Glycosylation sites of vesicular stomatitis virus glycoprotein.

Detailed analysis on DEAE-Sephadex of the tryptic digestion products of the glycoprotein from vesicular stomatitis virus grown in HeLa suspension cultures revealed the presence of two major and several minor sugar-labeled species. The minor tryptic glycopeptides were converted to one of the two major glycopeptide species by treatment with neuraminidase. Thus, vesicular stomatitis virus glycoprotein contains only two oligosaccharide side chains that are heterogeneous in their sialic acid content.     

Biologically active peptides of the vesicular stomatitis virus glycoprotein.

A peptide corresponding to the amino-terminal 25 amino acids of the mature vesicular stomatitis virus glycoprotein has recently been shown to be a pH-dependent hemolysin. In the present study, we analyzed smaller constituent peptides and found that the hemolytic domain resides within the six amino-terminal amino acids. Synthesis of variant peptides indicates that the amino-terminal lysine can be replaced by another positively charged amino acid (arginine) but that substitution with glutamic acid results in the total loss of the hemolytic function. Peptide-induced hemolysis was dependent upon buffer conditions and was inhibited when isotonicity was maintained with mannitol, sucrose, or raffinose. In sucrose, all hemolytic peptides were also observed to mediate hemagglutination. The large 25-amino acid peptide is also a pH-dependent cytotoxin for mammalian cells and appears to effect gross changes in cell permeability. Conservation of the amino terminus of vesicular stomatitis virus and rabies virus suggests that the membrane-destabilizing properties of this domain may be important for glycoprotein function.     

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.     

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).     

A fusion-defective mutant of the vesicular stomatitis virus glycoprotein.

We have recently described an assay in which a temperature-sensitive mutant of vesicular stomatitis virus (VSV; mutant tsO45), encoding a glycoprotein that is not transported to the cell surface, can be rescued by expression of wild-type VSV glycoproteins from cDNA (M. Whitt, L. Chong, and J. Rose, J. Virol. 63:3569-3578, 1989). Here we examined the ability of mutant G proteins to rescue tsO45. We found that one mutant protein (QN-1) having an additional N-linked oligosaccharide at amino acid 117 in the extracellular domain was incorporated into VSV virions but that the virions containing this glycoprotein were not infectious. Further analysis showed that virus particles containing the mutant protein would bind to cells and were endocytosed with kinetics identical to those of virions rescued with wild-type G protein. We also found that QN-1 lacked the normal membrane fusion activity characteristic of wild-type G protein. The absence of fusion activity appears to explain lack of particle infectivity. The proximity of the new glycosylation site to a sequence of 19 uncharged amino acids (residues 118 to 136) that is conserved in the glycoproteins of the two VSV serotypes suggests that this region may be involved in membrane fusion. The mutant glycoprotein also interferes strongly with rescue of virus by wild-type G protein. The strong interference may result from formation of heterotrimers that lack fusion activity.     

Transmembrane biogenesis of the vesicular stomatitis virus glycoprotein

Previous work has shown that the mRNA encoding the vesicular stomatitis virus (VSV) glycoprotein (G) is bound to the rough endoplasmic reticulum (RER) and that newly made G protein is localized to the RER. In this paper, we have investigated the topology and processing of the newly synthesized G protein in microsomal vesicles. G was labeled with [35S]methionine ([35S]met), either by pulse-labeling infected cells or by allowing membrane-bound polysomes containing nascent G polipeptides to complete G synthesis in vitro. In either case, digestion of microsomal vesicles with any of several proteases removes approximately 5% (30 amino acids) from each G molecule. These proteases will digest the entire G protein if detergents are present during digestion. Using the method of Dintzis (1961, Proc. Natl. Acad. Sci. U. S. A. 47:247--261) to order tryptic peptides (8), we show that peptides lost from G protein by protease treatment of closed vesicles are derived from the carboxyterminus of the molecule. The newly made VSV G in microsomal membranes is glycosylated. If carbohydrate is removed by glycosidases, the resultant peptide migrates more rapidly on polyacrylamide gels than the unglycosylated, G0, form synthesized in cell-free systems in the absence of membranes. We infer that some proteolytic cleavage of the polypeptide backbone is associated with membrane insertion of G. Further, our findings demonstrate that, soon after synthesis, G is found in a transmembrane, asymmetric orientation in microsomal membranes, with its carboxyterminus exposed to the extracisternal, or cytoplasmic, face of the vesicles, and with most or all of its amino-terminal peptides and its carbohydrate sequestered within the bilayer and lumen of the microsomes.     

Localization of two cellular forms of the vesicular stomatitis viral glycoprotein.

Two cell-associated forms of the glycoprotein (G) of vesicular stomatitis virus, termed G1 and G2, have been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. G1 has the higher electrophoretic mobility, but both forms migrate more slowly than G protein synthesized in a wheat germ cell-free system (G0), which presumably is the unglycosylated form. G1 is a kinetic precursor of the G2 form, and the apparent cause of the electrophoretic difference between the two species is the presence of N-acetylneuraminic acid on the G2 form. Conversion of G1 to G2 occurs 10 to 20 min prior to the appearance of the G2 form of the protein on the cell surface. This suggests that the G protein may be completely glycosylated several minutes prior to its migration to the cell surface and that glycosylation is not the limiting step in its maturation. No glycoprotein comigrating with G0 can be detected in the infected cells, even after 5-min labeling periods; this suggests that partial clycosylation of G occurs concomitantly with or immediately after its synthesis.     

Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein

Maturation of the vesicular stomatitis virus (VSV) glycoprotein (G) to the cell surface is blocked at the nonpermissive temperature in cells infected with temperature-sensitive mutants in the structural gene encoding for G. We show here that these mutants fall into two discrete classes with respect to the stage of post-translational processing at which the block occurs. In all cases the mutant glycoproteins are inserted normally into the endoplasmic reticulum membrane, receive the two-high-mannose oligosaccharides, and apparently lose the NH2-terminal signal sequence of 16 amino acids. In cells infected with one class of mutants, no further processing of the glycoprotein occurs, and we conclude that the mutant protein is blocked at a pre-Golgi stage. In cells infected with ts L511(V), however, addition of the terminal sugars galactose and sialic acid occurs normally. Thus the maturation of G proceeds through several Golgi functions but is blocked before its appearance on the cell surface. The oligosaccharide chain of ts L511(V) G, accumulated at either the permissive (where surface maturation occurs) or the nonpermissive temperature, lacks one saccharide residue, probably fucose. In addition, no fatty acid residues are added to the ts L511(V) G protein at the nonpermissive temperature, although addition does occur under permissive conditions.     

Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin.

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein.

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.     

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 mtein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the light-density internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars.     

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) binds 1–2 moles of fatty acid per mole of protein. The fatty acids cannot be released by repeated extractions of the protein with organic solvents, nor can they be released by denaturing the protein with ionic or nonionic detergents. Pronase digestion of G yields an organic extractable fragment that contains bound fatty acid. The fatty acid is quantitatively released from this fragment and from intact G by mild alkali treatment in methanol and is identified by gas-liquid and thin-layer chromatography as, predominantly, the methyl ester of palmitic acid. Insignificant amounts of phosphate are found in G, thus ruling out the presence of bound phospholipid. Chicken embryo fibroblast pre-labeled with ³H-palmitate and then infected with VSV for 4 hr show the presence of ³H label in G but not in other viral structural proteins. The ³H label is present only in the fatty acid moiety of the protein. Much smaller amounts of ³H fatty acid are bound to G protein formed by the VSV mutant ts045 grown at the nonpermissive temperature, and no ³H fatty acid is bound to G synthesized at 37°C in cells pretreated with tunicamycin, an inhibitor of glycosylation. However, infection with the VSV-Orsay strain at 30°C in the presence of tunicamycin allows for production of VSV particles with nonglycosylated G (Gibson, Schlesinger and Kornfeld, 1979), and this G has the same proportion of the fatty acid as does the normal glycosylated G. These data indicate that fatty acids become covalently attached to the G polypeptide chain during maturation of the protein—perhaps as the glycoprotein moves to the cell's plasma membrane.     

Screening procedure for complementation-dependent mutants of vesicular stomatitis virus.

To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.     

Noninfectious vesicular stomatitis virus particles deficient in the viral nucleocapsid.

Several temperature-sensitive mutants of vesicular stomatitis virus in complementation group III produce, at nonpermissive temperature, noninfectious particles which contain the viral M (matrix) and G (glycoprotein) proteins but less than 10% of the normal proportion of N protein or RNA. Since group III mutants are thought to be defective in the structural gene for the virus M protein, these findings demonstrate that an interaction between M and the nucleocapsid is of importance in virus budding. Taken together with earlier results, they suggest that M is the key protein in bud formation.     

Pressure-induced fusogenic conformation of vesicular stomatitis virus glycoprotein

Vesicular stomatitis virus (VSV) is composed of a ribonucleoprotein core surrounded by a lipid envelope presenting an integral glycoprotein (G). The homotrimeric VSV G protein exhibits a membrane fusion activity that can be elicited by low pH. The fusion event is crucial to entry into the cell and disassembly followed by viral replication. To understand the conformational changes involved in this process, the effects of high hydrostatic pressure and urea on VSV particles and isolated G protein were investigated. With pressures up to 3.0 kbar VSV particles were converted into the fusogenic conformation, as measured by a fusion assay and by the binding of bis-ANS. The magnitude of the changes was similar to that promoted by lowering the pH. To further understand the relationship between stability and conversion into the fusion-active states, the stability of the G protein was tested against urea and high pressure. High urea produced a large red shift in the tryptophan fluorescence of G protein whereas pressure promoted a smaller change. Pressure induced equal fluorescence changes in isolated G protein and virions, indicating that virus inactivation induced by pressure is due to changes in the G protein. Fluorescence microscopy showed that pressurized particles were capable of fusing with the cell membrane without causing infection. We propose that pressure elicits a conformational change in the G protein, which maintains the fusion properties but suppresses the entry of the virus by endocytosis. Binding of bis-ANS indicates the presence of hydrophobic cavities in the G protein. Pressure also caused an increase in light scattering of VSV G protein, reinforcing the hypothesis that high pressure elicits the fusogenic activity of VSV G protein. This "fusion-intermediate state" induced by pressure has minor changes in secondary structure and is likely the cause of nonproductive infections.     

Interference among defective interfering particles of vesicular stomatitis virus

Three defective interfering (DI) particles of vesicular stomatitis virus (VSV), all derived from the same parental standard San Juan strain (Indiana serotype), were used in various combinations to infect cells together with the parental virus. The replication of their RNA genomes in the presence of other competing genomes was described by the hierarchical sequence: DI 0.52 particles greater than DI 0.45 particles less than or equal to DI-T particles greater than standard VSV. The advantage of one DI particle over another was not due simply to multiplicity effects nor to the irreversible occupation of limited cellular sites. Interference, however, did correlate with a change in the ratio of plus and minus RNA templates that accumulated intracellularly and with the presence of new sequences at the 3' end of the DI genomes. DI 0.52 particles contained significantly more nucleotides at the 3' end that were complementary to those at the 5' end of its RNA than did DI-T or DI 0.45 particles. The first 45 nucleotides at the 3' ends of all of the DI RNAs were identical. VSV and its DI particles can be separated into three classes, depending on their terminal RNA sequences. These sequences suggest two mechanisms, one based on the affinity of polymerase binding and the other on the affinity of N-protein binding, that may account for interference by DI particles against standard VSV and among DI particles themselves.