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.     

Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.     

Assembly of viral membranes. I. Association of vesicular stomatitis virus membrane proteins and membranes in a cell-free system.

We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M andG proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations.     

Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex

We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.     

Transient activity of Golgi-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro

Previous reports demonstrated that the vesicular stomatitis viral glycoprotein (G protein), initially present in membranes of a Chinese hamster ovary mutant cell line (clone 15B) that is incapable of terminal glycosylation, can be transferred in vitro to exogenous Golgi membranes and there glycosylated (E. Fries and J. E. Rothman, 1980, Proc. Natl. Acad. Sci. U. S. A. 77:3870-3874; and J. E. Rothman and E. Fries, 1981, J. Cell Biol. 89:162-168). Here we present evidence that Golgi-like membranes serve as donors of G protein in this process. Pulse-chase experiments revealed that the donor activity of membranes is greatest at approximately 10 min of chase, a time when G protein has been shown to have arrived in Golgi stacks (J. E. Bergmann, K. T. Tokuyasu, and S. J. Singer, 1981, Proc. Natl. Acad. Sci. U. S. A. 78:1746-1750). Additional evidence that the G protein that is transferred to exogenous Golgi membranes in vitro had already entered the Golgi membranes in vivo was provided by observations that its oligosaccharides had already been trimmed, and that its distribution in a sucrose density gradient was coincident with that of enzymatic markers of Golgi membranes. The capacity of this Golgi-like membrane to serve as donor is transient, declining within 5 min after "trimming" in vivo as the G protein enters a "nontransferable" pool. The rapidity of the process suggests that both the "transferable" and "nontransferable" pools of G protein reside in Golgi-like membranes.     

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.     

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.     

Dynamic equilibrium between vesicular stomatitis virus glycoprotein monomers and trimers in the Golgi and at the cell surface.

Previous studies have shown that trimers of the vesicular stomatitis virus glycoprotein (VSV G protein) are in rapid equilibrium with monomeric subunits after folding and assembly in the endoplasmic reticulum (ER). To determine whether G protein trimers were in equilibrium with monomers in other cellular compartments, we studied heterotrimer formation between VSV G protein and a mutant G protein (G mu protein) containing a 3-amino-acid cytoplasmic domain replacing the normal 29-amino-acid domain. The G mu protein is transported from the ER much more slowly than G protein, although both G and G mu proteins form trimers rapidly in the ER. In coexpression experiments, we observed that VSV G protein molecules exited the ER about sixfold faster than G mu protein molecules, and we observed no heterotrimer formation in the ER, probably because of rapid reassortment of the mutant and wild-type trimers. However, heterotrimer formation between the two proteins was observed after long chase periods that allowed time for trimers of the mutant protein to reach the plasma membrane and reassort with the G protein subunits. Additional studies showed that heterotrimers of the two proteins could form in the Golgi or in the ER if exit of the G protein from either compartment was blocked.     

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.     

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.     

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.     

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.     

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).     

Shedding of the glycoprotein from vesicular stomatitis virus-infected cells.

In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.     

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.     

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.     

Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system

Rat liver Golgi stacks were incubated with mitotic cytosol for 30 min at 37 degrees C to generate mitotic Golgi fragments comprising vesicles, tubules, and cisternal remnants. These were isolated and further incubated with rat liver cytosol for 60 min. The earliest intermediate observed by electron microscopy was a single, curved cisterna with tubular networks fused to the cisternal rims. Elongation of this cisterna was accompanied by stacking and further growth at the cisternal rims. Stacks also fused laterally so that the typical end product was a highly curved stack of 2-3 cisternae mostly enclosing an electron-lucent space. Reassembly occurred in the presence of nocodazole or cytochalasin B but not at 4 degrees C or in the absence of energy supplied in the form of ATP and GTP. Pretreatment of the mitotic fragments and cytosol with N-ethylmaleimide (NEM) also prevented reassembly. GTP gamma S and A1F prevented reassembly when added during fragmentation but not when added to the reassembly mixture. In fact, GTP gamma S stimulated reassembly such that all cisternae were stacked at the end of the incubation and comprised 40% of the total membrane. In contrast, microcystin inhibited stacking so that only single cisternae accumulated. Together these results provide a detailed picture of the reassembly process and open up the study of the architecture of the Golgi apparatus to a combined morphological and biochemical analysis.