Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant.

We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.     

Requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus.

The cytoplasmic domains of viral glycoproteins are often involved in specific interactions with internal viral components. These interactions can concentrate glycoproteins at virus budding sites and drive efficient virus budding, or can determine virion morphology. To investigate the role of the vesicular stomatitis virus (VSV) glycoprotein (G) cytoplasmic and transmembrane domains in budding, we recovered recombinant VSVs expressing chimeric G proteins with the transmembrane and cytoplasmic domains derived from the human CD4 protein. These unrelated foreign sequences were capable of supporting efficient VSV budding. Further analysis of G protein cytoplasmic domain deletion mutants showed that a cytoplasmic domain of only 1 amino acid did not drive efficient budding, whereas 9 amino acids did. Additional studies in agreement with the CD4-chimera experiments indicated the requirement for a short cytoplasmic domain on VSV G without the requirement for a specific sequence in that domain. We propose a model for VSV budding in which a relatively non-specific interaction of a cytoplasmic domain with a pocket or groove in the viral nucleocapsid or matrix proteins generates a glycoprotein array that promotes viral budding.     

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.     

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.     

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity.

Nineteen independently isolated hybridomas producing monoclonal antibodies to the glycoprotein of vesicular stomatitis virus were isolated and studied for their capacity to neutralize viral infectivity. By measuring competitive binding of 125I-labeled monoclonal antibodies in a radioimmunoassay. 11 different, non-cross-reacting antigenic determinants were identified on the vesicular stomatitis virus G protein. All monoclonal antibodies reacting with determinants 1, 2, 3, and 4 resulted in viral neutralization, whereas those binding to the other seven determinants did not neutralize infectivity. The mixture of two monoclonal antibodies binding to different determinants resulted in a more rapid neutralization than either antibody alone, suggesting that different antibodies can exert a synergistic effect on viral neutralization. Kinetic experiments revealed biphasic neutralization curves similar to those expected for heterologous antibody. No evidence could be obtained to relate biphasic kinetics of viral neutralization to heterogeneous populations either of antibody molecules or of virus. The possible significance of the kinetic data with monoclonal antibodies is discussed.     

Accessibility to proteases of the cytoplasmic G protein domain of vesicular stomatitis virus is increased during intracellular transport

G1 and G2 are two forms of the membrane-integrated G protein of vesicular stomatitis virus that migrate differently in gel electrophoresis because G1 is modified by high-mannose and G2 by complex-type oligosaccharide side chains. The cytoplasmic domain in G1 is less exposed to cleavage by several proteases than in G2 molecules. Acylation by palmitic acid as well as inhibition of carbohydrate processing by swainsonine and deoxynojirimycin resulted in the same pattern of proteolytic sensitivity of both glycoproteins as in untreated cells. In contrast, accessibility of the cytoplasmic domain to proteases did not change when the intracellular transport of the G protein was blocked in carbonyl cyanide m-chlorophenylhydrazone- or monensin-treated BHK-21 cells, respectively. The results suggest that the increase in accessibility of the cytoplasmic tail of the G protein occurs after the monensin block in the trans-Golgi and might reflect a conformational change of functional significance--i.e., making the cytoplasmic domain of the viral spike protein competent for its interaction with the viral core, inducing thereby the formation of the budding virus particle.     

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.     

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.     

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.     

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.     

Interactions of vesicular stomatitis virus with murine cell surface antigens.

The process of maturation of vesicular stomatitis virus (VSV) results in the loss of 70% of the H-2k antigenic activity from L-cell plasma membranes. This phenomenon is also demonstrated during VSV infection of cells of the H-2d haplotype. Using the method of inhibition of immune cytolysis, VSV-infected L5178Y tissue culture cells and VSV-infected METH A fibrosarcoma cells grown in vivo show a loss of H-2d activity of 73 and 76%, respectively. Using monospecific antisera, it is seen that VSV infection results in a significant loss of antigenic activity of the gene products of both the H-2D and H-2K regions in cells of the H-2d and H-2k haplotypes. In hybrid cells expressing H-2k as well as H-2b, VSV infection results in the decrease of both H-2 antigenic activities to the same extent. VSV purified from L cells shows considerable H-2k activity, but the reaction of this virus with anti-H-2k serum does not prevent a normal subsequent infection with this virus. VSV may associate with H-2 antigen in the culture medium, but the results of mixing VSV with uninfected H-2-containing homogenates suggest that this association occurs only when the host cell and the cell homogenate share the same H-2 haplotype. Velocity sedimentation of VSV, which would remove contaminating cellular membrane fragments, does not separate H-2 activity from VSV. H-2 activity is also stably associated with VSV throughout sequential sucrose gradient centrifugation steps. It is possible that H-2 antigen is a structural component of VSV grown in murine cells.     

Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.     

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.     

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.     

Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein

We have characterized two stable transformed mouse cell lines (CG1 and CTG1) that express either the normal vesicular stomatitis virus glycoprotein (G) or a truncated form of the G protein (TG) that lacks the COOH-terminal anchor sequences and is secreted from the cells. These cell lines were obtained using a hybrid vector consisting of the transforming DNA fragment of bovine papilloma virus linked to a segment of the SV40 expression vector pSV2 containing cloned cDNA encoding either the normal or truncated form of the vesicular stomatitis virus G protein. Using indirect immunofluorescence we have found that greater than 95% of the cells in each line express the G protein(s), although the level of expression within the population is variable. The normal G protein expressed in these cells obtains its complex oligosaccharides in less than 30 min and is transported to the cell surface. In contrast, the TG protein obtains its complex oligosaccharides with a half-time of about 2.5 h. Immunofluorescence data show an apparent concentration of the TG protein in the rough endoplasmic reticulum. These data together suggest that transfer of this anchorless protein from the rough endoplasmic reticulum to the Golgi apparatus is the rate- limiting step in its secretion. We observed, in addition to normal G protein, two smaller G-related proteins produced in the CG1 cell line. We suggest that these proteins could result from aberrant splicing from sites within the G mRNA sequence to the downstream acceptor in the pSV2 vector.     

Influence of membrane anchoring and cytoplasmic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G.

Chimeric proteins in which the transmembrane anchoring sequence (TM) or both the TM and the cytoplasmic tail (CT) of vesicular stomatitis virus glycoprotein G were replaced with corresponding domains of viral or cellular integral membrane proteins were used to examine the influence of these domains on acidic-pH-induced membrane fusion by G protein. The TM and CT of G were also replaced with the lipid anchor glycosylphosphatidylinositol. Hybrids containing foreign TM or TM and CT sequences were fusogenic at acidic pH but glycosylphosphatidylinositol-anchored G was nonfusogenic at acidic pH. The results suggest that the fusogenic activity of G protein requires membrane anchoring by a hydrophobic peptide sequence and the specific amino acid sequence of the TM has no influence on fusogenic activity.     

Assembly of vesicular stomatitis virus: distribution of the glycoprotein on the surface of infected cells.

This study demonstrates that the glycoprotein of vesicular stomatitis virus clusters in the plasma membrane of infected Chinese hamster lung cells during morphogenesis and suggests that viral nucleocapsids are required for this clustering. A mutant virus (ts E-1) which is temperature sensitive for the synthesis of viral nucleocapsids but not viral membrane proteins was used. The surface distribution of the viral glycoprotein in cells infected by this virus was determined by a specific indirect immunoferritin stain. Early in infection at permissive temperatures, the glycoprotein was randomly distributed on membrane ghosts. Later, clusters of ferritin the size and shape of virus particles were seen. In contrast, ghosts prepared from virus-infected cells maintained at a restrictive temperature always had a random distribution of viral glycoprotein.