitis virus yield unsialylated virions of low infectivity.

Vesicular stomatitis virus propagated in and released from Aedes albopictus cells had the normal complement of viral proteins; the glycoprotein contained carbohydrate but no sialic acid. These virions had markedly reduced hemagglutinating activity and exhibited a very high ratio of physical particles to infectious virus. In vitro sialylation of vesicular stomatitis virions grown in mosquito cells resulted in a 100-fold increase in both infectivity and hemagglutination titers to levels approaching those of virus grown in BHK-21 cells. These experiments provide an example of host-controlled modification of viral infectivity.     

Restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components.

Noninfectious spikeless particles have been obtained from vesicular stomatitis virus (VSV, Indiana serotype) by bromelain or Pronase treatment. They lack the viral glycoprotein (G) but contain all the other viral components (RNA, lipid, and other structural proteins). Triton-solubilized VSV-Indiana glycoprotein preparations, containing the viral G protein as well as lipids (including phospholipids), have been extracted from whole virus preparations, freed from the majority of the detergent, and used to restore infectivity to spikeless VSV. The infectivity of such particles has been found to be enhanced by poly-L-ornithine but inhibited by Trition or homologous antiserum pretreatment. Heat-denatured glycoprotein preparations were not effective in restoring the infectivity to spikeless VSV. Heterologous glycoprotein preparations from the serologically distinct VSV-New Jersey serotype were equally capable of making infectious entities with VSV-Indiana spikeless particles, and the infectivity of these structures was inhibited by VSV-New Jersey antiserum but not by VSV-Indiana antiserum. Purified, detergent-free glycoprotein selectively solubilized from VSV-Indiana by the dialyzable detergent, octylglucoside, also restored infectivity of spikeless virions of VSV-Indiana and VSV-New Jersey.     

Permeability properties of the membrane of vesicular stomatitis virions

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

The Glycoprotein of Vesicular Stomatitis Virus Is the Antigen That Gives Rise to and Reacts with Neutralizing Antibody

The glycoprotein, but no other virion protein, of vesicular stomatitis virus was solubilized by the nonionic detergent Triton X-100 in low ionic strength buffer. The solubilized viral glycoprotein induced the synthesis of antibody that formed a single precipitin line with the glycoprotein and that neutralized the infectivity of the virus. The neutralizing activity of the antibody was efficiently blocked by purified glycoprotein.     

Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes

Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis-unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification.     

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.     

Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.     

Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus.

Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.     

Growth of enveloped RNA viruses in a line of chinese hamster ovary cells with deficient N-acetylglucosaminyltransferase activity.

Sindbis and vesicular stomatitis viruses were grown in a line (termed 15B) of Chinese hamster ovary (CHO) cells that is deficient in a specific UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosaminyltransferase. Both viruses replicated normally in the cell line, but the glycoproteins of the released virus migrated faster on sodium didecyl sulfate-polyacrylamide gels than did glycoproteins of virus grown in parent CHO cells. Digestion of the viral glycoproteins with Pronase followed by gel filtration demonstrated that the glycoproteins with Pronase followed by gel filtration demonstrated that the glycopeptides of Sinbis-15B virus were much smaller than the glycopeptides of Sindbis-CHO virus. In addition, Sindbis-15B viral glycopeptides but not Sindbis-CHO viral glycopeptides contained terminal alpha-mannose residues as shown by their susceptibility to alpha-mannosidase digestion. These findings demonstrate that the oligosaccharide units of the glycoproteins of vesicular stomatitis and Sinbis viruses are altered when the viruses are grown in 15B cells. We conclude that the N-acetylglucosaminyltransferase that is missing in 15B cells normally participates in the biosynthesis of the oligosaccharide units of the viral glycoproteins, and in the absence of this enzyme incomplete oligosaccharide chanis are produced. Viruses released from 15B cells appear to retain full infectivity; Sindbis-15B virus, however, showed a significant decrease in hemagglutination titer compared with that of Sindbis-CHO virus.     

Specificity of initiation factor preparations from poliovirus-infected cells.

Crude initiation factor preparations from poliovirus-infected cells stimulated the translation of poliovirus RNA in vitro, but were inactive for the translation of host cell or vesicular stomatitis virus mRNA's. In contrast, similar preparations from either uninfected or vesicular stomatitis virus-infected cells supported the initiation of translation of host cell mRNA's and both viral mRNA's. These results reflect a specific alteration of some components(s) of the initiation factor preparation from poliovirus-infected cells which is consistent with the ability of the virus to inhibit the translation of host cell and vesicular stomatitis virus-directed protein synthesis.     

Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus.

Vesicular stomatitis virus contains a single structural glycoprotein whose carbohydrate sequences are probably specified by the host cell. The glycopeptides derived by Pronase digestion of the glycoprotein of vesicular stomatitis virus grown in HeLa cells have an average molecular weight of 1,800. There are multiple oligosaccharide chains on the vesicular stomatitis virus glycoprotein with protein-carbohydrate linkages that are cleaved only by strong alkali under reducing conditions, suggesting that they contain asparagine and N-acetylglucosamine. The oligosaccharide moieties, in addition, appear to be heterogeneous in sequence on the basis of their mobilities during electrophoresis and their sensitivities to cleavage by an endoglycosidase. The carbohydrate-peptide linkage region of the major class of oligosaccharides of the vesicular stomatitis virus glycoprotein has the proposed sequence: (see article).     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.