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.     

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.     

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.     

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.     

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.     

Phosphorylation sites on phosphoprotein NS of vesicular stomatitis virus.

The phosphoprotein NS of vesicular stomatitis virus which accumulates within the infected cell cytoplasm is phosphorylated at multiple serine and threonine residues (G. M. Clinton and A. S. Huang, Virology 108:510-514, 1981; Hsu et al., J. Virol. 43:104-112, 1982). Using incomplete chemical cleavage at tryptophan residues, we mapped the major phosphorylation sites to the amino-terminal half of the protein. Analysis of phosphate-labeled tryptic peptides suggests that essentially all of the label is within the large trypsin-resistant fragment predicted from the sequence of Gallione et al. (J. Virol. 39:52-529, 1981). A similar result has been obtained for NS protein isolated from the virus particle by C.-H. Hsu and D. W. Kingsbury (J. Biol. Chem., in press). Analysis of phosphodipeptides utilizing the procedures of C. E. Jones and M. O. J. Olson (Int. J. Pept. Protein Res. 16:135-142, 1980) enabled us to detect as many as six distinct phosphate-containing dipeptides. From these studies, together with the known sequence data, we conclude that the major phosphate residues on cytoplasmic NS protein are located in the amino third of the NS molecule and most probably between residues 35 and 106, inclusive. The studies also provide formal chemical proof that NS protein has a structure consistent with a monomer of the sequence of Gallione et al. as modified by J. K. Rose (personal communication). The low electrophoretic mobility of this protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not therefore due to dimerization.     

Antigen processing and presentation of vesicular stomatitis virus glycoprotein

Antigen processing and presentation is the complex series of steps involving numerous compartments of cells derived from multiple lineages. This collective process is central to the initiation of specific cellular immune responses. Internalization of foreign proteins or infectious agents, degradation of proteins to peptides, and transfer to 1a heterodimers which are then translocated to the cell surface, comprise the pathway. This can be blocked by drugs which interfere with compartment acidification (ammonium chloride, methylamine, chloroquine), protein synthesis (emetine), vesicle trafficking (brefeldin A), and microtubule metabolism (taxol, colchicine, nocadazole).     

Interferon alters intracellular transport of vesicular stomatitis virus glycoprotein

Double-label immunofluorescence staining studies in virus-infected subclone 11 of LB cells indicated that almost all of the vesicular stomatitis virus (VSV) glycoprotein (G) was plasma membrane-associated during the logarithmic phase of virus replication. In contrast, treatment with interferon (IFN) resulted in inhibition of VSV-G transport, so that almost all of the G remained associated with the Golgi complex (GC) at comparable times after infection. In both IFN-treated and control cells, G was resistant to treatment with the enzyme endo-beta-N-acetylglucosamine H (endo H) indicating that the bulk of the G had reached the trans compartment of the GC.     

DBC2 is essential for transporting vesicular stomatitis virus glycoprotein

DBC2 is a tumor suppressor gene linked to breast and lung cancers. Although DBC2 belongs to the RHO GTPase family, it has a unique structure that contains a Broad-Complex/Tramtrack/Bric a Brac (BTB) domain at the C terminus instead of a typical CAAX motif. A limited number of functional studies on DBC2 have indicated its participation in diverse cellular activities, such as ubiquitination, cell-cycle control, cytoskeleton organization and protein transport. In this study, the role of DBC2 in protein transport was analyzed using vesicular stomatitis virus glycoprotein (VSVG) fused with green fluorescent protein. We discovered that DBC2 knockdown hinders the VSVG transport system in 293 cells. Previous studies have demonstrated that VSVG is transported via the microtubule motor complex. We demonstrate that DBC2 mobility depends also on an intact microtubule network. We conclude that DBC2 plays an essential role in microtubule-mediated VSVG transport from the endoplasmic reticulum to the Golgi apparatus.     

Shedding of vesicular stomatitis virus soluble glycoprotein by removal of carboxy-terminal peptide.

A comparison of partial NH2-terminal sequences of vesicular stomatitis viral glycoprotein G (molecular weight, 69,000) and the soluble extracellular glycoprotein antigen Gs (molecular weight, 57,000) shows that both of the sequences are identical. Tryptic fingerprint analyses show that Gs lacks the carboxy-terminal region containing the membrane-anchoring hydrophobic domain of G. These results suggest that Gs is formed by cleavage in the carboxy-terminal region of G.     

Characterization of the putative fusogenic domain in vesicular stomatitis virus glycoprotein G.

The envelope glycoprotein G of vesicular stomatitis virus induces membrane fusion at low pH. Site-directed mutagenesis of specific amino acids within a segment spanning amino acids 123 to 137 of G protein, which is highly conserved in vesiculoviruses and was previously shown by us to be involved in fusogenic activity (Y. Li, C. Drone, E. Sat, and H. P. Ghosh, J. Virol. 67:4070-4077, 1993), was used to determine the role of this region in low-pH-induced membrane fusion. The mutant glycoproteins expressed in COS cells were assayed for acid-pH-induced cell-cell fusion. Substitution of the variant Pro-123 with Leu had no effect on the fusogenic activity, while substitution of conserved Phe-125 and Asp-137 with Tyr and Asn, respectively, shifted the pH optimum of membrane fusion to a more acidic pH value and decreased the fusion efficiency. The deletion of amino acid residues 124 to 127, 131 to 137, or 124 to 137 produced mutants defective in transport. Mutation of the conserved residues Gly-124 and Pro-127 to Ala and to Gly or Leu, respectively, inhibited cell-cell fusion activity by about 90% without affecting transport of the mutant proteins to the cell surface, suggesting that these two residues may be present within the fusion peptide and thus may be directly involved in fusion. This highly conserved domain containing neutral amino acids of G protein may therefore represent the putative fusion domain of vesicular stomatitis virus G protein.     

Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein.

The vesicular stomatitis virus glycoprotein forms noncovalently linked trimers in the endoplasmic reticulum before being transported to the Golgi apparatus. The experiments reported here were designed to determine if the extracellular domain of the glycoprotein contains structural information sufficient to direct trimer formation. To accomplish this, we generated a construct encoding G protein with the normal transmembrane and anchor sequences replaced with the sequence encoding 53 C-terminal amino acids from the Thy-1.1 glycoprotein. We show here that these sequences were able to specify glycolipid addition to the truncated G protein, probably after cleavage of 31 amino acids derived from Thy-1.1. The glycolipid-anchored G protein formed trimers and was expressed on the cell surface in a form that could be cleaved by phosphoinositol-specific phospholipase C. However, the rate of transport was reduced, compared with that of wild-type G protein. A second form of the G protein was generated by deletion of only the transmembrane and cytoplasmic domains. This mutant protein also formed trimers with relatively high efficiency and was secreted slowly from cells.     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

Electrochemical modification of vesicular stomatitis virus glycoprotein by host cell transformation

Electrochemical properties of the glycoprotein of vesicular stomatitis virus (VSV) grown in Rous sarcoma virus (RSV)-transformed cells was compared with that of its counterpart grown in nontransformed cells. In DEAE-Sephadex column chromatography, the glycoproteins of VSV derived from transformed cells appeared more heterogeneous and had a tendency to elute with higher concentrations of NaCl than those from nontransformed cells. In isoelectric focussing, the glycoproteins of VSVs derived from transformed and nontransformed cells appeared as multiple components differing in the isoelectric point, and the glycoproteins from virus from transformed cells had isoelectric points that were more acidic than their counterparts from nontransformed cells. These results show that the glycoprotein of VSV consists of populations of molecules differing in charge and their isoelectric points were shifted to the acidic side by host cell transformation.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.