Glycosylation of Vesicular Stomatitis Virus Glycoprotein in

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1 JOURNAL OF VIROLOGY, Dec. 1976, p Copyright C) 1976 American Society for Microbiology Vol. 20, No. 3 Printed in U.S.A. Glycosylation of Vesicular Stomatitis Virus Glycoprotein in Virus-Infected HeLa Cells LAWRENCE A. HUNT* AND DONALD F. SUMMERS Department of Microbiology, University of Utah College of Medicine, Salt Lake City, Utah Received for publication 17 May 1976 Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 min) suggested that the sugar residues that are proximal to the protein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the lightdensity internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars. Vesicular stomatitis virus (VSV), a rhabdovirus that matures by budding through the host cell membrane (11, 23), contains a single species of glycoprotein (G) in association with a nonglycosylated membrane protein (M) and cellular lipids and glycolipids (14, 22). The G protein is glycosylated by host-specified transferases (5) and associates with HeLa plasma membranes in a manner similar to host cell membrane glycoproteins (1, 2). The purified VSV glycoprotein contains about 10% carbohydrate by weight, and the oligosaccharide moieties contain sugars normally found in plasma-type glycoproteins (6, 7, 21). The molecular weight of the carbohydrate side chains of the glycoprotein from VSV grown in HeLa suspension cultures is approximately 2,000, whereas the corresponding glycopeptides from VSV grown in monolayer cultures of a number of mammalian cells have a molecular weight of 3,000 to 4,000 (6, 7, 16, 17). Carbohydrate composition studies (6, 7) and enzymatic digestion studies (17; J. Etchison, submitted for publication) indicate that the carbohydrate side chains have an asparagine-linked core structure [ASPN-GlcNAc( ± FUCOSE)-GlcNAc- (MAN)j] and several distal carbohydrate branches linked to the mannose core (-GlcNAc- GAL-SIALIC ACID). In this study we utilized VSV-infected HeLa cells that were pulse-labeled with radioactive sugar precursors to examine the different steps in the glycosylation and maturation of the VSV glycoprotein. Cell fractionation studies and gel filtration of Pronase-digested glycopeptides suggested that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer in the rough endoplasmic reticulum, followed by the stepwise addition of the distal branch sugars in smooth internal membranes. MATERIALS AND METHODS Cells, virus infection, and radioactive labeling. VSV (Indiana serotype) was grown in HeLa cells as described in the accompanying paper (12). Radioactive virus was obtained by labeling VSV-infected cells from 4 to 16 h postinfection as described previously (17) with radioactive sugars at the following specific activities: L-[1-_4C]fucose, 0.1,uCi/ml (40 to 55 mci/mmol); and D_[1_-4C]glucosamine, 0.1,.Ci/ ml (45 to 55 mci/mmol), both from New England Nuclear. For short-term labeling, cells were infected and labeled at 10 times their normal concentration (4 x 106 to 8 x 106 cells per ml). Unless stated otherwise, cells were collected by centrifugation at 3 h postinfection and resuspended in growth medium minus glucose supplemented with 2 mm glutamine and 5% serum. At 4 h postinfection radioactive precursors were added at the following specific activities: D-[2-3H]mannose, 10 gci/ml (2 Ci/mmol), obtained from Amersham; D-[6-3H]glucosamine, 10 gci/ml (5 to 15 Ci/mmol); D-[1-14C]glucosamine, 2,uCi/ml (45 to 55 mci/mmol); D-[1-3H]galactose, 10,uCi/ml (5 to 10 Ci/mmol); L-[6-3H]fucose, 10,uCi/ml (10 to 15 Ci/mmol); or L-V3S]methionine, 15,tCi/ml 646

2 VOL. 20, 1976 (100 to 400 Ci/mmol), all from New England Nuclear. Fractionation of cell homogenates. Cell homogenates were prepared from radioactively labeled cells and separated into different cellular membrane fractions in discontinuous sucrose gradients as previously described (12). Digestion of glycoproteins with Pronase. Purified virus or cell homogenates were treated with 1% Nonidet P-40 (NP40; Shell Chemical Co.) for 15 min at room temperature and then centrifuged for 90 min at 40,000 rpm in a Beckman 5OTi or 65 rotor. The supernate containing the glycoprotein was extracted with 2 volumes of 1-butanol at room temperature to remove NP40 and glycolipids (J. Etchison, submitted for publication). Pelleted membrane fractions from discontinuous sucrose gradients were resuspended in E-T buffer (1 mm EDTA-10 mm Tris, ph 8.0) with 1% NP40 and directly extracted with butanol. The glycoprotein precipitate from the butanol extractions was digested with Pronase as previously described (16). Chromatography of glycopeptides. Pronase-digested glycopeptides were analyzed on either a Bio- Gel P-4 column (Bio-Rad; 1.5 cm by 90 cm) or a Sephadex G15/G50 column (Pharmacia; 1.5 by 34 cm [G15] and 1.5 by 58 cm [G50]). The following markers were added to the radioactive sample: dextran blue 2000 (Pharmacia), 0.2 mg; stachyose (Sigma; molecular weight, 667), 1 mg; and mannose, 2 mg. A 3H-labeled, acetylated ovalbumin glycopeptide (molecular weight, 1,554; gift of J. Etchison) was also utilized as a marker with some '4C-labeled samples. The columns were equilibrated, and glycopeptides were eluted at a flow rate of approximately 10 ml per h with 0.05 M ammonium acetate, ph 6.0, plus 1 mm NaN3. Fractions of approximately 0.7 or 0.9 ml were collected, samples were analyzed for radioactivity, and 0.1-ml portions were assayed for neutral sugars (to detect the stachyose and mannose markers) by the phloroglucinol method (J. Etchison, submitted for publication). Digestion of glycopeptides with exoglycosidases. Glycopeptides were lyophilized after gel filtration on Sephadex G-10 or G15/G50 columns and digested with 0.5 units of either Clostridium perfringens neuraminidase (Sigma) or Canavalia ensiformis (jack bean) a-mannosidase (Mannheim Boehringer) in a final volume of 1.0 ml. Digestion with neuraminidase was carried out in 0.1 M NaKPO4, ph 5.3, at 37 C for 48 h. Digestion with a-mannosidase was carried out in 0.1 M sodium acetate-0.15 M NaCl mm zinc acetate, ph 4.2, at 25 C for 48 h. A drop of chloroform was added to each digestion to prevent bacterial growth. At the end of the incubation period, the enzyme was inactivated at 100 C for 2 min, and the treated glycopeptides were analyzed by Bio- Gel P-4 chromatography. RESULTS Specific labeling of the virus glycoprotein in VSV-infected HeLa cells. The residual glycosylation of cellular glycoproteins was not significant after host protein synthesis had been GLYCOSYLATION OF VSV GLYCOPROTEIN 647 inhibited in VSV-infected HeLa cells, as shown by the gel electrophoresis of pulse-labeled cell fractions in Fig. 1. With either whole cell homogenates or individual membrane fractions from discontinuous sucrose gradients (see below), the VSV glycoprotein was the only major species that was labeled with radioactive sugar precursors. Identical results were obtained from pulse-labeling with [3H]fucose or from long-term labeling with 3H- or '4C-labeled sugar precursors. Two minor proteins (gpl40, migrating ahead of the L protein; and gp62, migrating slightly ahead of the G protein) could be detected in the mannose-labeled samples and in the light-density membrane fraction of [35S]methionine-labeled cells. The host or virus origin of these glycoprotein species is currently being investigated. Fractionation of cell membranes after pulse-labeling with radioactive sugar precursors. The fractionation of HeLa cell homogenates by equilibrium centrifugation in discontinuous sucrose gradients resulted in three major membrane fractions, which were designated heavy, medium and light on the basis of their relative densities in sucrose. The heavy fraction was shown to be enriched for rough endoplasmic reticulum (RER), and the medium fraction was shown to be enriched for plasma membranes (12). The light fraction was presumed to be enriched for smooth internal membranes. Figure 2 displays the radioactive patterns of the membrane fractions from VSV-infected cells that were pulse-labeled with fucose and glucosamine; Fig. 3 shows the corresponding patterns obtained with radioactive mannose or galactose. Initial incorporation of [3H]fucose and [3H]glucosamine into glycoprotein was observed primarily in the light-density membranes, although some [3H]glucosamine was incorporated into the heavy fraction after the shortest pulse time (Fig. 2). Incorporation into the medium-density membranes was observed with the longer pulse-labeling periods. After a 60-min pulse with either [3H]fucose or [14C]glucosamine, the medium-density fraction was the most heavily labeled membrane fraction. Incorporation of [3H]mannose was observed primarily in the heavy-density membrane fraction after 5- to 10-min pulses (Fig. 3). The medium fraction was more heavily labeled than the light fraction after longer pulses with [3H]mannose. The labeling pattern with [3H]galactose (Fig. 3) was similar to the results with [3H]fucose. These results suggest that fucose, glucosamine, and galactose were incorporated predominantly in the light-density internal mem-

3 648 HUNT AND SUMMERS J. VIROL.,:%m! nw 'Ili Downloaded from CS. -, C A... 7_:ZF f_.. i,_s. 1_ - C * -._l....:. -ti. on August 25, 2018 by guest FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel profiles of VSV-infected HeLa cells pulse-labeled with radioactive sugar precursors or amino acids. Cell homogenates or cell membrane fractions were subjected to electrophoresis on a 1 0% polyacrylamide-sodium dodecyl sulfate slab gel as previously described (12), and the gel was subjected to fluorography (3). The labeling time, radioactive precursor, and cell membrane fraction are indicated below each gel profile. Abbreviations: GlcNH2, [3H]glucosamine; GAL, [3H]galactose; MAN, [3H]mannose; MET, [35S] methionine; H, heavy-density membrane fraction; and L, light-density membrane fraction (all samples without H or L refer to cell homogenates). L, N, and NS are components of the nucleocapsid along with the viral RNA genome, and M and G are viral envelope proteins (22). branes during the initial pulse-labeling periods, whereas mannose was incorporated predominantly in the heavy-density, RER-enriched membranes. All four radioactive labels apparently accumulated in the medium-density, plasma membrane-enriched fraction with

4 VOL. 20, 1976 E CD 00 co C\J 0 4- f FUCOSE A. H M L i i GLYCOSYLATION OF VSV GLYCOPROTEIN 649 B. GLUCOSAMINE H M L i -1.5 E co -05ci j 5 2"-2zo (1-) (CL 1.0< 30 -v u-, 0.5=Bd\^ C-) J 4- bottom top bottom ' 6-4 ID 6O'6d 25 top ' FIG. 2. Distribution of radioactive incorporation into VSV-infected HeLa cells after pulse-labeling with radioactive fucose and glucosamine. In the optical density profiles at the top ofthe figure, H, M, and L refer to the three light-scattering bands at the interfaces of the 44 to 39% sucrose, 39 to 34% sucrose, and 34 to 29% sucrose layers, respectively. (A) VSV-infected cells were pulse-labeled with [3H]fucose in medium with unlabeled glucose for 10, 30, or 60 min, and cell homogenates were fractionated in 29 to 44% discontinuous sucrose gradients. Aliquots of each gradient fraction (0.5 ml for the 10-min sample; 0.2 ml for the 30-min sample; 0.1 ml for the 60-min sample) were assayed for trichloroacetic acid- and ethanol-insoluble radioactivity (1). (B) VSV-infected cells were pulse-labeled with [3H]glucosamine for 5, 10, or 20 min and with ['4C]glucosamine for 60 min. Cell homogenates were fractionated on 29 to 49% discontinuous sucrose gradients (0.2-ml portions ofeach gradient fraction were assayed). The polyacrylamide gel profile of the 20- min [3H]glucosamine-labeled homogenate is displayed in Fig. 1. longer labeling periods. Analysis of pulse-labeled glycopeptides by Sephadex G15/G50 chromatography. In an attempt to detect the smallest intracellular precursors to the completed carbohydrate side chains of G, homogenates of VSV-infected cells that had been pulse-labeled for 5 min with either [3H]glucosamine or [3H]mannose were digested with Pronase and analyzed by gel filtration on a column of Sephadex G15/G50 along with glycopeptides from [14C]glucosamine-labeled VSV. [3H]mannose and [3H]glucosamine were selected because these sugar residues are proximal to the asparagine residues in the G polypeptide (see diagram in Discussion) and should selectively label the smallest Pronasedigested glycopeptides present in the VSV-infected cell. Figure 4 shows that the 14C-labeled virus glycopeptides eluted as a single included peak with an apparent molecular weight of approximately 2,000, as previously reported (17). The glycopeptides from the 5-min pulse-labeled VSV-infected cells also eluted as single major peaks, with apparent molecular weight values of approximately 1,800 for the [3H]glucosamine peak and 1,600 to 1,700 for the [3H]mannose peak. Both of the 3H-labeled glycopeptide peaks eluted ahead of the position of an ovalbumin glycopeptide containing a single amino acid and eight sugar residues. The two extra peaks obtained with the [3H]glucosamine label migrated in the positions expected for sialic acid (fractions 124 to 132) and free glucosamine (fractions 136 to 142). Significant amounts of 3H-labeled glycopeptides were not observed at the lower molecular weight expected for a newly synthesized G peptide containing several amino acids and only a few sugar residues. [3H]- and [14C]glucosamine-labeled glycopeptides from individual membrane fractions of x

5 650 HUNT AND SUMMERS J. VIROL. c\j 0 x L LLJ,) 0, ze z r4') ) 30 top 10 FIG. 3. Distribution of radioactive incorporation into VSV-infected HeLa cells after pulse-labeling with radioactive mannose and galactose. A culture of VSV-infected cells was divided into two equal fractions at 4 h postinfection; one subculture was pulse-labeled for 5, 10, or 20 min with [fhlmannose, and the other subculture was pulse-labeled for 5, 10, or 20 min with [3H]galactose. The experimental conditions of cell fractionation were identical to those in Fig. 2B except that 0.1-ml portions of each gradient fraction were assayed for trichloroacetic acid- and ethanol-insoluble radioactivity. The three major membrane fractions are indicated by vertical arrows (H, M, and L from left to right). The polyacrylamide gel profiles of the 20-min mannose sample (homogenate and heavy-density membrane fraction) and the 20-min galactose sample (homogenate and light-density membrane fraction) are displayed in Fig H-GIcNH2 A Viral 14C-GIcNH2 ie '3H-MAN Viral Virl 14C-GIcNH2 t II I I FIG. 4. Sephadex G151G50 chromatography of Pronase-digested glycopeptides from glucosamine-labeled VSV and VSV-infected cells pulse-labeled with either glucosamine or mannose. The 3H-labeled cellassociated and '4C-labeled virus samples were extensively digested with Pronase, mixed, and analyzed by gel filtration. The fraction volumes were approximately 0.9 ml in the top profile and 0.7 ml in the bottom profile. The elution positions of the carbohydrate markers chromatographed with the radioactive VSV-infected HeLa cells were also chromatographed on Sephadex G15/G50 to follow the stepwise growth ofthe carbohydrate side chains -1.0 of the G protein through the different classes of cell membranes. Figure 5 shows the gel filtration patterns for the glycopeptides from the -0.5 c\ heavy-density membranes (Fig. 5A) and light- O density membranes (Fig. 5B) that were labeled x for 20 min with [3H]glucosamine compared with the glycopeptides from the medium-density Q c- membranes that were labeled for 60 min with I1[4C]glucosamine. Glycopeptides from all three membrane fractions eluted as single major peaks. Glucosamine-labeled material also eluted in the position of sialic acid and free glucosamine. The :3H-labeled glycopeptides from the heavy fraction had a slightly lower apparent molecular weight than the '4C-labeled glycopeptides from the medium fraction. The profiles for the 3H-labeled light fraction and the '4C-labeled medium fraction glycopeptides were samples are indicated by solid vertical arrows, from left to right: blue dextran, stachyose (molecular weight, 666) and mannose (molecular weight, 180). The dotted vertical arrow indicates the peak elution position of a Hd-labeled acetylated ovalbumin glycopeptide (molecular weight, 1,553), which was determined on the same column in a separate doublelabeled experiment with the f'4c]glucosame-labeled virus glycopeptides.

6 VOL. 20, FIG. 5. Sephadex G151G50 chromatography of Pronase-digested glycopeptides from membrane fractions of glucosamine-labeled VSV-infected HeLa cells. Peak radioactive fractions of each membrane class in the 20-min [:H]glucosamine- and 60-min ["Clglucosamine-labeled samples shown in Fig. 2B were pooled, concentrated by high-speed centrifugation, and digested with Pronase. Gel filtration of glycopeptides was performed as described in Fig. 4, with 0.9-ml fraction volumes. almost identical, with a peak molecular weight similar to the value for virus glycopeptides shown in Fig. 4. The combined results of cell membrane fractionation and gel filtration of Pronase-digested glycopeptides suggest that G is initially glycosylated in the heavy-density membranes, the apparent site of mannose incorporation (Fig. 3), and additional glycosylation takes place in the light-density membranes, where fucose and galactose are initially incorporated (Fig. 2 and 3). Comparison of cell-associated and virus glycopeptides by Bio-Gel P-4 chromatography. An alternative method of chromatography was tried next in an attempt to obtain better resolution between intracellular precursor glycopeptides and virus-sized glycopeptides. Figure 6 presents the Bio-Gel P-4 gel filtration profile of [E4C]glucosamine-labeled glycopeptides from purified virus (Fig. 6A) and the residual cell-associated glycopeptides from the same 16-h labeling of VSV-infected HeLa cells. Chromatography of virus glycopeptides on this resin gave a significantly altered profile compared to the Sephadex G15/G50 profile in Fig. 4. In addition to material eluting with the void volume, there were two minor peaks, desig- GLYCOSYLATION OF VSV GLYCOPROTEIN 651 nated S1 and S2, and one major peak, S3. The virus S3 peak appeared to elute as a doublet, but the same [14C]glucosamine-labeled glycopeptides sometimes eluted as a single broad peak in other gel filtration experiments (see t Fig. 9). The width of these glycopeptide peaks 5 i was relatively broad compared to the 3H-lac\J beled ovalbumin marker glycopeptide, indicat- Q ing a possible heterogeneity in the number of x sugar residues and/or the number of amino 2 acids. Extensive digestion of the G protein from u purified virus resulted in glycopeptides cons taining approximately two to four amino acids t each (J. Etchison, submitted for publication). The profile of cell-associated glycopeptides (Fig. 6B) contained the same major peaks (void v 11 III II,I 2- Cellular 14C-GIcNH2 + c Siolidose FIG. 6. Bio-Gel P-4 chromatography of Pronasedigested glycopeptides from glucosamine-labeled VSV and VSV-infected HeLa cells. ['4C]glucosamine-labeled glycopeptides were obtained from both released VSV and VSV-infected cells after labeling from 4 to 16 h postinfection and co-chromatographed on Bio-Gel P-4 with a 3H-labeled ovalbumin glycopeptide marker (molecular weight, 1,553). The void volume is indicated by the vertical arrow, and the peak elution positions for unlabeled stachyose and mannose were fractions 165 and 190, respectively (fraction volume of 0.7 ml). The three major peaks of virus glycopeptides are designated S,, S2, and S3 by their order ofelution. (C) Elution profile ofportion of the cell-associated glycopeptides that was desalted on a Sephadex G-10 column and digested with neuraminidase (sialidase) prior to Bio-Gel P-4 chromatography.

7 652 HUNT AND SUMMERS material, S,, S2, and S3) as the virus glycopeptides. However, the S3: peak was broader and contained additional material eluting after the ovalbumin glycopeptide. Treatment of these cell-associated glycopeptides with neuraminidase converted most of the S, glycopeptides and a large fraction of the S2 glycopeptides to species which eluted ahead of the ovalbumin glycopeptide (Fig. 6C). The '4C-labeled material in fractions 130 to 150 probably corresponded to enzymatically released sialic acid. This comparison of virus glycopeptides and residual cellassociated glycopeptides indicated that some of the G protein (represented by the more slowly eluting S3-type glycopeptides) in VSV-infected HeLa cells was incompletely glycosylated and did not associate with released virus. The chromatography of untreated and neuraminidase-treated glycopeptides from a 30-min pulse-label of VSV-infected HeLa cells with [LH]galactose demonstrated more clearly that glycopeptides S, and S2 eluted ahead of glycopeptide S3 on Bio-Gel P-4 because they contained negatively charged sialic residues (Fig. 7). Keegstra et al. (13) have reported a similar conversion of Sindbis virus glycopeptides (similar in size to VSV glycopeptides S,-S,) S, and S2 to glycopeptide S, after neuraminidase treatment and re-chromatography on Bio-Gel P-6 columns. The '4C-labeled virus S,-S: glycopeptides that co-migrated on Bio-Gel P-4 with the galactose-labeled, cell-associated glycopeptides (Fig. 7A) were all derived from the single, apparently homogeneous peak obtained from gel filtration on Sephadex G15/G50 (see' Fig. 4). Thus, the elution of glycopeptides on Bio-Gel P- 4 was affected by factors in addition to molecular size (presumably an exclusion due to the negative charges of the terminal sialic acid residues), and reliable values for the molecular weight of the S,,, S,, and S glycopeptides could not be obtained from their relative elution positions. Analysis of sugar-labeled glycopeptides from VSV-infected cells after different pulselabeling periods. The elongation of the carbohydrate side chains of the VSV glycoprotein could be followed in more detail by varying both the length of pulse-labeling and the species of radioactive sugar and assaying the progressive increase in the size of the resulting glycopeptides. VSV-infected HeLa cells were, therefore, labeled with one of four different sugar precursors (3H-labeled glucosamine, mannose, galactose, or fucose) for periods ranging from 5 to 60 min, and Pronase-digested glycopeptides were obtained from whole cell homogenates or specific membrane fractions. Figure 8 shows the Bio-Gel P-4 elution profile J. VIROL. FIG. 7. Bio-Gel P-4 chromatography of untreated and sialidase-treated glycopeptides from galactoselabeled VSV-infected cells. Pronase digests from cells that were pulse-labeled for 30 min with [3H]galactose were prerun on a Sephadex G151G50 column to eliminate undigested material and free radioactive galactose. These 3H-labeled cell-associated glycopeptides (either untreated or sialidase treated) were co-chromatographed with Pronase-digested glycopeptides from [f4cjglucosamine-labeled VSV (also prerun on Sephadex G151G50). The fraction volumes for both profiles were 0.9 ml, and the peak elution positions of the carbohydrate markers were fractions 138 (stachyose) and 158 (mannose). The void volume is indicated by a vertical arrow, and the major 3H-labeled glycopeptides peaks by S,,-S3. of glycopeptides from cells that were pulse-labeled with [3H]glucosamine for 5, 15, and 30 min. The 5-min sample was identical to the 3Hlabeled sample in the top profile of Fig. 4. This sample eluted on Bio-Gel P-4 with a single major glycopeptide peak in addition to material in the void volume and in the positions of sialic acid (fractions 120 to 135) and glucosamine (fractions 146 to 152). The elution profiles of the 15- and 30-min samples were essentially identical to the profile of the Pronase digest from VSV-infected cells that were labeled for 23/4 h with [14C]glucosamine. Small amounts of S,- and S2-type glycopeptides (fractions 68 to 80) were observed in these :H- and '4C-labeled samples in addition to the major glycopeptide peak observed in the 5-min pulse-labeled sample. The comparison of the 2.75-h sample with the ovalbumin glycopeptide (Fig. 8D) indicated that the major ['4C]glucosamine-labeled glycopeptide peak from cells that were pulse-labeled for several hours eluted with an apparently lower molecular weight than the S:, glycopep-

8 VOL. 20, 1976 D 23/4hr 14C-GIcNH2 40- r,1xy /Y J '. LI- x FIG. 8. Bio-Gel P-4 chromatography of Pronasedigested glycopeptides from VSV-infected HeLa pulse-labeled with radioactive glucosamine. Glycopeptides were obtained from a culture of VSV-infected cells that was pulse-labeled with [3H]glucosamine for 5, 15, or 30 min and with ['4C]glucosamine for 2.75 h. The elution positions of the three carbohydrate markers are indicated with vertical arrows as described in the legend to Fig. 4. The 3H-labeled, pulse-labeled, cell-associated glycopeptides were co-chromatographed with the "4C-labeled cell-associated glycopeptides labeled for 2.75 h, with a fraction volume of0.9 (A, B, and C). Elution ofthe 3H-labeled ovalbumin glycopeptide with cell-associated glycopeptides (2.75 h labeling period), which were. prerun on Sephadex G15/G50, with a fraction volume of 1.1 ml, is shown in (D). tides from purified virus or the long-term labeled cells shown in Fig. 6. These results suggested that the glycosylation of intracellular G protein, which was monitored by radioactive glucosamine labeling, was either slow or inefficient. The results of the analogous experiment using [3H]mannose as the specific sugar precursor are shown in Fig. 9. The glycopeptides from the VSV-infected cells that were pulse-labeled for either 5 or 20 min eluted as a broad peak with a lower apparent molecular weight than the S3- type glycopeptides from the [14C]glucosaminelabeled virus glycopeptides. This mannose-labeled peak also eluted after the expected posi- -2 -I GLYCOSYLATION OF VSV GLYCOPROTEIN 653 tion of the ovalbumin marker (compare Fig. 6A and Fig. 9A), in contrast to elution of the 5-min mannose-labeled glycopeptides ahead of the ovalbumin glycopeptide on Sephadex G15/G50 in Fig. 4 (the double-labeled sample displayed in Fig. 9A was identical to the sample analyzed in Fig. 4B). Virus-like glycopeptides (Si, S2, and S3) were observed only after longer labeling periods with [3H]mannose, as shown in the gel filtration profile of the glycopeptides from the 60-min light membrane fraction. The major glycopeptide peak still eluted with the glycopeptides from the 5- and 20-min pulses, but small S, and S peaks were observed along with a "shoulder" (fractions 85 to 90) on the major peak that eluted with the virus S3 glycopeptides. Treatment of [3H]mannose-labeled glycopeptides from cells that were pulse-labeled for 5 min with a-mannosidase converted over c 3- ~~~60' 3H- MAN - Iv Light 2- I I:b FIG. 9. Bio-Gel P-4 chromatography of Pronasedigested glycopeptides from VSV-infected HeLa cells pulse-labeled with radioactive mannose. [3H]mannose-labeled glycopeptides from homogenates or membrane fractions of VSV-infected cells were cochromatographed with the glycopeptides from ['4C]glucosamine-labeled VSV (analysis shown in Fig. 6). The fraction volume was approximately 0.7 ml for the 5- and 20-min samples (from the labeling experiment described in the legend to Fig. 3), and the peak elution positions ofstachyose and mannose were fractions 164 and 190, respectively. (C) Glycopeptides from the light-density membrane fraction. Fraction volume was 0.9 ml, and the peak elution of stachyose and mannose was at fractions 140 and 160.

9 654 HUNT AND SUMMERS two-thirds of the radioactivity in the single major peak to free mannose (data not shown). The exoglycosidase treatment of the 60-min pulselabeled sample did not affect the S,-S:,-type glycopeptides but converted the majority of the radioactivity in the major glycopeptide peak to free mannose (data not shown). These results indicated that most of the LH]mannose residues eluting in the major broad peak were in a terminal position in the carbohydrate side chains of the pulse-labeled, cell-associated G protein. In contrast to the results with radioactive glucosamine and mannose, the distribution of [PH]galactose-labeled glycopeptides was shifted toward more completely glycosylated carbohydrate side chains, as demonstrated in Fig. 7 for a 30-min pulse-labeled sample. The galactoselabeled glycopeptides from shorter pulse-labeling periods also had a profile similar to [14C]glucosamine-labeled glycopeptides from purified virus (Fig. 10). S,- and S,-type glycopeptides were apparently present after a 5-min pulse, when most of the [:H]galactose-labeled glycoprotein was present in the light-density membrane fraction (Fig. 3). The 15-min galactose-labeled sample was almost identical to the 30-min sample in Fig. 7. However, the galactose-labeled glycopeptides differed dramatically from the 2.75-h [14C]glucosamine-labeled sample with respect to both the elution position of the major glycopeptide peak and the relative amounts of the sialic acid-containing glycopeptides. The presence of the sialic acid-containing glycopeptides (Se,, S,, and S,) in the Pronase digest of the light membrane fraction after a 20-min pulse-label with [:1H]galactose is clearly demonstrated in Fig. 10C, suggesting that the addition of galactose and sialic acid may occur at the same intracellular membrane site. The gel filtration profiles of [3H]fucose-labeled glycopeptides from VSV-infected cells are presented in Fig. 11 in comparison with the corresponding profile of [ 14C]fucose-labeled glycopeptides from purified virus. The glycopeptides from the 10-min pulse-labeled sample differed from the virus glycopeptides in two respects: (i) the major 3H-labeled peak eluted slightly behind the S3-type glycopeptide peak from ['4C]fucose-labeled virus; and (ii) the 'Hlabeled sample contained relatively fewer Sitype glycopeptides (fractions 75 to 83). The glycopeptides from the VSV-infected cells labeled for 40 min with [3H]fucose had a gel filtration profile that was almost identical to that of the virus glycopeptides. The glycopeptides from the light membrane fraction of pulse-labeled VSVinfected cells also co-chromatographed with the [14C]fucose-labeled virus glycopeptides, al- C\J I0 -' 2 N.~~~~~~~~~C. 7 0_, c 20' 3H-GAL ' 8 Light ~ FIG. 10. Bio-Gel P-4 chromatography of Pronasedigested glycopeptides from VS V-infected HeLa cells pulse-labeled with radioactive galactose. [PHigalactose-labeled glycopeptides from cell homogenates or membrane fractions of pulse-labeled VSV-infected cells were co-chromatographed with [14C]glucosamine-labeled glycopeptides from VSV-infected cells that were labeled for 2.75 h (fraction volume of 0.9 ml). The void volume is indicated by a vertical arrow, and the peak elution positions of stachyose and mannose were fractions 140 and 160. The "Clabeled glycopeptides displayed in the middle profile were prerun over Sephadex G151G50 (identical to the "4C-labeled sample at the bottom of Fig. 8). The 5- and 15-min samples were from the same labeling experiment as the 30-min sample in Fig. 7. though the sialic acid-containing glycopeptides were not as well resolved. A direct comparison of the fucose-labeled virus glycopeptides with the ovalbumin marker glycopeptide indicated that the fucose-labeled S:3 glycopeptides did not contain the lower-molecular-weight component of the ['4C]glucosamine-labeled S,-type glycopeptides which co-eluted with the ovalbumin glycopeptide (compare Fig. 6A with Fig. lid). DISCUSSION VSV-infected HeLa cells were used to monitor the different steps in the glycosylation of a single species of membrane-like glycoprotein. We have demonstrated that the incorporation of radioactive sugar precursors into glycoprotein in virus-infected cells represents a relatively pure population of a single virus-specific species, protein G (Fig. 1). The cell fractiona- J. VIROL

10 VOL. 20, 1976 tion procedures used in the present study can be valuable in deciphering the intracellular localization of specific glycosylation events. The distinct pulse-labeling patterns obtained with different radioactive sugar precursors (Fig. 2 and 3) indicated that more than one subcellular organelle was involved in the synthesis of complete carbohydrate side chains with the following putative structure (17; J. Etchison, submitted for publication): GLYCOSYLATION OF VSV GLYCOPROTEIN 655 structures were added in a glycosylation step separate from the addition of mannose residues in VSV-infected HeLa cells. The site of addition of these N-acetylglucosamine residues was difficult to determine, since radioactive glucosamine was added to three distinct sites on the carbohydrate side chains: (i) proximal to the asparagine residue in the polypeptide backbone; (ii) in the branch structures adjacent to mannose; and (iii) at the terminal nonreducing a.a. fucose -GlcNAc - gal + sialic acid I (+) / aspn -GlcNAc - GlcNAc - (man)4-glcnac - gal + sialic acid a.a. Mannose residues were apparently added to the VSV G protein in the heavy-density, RERlike membranes (Fig. 3). This membrane fraction has been implicated as the biosynthetic site for the polypeptide chain of the virus glycoprotein (8, 9). The initial glycosylation event(s) may have occurred while G was still bound to ribosomes as a nascent polypeptide chain or shortly after biosynthesis of the polypeptide was completed. The gel filtration analysis of pulse-labeled glycopeptides in Fig. 4 suggested that a glucosamine- and mannose-labeled core region (see above diagram) was added en bloc to the specific asparagine residues in the G protein, since the smaller precursor glycopeptides of heterogeneous size that would be expected from the stepwise addition of single N-acetylglucosamine and mannose residues were not observed. This glycosylation step is consistent with the putative preassembly of oligosaccharides on a specific class of membrane-bound lipids (dolichols) and the single-step transfer of the oligosaccharides to acceptor polypeptides (15, 20). An alternative, but less likely, glycosylation model is the addition of the first two N-acetylglucosamine residues in a slow step, with subsequent addition of the mannose core in a rapid step. The broad peak of mannose-labeled glycopeptides obtained after pulse-labeling periods of 5 to 20 min (Fig. 9) may have been indicative of some heterogeneity in the number of mannose residues per carbohydrate side chain. Virus-associated glycoprotein apparently has four mannose residues per carbohydrate side chain (6, 7), but a heterogeneous population of oligomannose structures has been found in total cellular glycoproteins from a single cell type (19). The a-mannosidase digestion of pulse-labeled glycopeptides suggested that the N-acetylglucosamine residues in the distal branch GlcNAc - gal ± sialic acid positions as sialic acid. Galactose and fucose residues were apparently added in light-density structures that were presumably enriched for smooth internal membranes (Fig. 2 and 3). These membranes probably included the Golgi apparatus, which has been reported to be enriched for specific glycosyltransferase activities (4, 10, 18). The presence of sialic acid in pulse-labeled glycopeptides from the light-density membranes (Fig. 10 and 11) suggested that these terminal sugar residues were added at the same intracellular membrane site as fucose and galactose. The glycosylation process was probably completed before the G protein associated with the medium density membranes, but our evidence did not rule out the possible addition of some sugar residues at the plasma membrane. The branch sugars (N-acetylglucosamine, galactose, and sialic acid) were not necessarily added to the growing carbohydrate side chains as oligosaccharide units, since many of the glycopeptides contained galactose but not sialic acid (Fig. 7 and 10). These sugar residues, like fucose, were probably added as individual units. The order of addition of the branchedchain sugar species could not be determined from the pulse-labeling studies, presumably because their sequential addition occurred independently for each of the three postulated branch structures for any given carbohydrate side chain. The addition of fucose occurred late in the glycosylation process as noted earlier (2), but possibly before all of the galactose and sialic acid had been added (Fig. 11). Our results also suggested that the addition of sialic acid residues to the VSV G protein was less efficient in HeLa cells grown in suspension culture than with cells grown in monolayer cultures: a large fraction of the virus glycopeptides contained no sialic acid (indicated by the neuraminidase-resistant S3 peak in Fig. 7), whereas the glycopeptides from VSV grown in

11 656 HUNT AND SUMMERS J. VIROL. been greater than 20 min, since [3VH]mannose 0.6 A 10' 3H-Fucose 8 was added in an early glycosylation step but did not appear in full-size carbohydrate side chains 04 (detected as Si-S:3 glycopeptides on Bio-Gel P-4) until pulse-labeling periods longer than 20 min 0.2 and shorter than 60 min (Fig. 9). One contribution to this long processing time was the transit time between the biosynthetic site of the polypeptide (RER) and the light-density internal membranes, where fucose and the branchedchain sugar residues were added. The glyco- 4 B. 40' 3H- e< Fucose -.0 sylation process at this second major intracellular site may have been relatively rapid. Most of the [3H]galactose and [3H]fucose that was incorporated into glycoprotein during 5- to 10-min 2 -~~~~~~~~~~~.5 pulses appeared in full-size carbohydrate side c\j 2 C 20' 3H-Fucose -2cj I0 -ucose /tQj_.5 FIG. 11. Bio-Gel P-4 chromatography of Pronasedigested glycopeptides from VSV and VSV-infected HeLa cells labeled with radioactive fucose. [3H]fucose-labeled cell-associated glycopeptides and f'4c]fucose-labeled virus glycopeptides were co-chromatographed on Bio-Gel P-4. The carbohydrate markers in (A) (fraction volumne of 0.7 ml) eluted at peak fractions of 165 (stachyose) and 190 (mannose). The fraction volume was 0.9 ml for (B, C, and D), with peak elution positions ofstachyose and mannose at fractions 140 and 160. (D) Comparison of the elution of the :H-labeled ovalbumin glycopeptide with the ['4Clfucose-labeled virus glycopeptides. a number of monolayer cultures contain approximately two to three sialic residues (7). Virus-associated glycoprotein may also have been deficient in galactose, since the pulselabeling of VSV-infected cells with [3:H]galactose apparently selected for a population of glycopeptides that was more completely glycosylated than ["4Cifucose- or [14C]glucosaminelabeled virus glycopeptides (Fig. 7). The total time required to glycosylate the G protein in VSV-infected HeLa cells must have chains (So,-S:, glycopeptides; Fig. 10 and 11). The processing time for the VSV G protein once it associated with the plasma membrane was also expected to be minimal, since the glycosylation was nearly complete and there was no apparent lag between the accumulation of fucose-labeled G at the plasma membrane and its appearance in released virus (2). A fraction of the VSV glycoprotein (preferentially labeled with mannose) apparently was not processed through the light-density internal membranes but associated directly with the plasma membrane as incompletely glycosylated G (Fig. 3 and 9). This population of VSV glycoprotein lacked the branched-chain sugars that were apparently added in the smooth internal membranes, and these incompletely glycosylated G polypeptides were not detected in released virus. This abortive process was consistent with the rapid association of some [35S]methionine-labeled G with plasma membranes prior to both the high rate of accumulation observed after a 20-min delay (2) and the appearance of newly synthesized glycoprotein in released virus after a delay of approximately 45 min (12). The experiments reported here and the previous pulse-labeling studies with [3H]fucose and [35S]methionine (2, 12) suggested that there were at least three major steps in the synthesis and maturation of the VSV glycoprotein prior to its appearance in released virus: (i) biosynthesis of the G polypeptide and addition of the proximal sugar residues (glucosamine and mannose core) in the rough endoplasmic reticulum; (ii) transport to the smooth internal membranes where the distal sugar residues were added (fucose and branch sugars); and (iii) association with the plasma membrane where the glycoprotein was assembled into virus particles along with the other envelope and nucleocapsid components.

12 VOL. 20, 1976 ACKNOWLEDGMENTS This research was supported by Public Health Service grant no. 1 R01 A from the National Institute of Allergy and Infectious Diseases and by National Science Foundation grant no. BMS A01 to D.F.S. L.A.H. is a postdoctoral Research Fellow of the Cystic Fibrosis Foundation. D.F.S. is a recipient of an American Cancer Society Faculty Award. LITERATURE CITED 1. Atkinson, P.H Synthesis and assembly of HeLa cell plasma membrane glycoproteins and proteins. J. Biol. Chem. 250: Atkinson, P. H., S. A. Moyer, and D. F. Summers Assembly of vesicular stomatitis virus glycoprotein and matrix protein into HeLa cell plasma membranes. J. Mol. Biol. 102: Bonner, W. M., and R. A. Laskey A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46: Bosmann, H. B., A. Hagopian, and E. H. Eylar Glycoprotein biosynthesis: the characterization of two glycoprotein:fucosyl transferases in HeLa cells. Arch. Biochem. Biophys. 128: Burge, B. W., and A. S. Huang Comparison of membrane protein glycopeptides of Sindbis virus and vesicular stomatitis virus. J. Virol. 6: Etchison, J. R., and J. J. Holland Carbohydrate composition of the membrane glycoprotein of vesicular stomatitis virus. Virology 60: Etchison, J. R., and J. J. Holland Carbohydrate composition of the membrane glycoprotein of vesicular stomatitis virus grown in four mammalian cell lines. Proc. Natl. Acad. Sci. U. S. A. 71: Grubman, M. J., E. Ehrenfeld, and D. F. Summers In vitro synthesis of proteins by membranebound polyribosomes from vesicular stomatitis virus infected HeLa cells. J. Virol. 14: Grubman, M. J., S. A. Moyer, A. K. Banerjee, and E. Ehrenfeld Subcellular localization of vesicular stomatitis virus messenger RNAs. Biochem. Biophys. Res. Commun. 62: Hagopian, A., H. B. Bosmann, and E. H. Eylar Glycoprotein biosynthesis: the localization of polypeptidyl:n-acetylgalactosaminyl, collagen:glucosyl, and glycoprotein:galactosyl transferases in HeLa cell membrane fractions. Arch. Biochem. Biophys. GLYCOSYLATION OF VSV GLYCOPROTEIN : Howatson, A. F., and G. F. Whitmore The development and structure of vesicular stomatitis virus. Virology 16: Hunt, L. A., and D. F. Summers Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus. J. Virol. 20: Keegstra, K., B. Sefton, and B. W. Burge Sindbis virus glycoproteins: effect of the host cell on the oligosaccharides. J. Virol. 16: Klenk, H.-D., and P. W. Choppin Glycolipid content of vesicular stomatitis virus grown in baby hamster kidney cells. J. Virol. 7: Lucas, J. J., C. J. Waechter, and W. J. Lennarz The participation of lipid-linked oligosaccharides in synthesis of membrane glycoproteins. J. Biol. Chem. 250: Moyer, S. A., and D. F. Summers Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation. Cell 2: Moyer, S. A., J. M. Tsang, P. H. Atkinson, and D. F. Summers Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus. J. Virol. 18: Munro, J. R., S. Narasimhan, S. Wetmore, J. R. Riordan, and H. Schachter Intracellular localization of GDP-L-fucose:glycoprotein and CMP-sialic acid:apolipoprotein glycosyl-transferases in rat and pork livers. Arch. Biochem. Biophys. 169: Muramatsu, T., N. Koide, and M. Ogata-Arakawa Analysis of oligomannosyl cores of cellular glycopeptides by digestion with endo-,3-n-acetylglucosaminadases. Biochem. Biophys. Res. Commun. 66: Oliver, G. J. A., J. Harrison, and F. W. Hemming The mannosylation of dolichol-diphosphate oligosaccharides in relation to the formation of oligosaccharides and glycoproteins in pig liver endoplasmic reticulum. Eur. J. Biochem. 58: Spiro, R. G Studies on fetuin, a glycoprotein of fetal serum. II. Nature of the carbohydrate units. J. Biol. Chem. 237: Wagner, R. R., L. Prevec,, F. Brown, D. F. Summers, F. Sokol, and R. MacLeod Classification of rhabdovirus proteins: a proposal. J. Virol. 10: Zee, Y. C., A. J. Hackett, and L. Talens Vesicular stomatitis virus maturation in six different host cells. J. Gen. Virol. 7: