Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes

In a previous communication we reported that the newly synthesized membrane glycoprotein of vesicular stomatitis virus could be transported in crude extracts of CHO cells from endoplasmic reticulum-derived membranes to membranes of the Golgi complex. This conclusion was an indirect one, based on the terminal glycosylation of this glycoprotein, a reaction that was dependent upon a Golgi-specific enzyme, UDP-GlcNAc transferase I. We show here that the Golgi fraction of rat liver will substitute for members of CHO cells as a source of transferase I in this reaction. The use of highly purified fractions of liver Golgi membranes, coupled with the ability to recover these membranes from incubations, has now permitted a direct demonstration of net transport of G protein to these heterologous Golgi membranes. This transport reaction is specific, in that the smooth endoplasmic reticulum fraction will not substitute for the Golgi fraction, is quantitatively significant, involving at least 30% of the viral glycoprotein, and is sustained only in the presence of both ATP and a soluble, cytosol fraction of liver cells.     

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.     

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.     

Transport of the vesicular stomatitis glycoprotein to trans Golgi membranes in a cell-free system

Terminal steps in the transport of the vesicular stomatitis virus glycoprotein (G protein) in the Golgi stack have been reconstituted in a cell-free system. Incorporation of sialic acid into the oligosaccharide chains of G protein was used to monitor transport into the trans Golgi compartment. Transport-coupled sialylation required cytosol, ATP, an N-ethylmaleimide-sensitive factor extractable from Golgi membranes, and long chain acyl coenzyme A. The G protein receiving sialic acid in the cell-free system begins its in vitro transport bearing galactose residues acquired in vivo. Earlier reports (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984a) Cell 39, 405-416) documented that transport of G protein into the medial (GlcNAc Transferase-containing) compartment is reconstituted under the same conditions. On the basis of the results reported here, it now appears that a more complete set of transport operations of the Golgi stack may be simultaneously reconstituted.     

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.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Assembly of viral membranes: nature of the association of vesicular stomatitis virus proteins to membranes.

To explore the interaction of vesicular stomatitis virus (VSV) proteins with cellular membranes, we have isolated membranes from infected cells that have been radioactively pulse-labeled. We have found conditions of isolation that result in membrane preparation which contain primarily the VSV membrane protein (M) and glycoprotein (G). Both of these proteins are very firmly attached to membranes: conditions known to release peripherally associated membrane proteins from membranes (S. Razin, Biochim, Biophys. Acta 265:241-246, 1972; S. J. Singer, Annu. Rev. Biochem. 43:805-826, 1974; S. J. Singer and G. L. Nicholson, Science 175:720-731, 1972) are ineffective in detaching either the G or the M protein. The results of trypsin digestion of these membrane fractions suggest that the M protein resides primarily on one side, the cytoplasmic side of cellular membranes, whereas the glycoprotein has been transported to the lumen of the membrane vesicle. However, we present evidence that the glycoprotein is transmembranal and that approximately 3,000 daltons of one end of the molecule is on the cytoplasmic side of the membrane. We have also found that undenatured VSV M protein contains a trypsin-resistant core with a molecular weight of 22,000. This region of the M protein is trypsin-resistant regardless of its association with membranes.     

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.     

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.     

Assembly of vesicular stomatitis virus glycoprotein and matrix protein into HeLa cell plasma membranes.

The kinetics of the incorporation of the proteins of vesicular stomatitis virus into the HeLa cell plasma membrane have been studied. The virus M and NS proteins become associated with the plasma membrane very rapidly (< 5 min) while the glycoprotein G shows a lag of about 20 minutes. A similar lag is observed for the incorporation of the G protein into released virus. By pulse-chase experiments the transit time for the G protein from the site of completion to the plasma membrane was also calculated to be about 20 minutes although not all of the G protein could be chased into the plasma 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.     

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).     

Organization of the vesicular stomatitis virus glycoprotein into membrane microdomains occurs independently of intracellular viral components

The glycoprotein (G protein) of vesicular stomatitis virus (VSV) is primarily organized in plasma membranes of infected cells into membrane microdomains with diameters of 100 to 150 nm, with smaller amounts organized into microdomains of larger sizes. This organization has been observed in areas of the infected-cell plasma membrane that are outside of virus budding sites as well as in the envelopes of budding virions. These observations raise the question of whether the intracellular virion components play a role in organizing the G protein into membrane microdomains. Immunogold-labeling electron microscopy was used to analyze the distribution of the G protein in arbitrarily chosen areas of plasma membranes of transfected cells that expressed the G protein in the absence of other viral components. Similar to the results with virus-infected cells, the G protein was organized predominantly into membrane microdomains with diameters of approximately 100 to 150 nm. These results indicate that internal virion components are not required to concentrate the G protein into membrane microdomains with a density similar to that of virus envelopes. To determine if interactions between the G protein cytoplasmic domain and internal virion components were required to create a virus budding site, cells infected with recombinant VSVs encoding truncation mutations of the G protein cytoplasmic domain were analyzed by immunogold-labeling electron microscopy. Deletion of the cytoplasmic domain of the G protein did not alter its partitioning into the 100- to 150-nm microdomains, nor did it affect the incorporation of the G protein into virus envelopes. These data support a model for virus assembly in which the G protein has the inherent property of partitioning into membrane microdomains that then serve as the sites of assembly of internal virion components.     

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.     

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.     

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.     

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.