5 Recipient of Public Health Service Grant R01 A from. Sondra SchlesingerS, Carol Malfer, and Milton J. Schlesingert

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19% by The American Society of Biological Chemists, Inc. Vol. 259, No. 12, Issue of June 25. pp ,19&2 Printed in U.S.A. The Formation of Vesicular Stomatitis Virus (San Juan Strain) Becomes Temperaturesensitive When Glucose Residues Are Retained on the Oligosaccharides of the Glycoprotein* (Received for publication, January 27, 1984) Sondra SchlesingerS, Carol Malfer, and Milton J. Schlesingert From the Department of Microbiology and Immunology, Washington Uniuersity School of Medicine, St. Louis, Missouri We have studied the effects of inhibiting the initial steps in processing of asparaginelinked oligosaccharides on the formation of vesicular stomatitis virus (VSV). Our data show that conditions which prevent the removal of glucose can block the growth of this virus. Our conclusion that inhibition of VSV synthesis is due specifically to an effect on the ability of the virus glycoprotein, G, to mature to a correct functional conformation is based on the following observations: (i) two drugs, deoxynojirimycin and castanospermine, both of which selectively inhibit the processing glucosidases, affected virus growth; (ii) only one of the two strains (San Juan and ) of VSV tested was affected and that strain, VSV(San Juan), is known to have a G protein highly sensitive to alterations in oligosaccharide structure; (iii) the effect was to make the formation of VSV(San Juan) temperaturesensitive, a result previously observed with alterations in the oligosaccharides on G protein; (iv) a cell variant missing glucosidase I1 activity also became temperaturesensitive in its ability to produce VSV(San Juan) but not VSV(0rsay). Although inhibition of glucosidase activity by ldeoxynojirimycin caused a 10fold drop in virion formation, transport of G protein to the plasma membrane was not altered. The growth of VSV(San Juan) at 40 C was not affected when subsequent steps in the processing pathway were blocked. These data indicate that by the time the glucose residues are removed G has attained a stable conformation. Glycosylation of asparagine residues in glycoproteins normally begins with the transfer of the precursor oligosaccharide, Glc3Man9GlcNAc2, from the carrier lipid, dolichol phosphate, to those nascent polypeptide chains destined to become glycoproteins (1, 2). The extent to which this precursor is processed determines the final structure of the asparaginelinked oligosaccharides on mature glycoproteins (3). The first step in processing is the removal of the three glucose residues which occurs shortly after completion of the polypeptide chain while the protein is still in the rough endoplasmic reticulum (2,4). The synthesis of complex oligosaccharides involves the removal of all but three of the mannose residues and the addition of Nacetylglucosamine, galactose, sialic acid, and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked nduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of Public Health Service Grant R01 A from the National Institute of Allergy and Infectious Diseases. 5 Recipient of Public Health Service Grant R01 A from the National Institute of Allergy and Infectious Diseases fucose (2, 3). These steps take place in the Golgi vesicles. The isolation of cell variants blocked at a particular stage in oligosaccharide processing and the identification of drugs able to inhibit either the synthesis or processing of oligosaccharides have made it possible to investigate the role of glycosylation in the synthesis and localization of glycoproteins. One of the first drugs to be described was tunicamycin, an antibiotic which inhibits the synthesis of Nacetylglucosaminylpyrophosphorylpolyisoprenol and prevents the addition of any carbohydrate to asparagine residues of potential glycoproteins. Studies with tunicamycin demonstrated that some proteins are able to reach their correct cellular location and function properly in the absence of carbohydrate, whereas other proteins are greatly affected by the lack of carbohydrate (5). These diverse results led to the proposal that covalently bound oligosaccharides can affect the conformation or stabil ity of a protein; thus proteins with different amino acid sequences would have different requirements for carbohydrate. This point is illustrated clearly with the glycoprotein, G, of VSV (68). There are two strains of the VSV Indiana serotype, VSV(San Juan) and VSV(), which have related but distinct G polypeptides. In the presence of tunicamycin, virus yields for both strains ofvsv are severely inhibited at 38 C. At C the growth of VSV(San Juan) is still inhibited by tunicamycin but the yield of VSV(0rsay) containing unglycosylated G protein is nearly equal to that of the control. The increase in virus production at C corre lates with the ability of unglycosylated G(0rsay) to fold correctly at the lower temperature (7). Thus, in the absence of carbohydrate the G protein becomes temperaturesensitive during folding, and the sensitivity depends on the amino acid sequence of the protein. The importance of the structure of the oligosaccharides to the proper folding of G is also seen in a mutant cell line which transfers Glc3Man6GlcNAc2 instead of the normal precursor to nascent polypeptides (9). Growth of VSV is temperaturesensitive in this mutant and VSV(San Juan) is more temperaturesensitive than VSV(0rsay) (10). Recently, two drugs, ldeoxynojirimycin and castanospermine, have been shown to inhibit the glucosidases which act at the initial stages of oligosaccharide processing (1113). To determine if these and some of the following steps in oligosaccharide processing are critical for certain proteins we compared the growth of VSV(San Juan) and VSV(0rsay) in the presence of these drugs and in cell variants altered in the processing pathway. Our approach stems from the earlier work of Gibson et al. (7, 10) and is based on the knowledge and the San Juan G protein should be more sensitive to The abbreviations used are: VSV, vesicular stomatitis virus; G, glycoprotein of VSV; SDS, sodium dodecyl sulfate, Endo H, endop Nacetylglucosaminidase H.

2 7598 Glucosidase Activity Is Required for VS V Formation alterations in oligosaccharide structure than the G protein. EXPERIMENTAL PROCEDURES Cells and VirusSecondary chicken embryo fibroblasts were used in most experiments with deoxynojirimycin and castanospermine. The mutant PHAR2.7 lacking glucosidase I1 activity has been described in detail (9) as have the other cell variants (14, 15). The distinctive properties of the G protein of VSV(San Juan) and VSV(0rsay) have been reported (68, IO). Growth of Radiolabeled VS VCells infected with VSV were labeled with [%]methionine or in some experiments [%]cysteine in medium lacking the equivalent nonradioactive amino acid. The procedures have been described previously (7, IO), and specific details are included in the figure legends. Cell Surface LabelingChicken embryo fibroblasts infected with VSV(San Juan) or VSV(0rsay) were pulselabeled with [36S]methionine 4 h postinfection. After 15 min the labeled medium was removed. The cells were either harvested or washed and prewarmed medium containing 1.5 mm methionine added for a 60min chase period (16). We followed the procedures described by Strous and Lodish (17) and Lodish and Kong (18) for the trinitrobenzenesulfonate treatment of cells and immunoprecipitation with antitrinitrophenol antisera. MaterialsIDeoxynojirimycin was obtained from Dr. H. D. Schlumberger and Dr. D. Schmidt (Bayer A. G., Wuppertal, West Germany). Castanospermine was a gift from Dr. Philip Stahl (Washington University) who obtained it from Dr. Alan Elbein (University of Texas, San Antonio). Antitrinitrophenol antisera was a gift from Dr. J. R. Little (Washington University). Endo H was a gift from Dr. P. W. Robbins (Massachusetts Institute of Technology). RESULTS The Effect of Deoxynujirimycin and Temperature on the Growth of VSV(San Juan) and VSV(0rsay)1Deoxynojirimycin is an inhibitor of the processing glucosidases (11). We determined the effect of this compound on the growth of VSV by labeling cells with a radioactive amino acid and measuring the production of radioactive particles released into the medium of infected cells. The addition of 1 mm deoxynojirimycin to chicken embryo fibroblasts did not affect the growth of VSV(San Juan) or VSV(0rsay) in these cells at "C. At 40 "C, however, deoxynojirimycin inhibited the production of VSV(San Juan) almost 10fold whereas the growth of VSV(0rsay) appeared normal (Fig. 1). 1Deoxynojirimycin inhibited the formation of infectious virions (plaqueforming units) of VSV(San Juan) at 40 "C to the same extent as particle formation (Table I). Under these conditions there was a small but reproducible inhibition of proiein synthesis in the presence of deoxynojirimycin in cells infected with VSV(San Juan). The synthesis of all of the VSV(San Juan) proteins was inhibited to the same extent, based on an analysis of the proteins after SDSpolyacrylamide gel electrophoresis (data not shown). Because this inhibition was only observed with VSV(San Juan) at 40 "C it may be a consequence of the effects on G which result from the block in processing of the oligosaccharides. A recent report (19) indicated that under some conditions deoxynojirimycin may inhibit the formation of the dolichollinked oligosaccharide. Our analyses of radioactively labeled proteins in infected chicken embryo fibroblasts did not show any evidence that this step was affected; no G protein could be detected migrat ing in an acrylamide gel at the position of the nonglycosylated polypeptide. Furthermore, the growth of VSV(0rsay) is greatly reduced at 40 "C when glycosylation is inhibited by tunicamycin (7, 8), and we saw no effect of deoxynojirimycin on the growth of this virus at 40 "c. Susceptibility of G Synthesized in the Presence of 1 Deoxynojirimycin to Endo IfThe enzyme Endo H cleaves high mannosetype asparaginelinked oligosaccharides between TUBE NUMBER FIG. 1. The growth of VSV(San Juan) and VSV(0rsay) in the presence or absence of 1deoxynojirimycin as measured by the release of radioactive particles detected in a sucrose gradient. Secondary chicken embryo fibroblasts in 60mm dishes were infected with either VSV(San Juan) or VSV(0rsay) at a multiplicity of infection of 50. After a 1h adsorption period the inoculum was removed and cysteinefree medium added to the dishes. One dish for each strain and each temperature also contained 1 mm Ideoxynojirimycin added after the adsorption period. The medium was harvested 7 h postinfection for the cells incubated at 40 "C and 8 h postinfection for the cells incubated at "C. Three h postinfection 16pCi of [35S]cysteine were added to each dish. The virus was sedirnented to equilibrium in a preformed 2550% sucrose gradient. Fractions of 0.5 ml were collected and a sample from each fraction was precipitated with tricholoroacetic acid to determine radioactivity. 0 0, particles obtained from cells incubated with ldeoxynojirimycin; M, the controls. the two Nacetylglucosamine residues at the reducing end of the molecule. Only newly synthesized G is normally susceptible to Endo H as the two oligosaccharide chains are usually processed to complextype structures and as such are resistant to the enzyme. Under conditions in which processing is inhibited, treatment of G with Endo H removes most of the oligosaccharides and leads to a detectable increase in the mobility of the protein in an SDSpolyacrylamide gel. At both and 40 "C the presence of 1deoxynojirimycin in the medium of cells infected with VSV converted G from a mostly Endo Hresistant form to one which was sensitive to the enzyme (Fig. 2). These results demonstrated that deoxynojirimycin was interfering with normal oligosaccharide processing under our experimental conditions. We did observe some heterogeneity of G protein after treatment with Endo H. This was not surprising since it has been reported that deoxynojirimycin does not completely block oligosaccharide processing (20). The G protein of VSV(San Juan) produced in chicken embryo fibroblasts retained some high mannose oligosaccharide chains even in the absence of deoxynojirimycin (Fig. 2, lane 1). The presence of Endo Hsensitive oligosaccharide chains on mature G proteins was not found when VSV(San Juan) was grown in mammalian cells (14, 151.' The Effect of Castanospermine and Temperature on the S. Schlesinger, C. Malfer, and M. J. Schlesinger, unpublished results.

3 Glucosidase Activity Is Required for VSV Formation TABLE I The effect of deoxynojirimycin and temperature on protein synthesis and yield of VSV Chicken embryo fibroblasts (5 X 10' cells/35mm dish) were infected with VSV, and after a 1h adsorption period the cells were incubated in methioninefree medium with or without 1 mm deoxynojirimycin, as indicated. One pci of [%S]methionine was added to the cells a t 40 "C 4 h postinfection and to the cells a t "C 6 h postinfection. One h after addition of the radioactive amino acid the cells were harvested in cold 10% trichloroacetic acid and the total acidinsoluble radioactivity determined. The plaqueforming units were determined by harvesting the medium from dishes 6 h postinfection from cells incubated at 40 'C and 8 h postinfection from cells incubated a t "C. The virus was titered on secondary chicken embryo fibroblasts. Strain of vsv Juan San Counts per min TemperDeoxyincorporated Plaqueforming ature nojirimycin protein into units per ml per h "C x 105 x VSV [Son Juan) V S V () Deoxynoiirimycin Endo H FIG. 2. The effect of Endo H treatment on VSV produced in the presence or absence of 1deoxynojirimycin. Samples of radioactive virus prepared by growth a t 40 "C were purified by sucrose gradient centrifugation as described in Fig. 1. The virus samples were concentrated by pelleting at 100,000 X g and then were incubated with or without Endo H at 37 "C for 18 h (23). The samples were then reduced and analyzed by electrophoresis in a SDSpolyacrylamide gel. " Synthesis of VSVfSan Juan) and VSV(0rsay)The plant alkaloid, castanospermine, is another compound known to inhibit the processing glucosidases (12, 13). Recent studies showed that in the presence of castanospermine the major oligosaccharide found on the hemagglutinin of influenza virus is Glu3Man7GlcNAcp (13).Castanospermineinhibitedthe growth of VSV(San Juan) but not that of VSV(0rsay) at 40 "C (Fig. 3). The G protein synthesized in chicken embryo fibroblasts in the presenceof castanospermine was susceptible to Endo H (data not shown). The Effect of Temperature on the Growth of VSV in Cell Variants Alteredin Oligosaccharide ProcessingThe data presented show that inhibitors of glucosidases can affect the VSV ( Son 4(r Jwn) 7599 VSV () 40 3 w 2 1 TUBENUMBER FIG. 3. The growth of VSV(San Juan) and VSV(0rsay) in the presence or absence of castanospermine as measured by the release of radioactive particles detected in a sucrose gradient. The experimental protocol was identical to that described in Fig. 1 except that 10 pci of [35S]methioninewere added to each dish 3 h postinfection. Castanospermine a t a final concentration of 20 pg per ml (13) was added 1 h postinfection. 0 0, particles obtained from cells incubated with castanospermine; W,the controls. formation of VSV(San Juan). It is also possible to prevent the removal of glucose from the oligosaccharide chains of G by growing VSV in a mouse lymphoma cell line (PHAR2.7) which is deficient in glucosidase I1 activity and is unable to remove the two inner glucose residues from the oligosaccharide chains (9). We tested the growth of the two strains of VSV in this cell line. VSV(San Juan) produced 7fold less virus at 40 "C than at "C, whereas VSV(0rsay) was only minimally affected at 40 "C (Table 11). The G protein of these viruses grown in PHARcells was sensitive to EndoH. Thestrains ofvsv grew well intheparent cell line (BW5147) except in the presenceof deoxynojirimycin (Table 11). As expected in the presence of this drug the growth of VSV(San Juan), but not that of VSV(), was inhibited a t 40 "C. We wished to determine if inhibition of any of the later steps in processing could also affect the growth of this virus. Inhibitors able toblock some of the subsequent steps are not available, but it was possible to examine cells in which some of these steps do notoccur because of mutational alterations. In addition to the variant lacking glucosidase I1 activity we tested clone 6 which accumulates high mannose oligosaccharides, most of which have the compositionman8glcnac, (15) and15b which lacksan Nacetylglucosamine transferase activity (14). Theasparaginelinked oligosaccharides produced by the latter mutant are MansGlcNAc2. Cells blocked in steps in processing after the removal of glucose produced similar orhigher yields of VSV(San Juan) at 40 than at "C (Table 11). In each case, however, the yield of this strain of VSV but not that of VSV(0rsay) was inhibited at 40 "C by deoxynojirimycin. Cell Surface Labeling of G Protein a t 40 "C in the Presence and Absence of 1DeoxynojirimycinWhen cells infected with VSV(San Juan) are treated with tunicamycin G protein is synthesized without oligosaccharide chains and does not localize to the outer surface of the cell membrane (7, 21). T o determine if the retentionof glucose residues also affected the ability of G protein to migrate to thecell surface, we measured the amount of G which reached the plasma membrane in a pulsechase experiment. The amountof G on thesurface was measured using the trinitrobenzenesulfonate method described by Strous andlodish (17) andlodish and Kong (18).

4 L Glucosidase Activity Is Required for VSV Formation Cell line TABLE I1 The effect of temperature and deoxynojirirnycin on the growth of VSV(San Jwn) and VSV(0rsay) in cell lines altered in oligosaccharide processing Infection and labeling of these cells were carried out as described for chicken embryo fibroblasts. The medium was harvested for cells incubated at 40 "C 8 h postinfection and for cells incubated at "C 10 h postinfection. The samples were subjected to centrifugation in a sucrose gradient as described in the legend to Fig. 1. Enzyme SffeCbd VSV(strain) L L PHAR2.7 Glucosidase I1 San Juan BW5147 Clone 6 Multiple Deoxynojirimycin Temperature San Juan San Juan f 40 f 40 Counts per min incorporated into particlea" x "C/ "Cb 15B GlcNAc trans San Juan 4.2 fersse I O a The fractions from the sucrose gradients containing the radioactive particles were pooled and samples were taken to determine the amount of acidinsoluble radioactivity. The counts per min reported are the total counts per min in the peak fractions. The numbers in this column are the total counts per min obtained at 40 "C divided by the total counts per min obtained at "C. c These numbers are the total counts per rnin obtained at 40 "C in the presence of deoxynojirimycin divided by the total counts per min obtained at 40 "C in the absence of the drug "C()/ 40 'C(Y At 40 "C in the presence of 1deoxynojirimycin the production of VSV(San Juan) was inhibited about 10fold, yet the fraction of G on the surface was essentially the same as that detected in the absence of the inhibitor (Table 111). Labeling of G Protein with [3H]PulmitateMany viral and cellular membrane glycoproteins contain covalently bound fatty acid residues (22). An analysis of the addition of fatty acid to the G protein demonstrated that the fatty acid is added at approximately the same time as the protein enters the Golgi vesicles (23) and that it is added to the cytoplasmic tail (24, 25). Schlesinger and Malfer showed that if the addition of fatty acid to G protein is inhibited by cerulenin the protein reaches the cell surface, but virion formation is blocked (16). To determine if deoxynojirimycin, which inhibited VSV(San Juan) formation at 40 "C, affected the addition of fatty acid to G, we labeled infected chicken embryo fibroblasts 4 h postinfection with [3H]palmitate for 2 h. There was a decrease in the incorporation of palmitate at 40 "C, but it was not significantly different from that noted earlier (Table I) for the decrease in incorporation of [35S]methionine. Therefore, the G(San Juan) protein synthesized at 40 "C in the presence of deoxynojirimycin did contain covalently bound fatty acid. DISCUSSION Once the processing pathway for asparaginelinked oligosaccharides had been described the question of whether or not any of the structures along that pathway have a specific function became a focus of interest. Cell variants blocked in a particular step and drugs able to inhibit one of the steps have provided tools with which to answer this question. We have made use of both of these to show that conditions which inhibit the removal of glucose from asparaginelinked oligosaccharides can block the growth of the enveloped virus, VSV. The conclusion that inhibition ofvsv synthesis was due specifically to an effect on the ability of the G protein to mature to a correct functional conformation is based on the following observations: (i) two drugs, deoxynojirimycin and castanospermine, both of which selectively inhibit the processing glucosidases, affected virus growth; (ii) only one of the two strains ofvsv tested was affected and that strain, VSV(San Juan), is known to have a G protein highly sensitive to alterations in oligosaccharide structure; (iii) the effect was to make the formation of VSV(San Juan) temperaturesensitive, a result previously observed with alterations in the oligosaccharides on G protein (68, 10); (iv) a cell variant missing glucosidase I1 activity also became temperaturesensitive in its ability to produce VSV(San Juan) but not VSV(0rsay). Apparently, once the glucose residues are re moved the G protein is no longer sensitive to changes in oligosaccharide structure, since blocking later steps in the processing pathway did not affect the growth of VSV(San Juan) at 40 "C. The most likely interpretation of these results is that the time at which the protein attains a stable conformation coincides with the time the glucose residues have been removed. The synthesis of G(San Juan) under conditions in which glycosylation is totally inhibited leads to the formation of a G protein which does not reach the cell surface and is aggregated when extracted with nonionic detergents (7, 21). In

5 TABLE 111 Percentage of lobeled G protein on the cell surface at 40 "C in the Dresence and absence of deoxvmiirimvcin Strain of VSV and pulsechase conditions San Juan 60min chase 60min chase 60min chase 60min chase Glucosidase Activity Is Required for VSV Formation 7601 Counts Deoxy Counts per min G protein nojiri per rnin immuno on the mycin in extract" prec cell surface ipitatedb x % These numbers are the total counts per min in G obtained in each extract after the pulse with [Y3]methionine or after the pulse and chase periods. The details for measuring the amounts of radiolabeled G have been described (16). * These numbers are the total counts per min in G obtained after treatment of the cells with trinitrobenzenesulfonate and rabbit antitrinitrophenol serum (17, 18). contrast, the G(San Juan) containing oligosaccharides with glucose residues still present moved to the cell surface but was unable to function properly in virion formation. This G protein may have properties similar to those of a temperaturesensitive G protein which reaches the cell surface at nonpermissive temperature but does not participate in virion formation (18). One possible explanation for the malfunctioning of such G proteins is that they are unable to achieve the correct quaternary structure. Recently, however, studies with a soluble form of G lacking the COOHterminal and membranespanning domains suggest that G is a monomer (26). The effects of both 1deoxynojirimycin and castanospermine on several other glycoproteins have been reported (13, 19,20,27). Neither drug alters the growth of influenza viruses (13, 19). In contrast, the compound bromoconduritol, which inhibits the release of the innermost glucose residue on the oligosaccharides, does block the growth of fowl plague virus (28). The removal of that particular glucose moiety may be critical for the proper structure of the hemagglutinin to be attained (28). 1Deoxynojirimycin affects the secretion of some, but not all, cellular glycoproteins. The secretion of IgD, but not that of IgM, is inhibited by 1deoxynojirimycin (27). This drug also prevents the secretion of alproteinase inhibitor (20). These initial results with deoxynojirimycin are reminiscent of the variety of results obtained with tunicamycin (5) and point to differences in protein structure as an explanation for why the retention of the glucose residues on the oligosaccharides is detrimental only for some proteins. Gibson et al. (10) had provided evidence that during the synthesis of the G(San Juan) protein the large precursor oligosaccharide stabilizes the nascent polypeptide. They proposed that the existence of a single major pathway for the synthesis and processing of asparaginelinked oligosaccharides requires that the structure of the oligosaccharide transferred to nascent polypeptides meets the needs of the most sensitive glycoproteins. Based on the results presented here that proposal can be extended to include the requirement for removal of the glucose residues from the oligosaccharide for the proper maturation of some glycoproteins. In addition, our data suggest that the removal of glucose is crucial for the protein to achieve a functional conformation and not for any type of receptor recognition involved in localization of the protein. REFERENCES 1. Struck, D., and Lennarz, W. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W., ed) pp. 3583, Plenum Press, New York 2. Hubbard, S. C., and Ivatt, R. J. (1981) Annu. Rev. Biochem. 50, Kornfeld, R., and Kornfeld, S.. 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