Role of Glycosylation in Transport of Vesicular Stomatitis Virus

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 hy The American Society of Biological Chemists, Ine.

Vol. 261, No. 19, Issue of July 5, pp. 8936-8943, 1986 Printed in U.S.A.

Role of Glycosylation in Transport of Vesicular Stomatitis Virus Envelope Glycoprotein A NEW CLASS OF MUTANT DEFECTIVE IN GLYCOSYLATION AND TRANSPORT OF G PROTEIN*

(Received for publication, July 7, 1985)

Girish J. Kotwal$§, R. Mark L. Bullerg, William H. Wunnerll, Craig R. PringleII, and Hara P. GhoshS $$ From the $Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 325 and the Medical Research Center Virology Unit, Church Street, Gkzsgow GI1 5JR, Scotland

A temperature-sensitive mutant (ts yl) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 "C) a glycoprotein G whose size is smaller (M, 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 "C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NHz-terminal sequence analysis of the various glycoproteins suggest that at 32 "C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 "C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.

Enveloped animal viruses such as vesicular stomatitis virus (VSV') provide an elegant system for examining the biosynthesis of membrane glycoproteins (1). Biosynthesis of a membrane glycoprotein molecule is a complex process involving intracellular transport of the molecule and sequence-specific attachment of the oligosaccharide or fatty acid molecules occurring in precise organelles (1-6).

Temperature-sensitive (ts) mutants assigned to complementation group V of VSV Indiana, which include the mu-

* This work was supported by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§Present address: Lab. of Viral Diseases, NIH, Bethesda, MD 20205.

n Present address: Wistar Inst. 36th St. at Spruce, Philadelphia, PA 19104.

11 Present address: Dept. of Biological Sciences, Univ. of Warwick, Coventry, Warwickshire CV4 7AL, England.

$$To whom all correspondence should be addressed Dept. of Biochemistry, McMaster Univ., 1200 Main St. W., Hamilton, On- tario, Canada L8N 325.

The abbreviations used are: VSV, vesicular stomatitis virus; ts, temperature-sensitive; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; m.o.i., multiplicity of infection.

tants with specific lesion(s) in the structural gene coding for the viral glycoprotein (G protein), have been isolated on the basis of their ability to grow at the permissive temperature of 32 "C but not at 39 "C (nonpermissive) (7-9). These all appear to be glycosylation-defective mutants and are being used to elucidate the steps involved in the biosynthesis of a glycoprotein molecule and to study the role of post-translational modifications at different stages in maturation of viral glycoproteins (5, 10-12). Two classes of group V ts mutants derived from the VSV Indiana serotype have been character- ized. One class of mutants includes ts 045 and ts L513, which, at the nonpermissive temperature, accumulates G protein containing only the high mannose core oligosaccharide chain at the endoplasmic reticulum (lo, 11); whereas mutants of the other class, represented by ts L511, at the nonpermissive temperature are blocked in the fatty acid acylation of G protein (11, 12).

The importance of glycosylation in transport of viral envelope glycoprotein has been studied using the antibiotic tunicamcyin, which inhibits glycosylation of nascent glycoproteins (13). Studies with a number of viruses, such as the Orsay strain of VSV Indiana (14, 15) and influenza virus (16, 40), showed that the nonglycosylated glycoprotein can reach the cell surface. In contrast, inhibition of glycosylation of glycoproteins of the San Juan strain of VSV Indiana (17), Sindbis virus (17), and herpes simplex virus type 1 (18) with tunicamycin resulted in blocking of their intracellular transport to and expression at the cell surface. We have, however, studied, without the use of any glycosylation inhibitor, the role of glycosylation of proteins by using a new class of glycosylation mutant o f VSV which is defective at both the permissive and nonpermissive temperatures.

In this study, we describe a new class of ts mutant from the Cocal strain of VSV belonging to complementation group y l (13). This mutant, Cocal ts y l G (13) (ts yl) , is defective in the glycosylation of G protein such that at the permissive temperature only one of the two available glycosylation sites is glycosylated. The G protein containing only one oligosaccharide residue is transported to the cell surface and incorporated in infectious virus particles. At the nonpermissive temperature, neither of the two sites is available for glycosylation, and a nonglycosylated G protein is synthesized. The nonglycosylated G protein is not transported to the plasma membrane, suggesting the requirement of glycosylation of G protein for cell surface transport.

8936

Role of Glycosylation in G Protein Transport 8937 TABLE I

Assay of infectivity of wild-type VSV Cocal and ts y l viral particles L cells were infected with VSV Cocal wild-type or mutant ts y l at

either 32 or 39 "C. After adsorption at 32 "C, the cells were grown at 32 or 39 "C, and plaques produced were counted as described under "Materials and Methods."

virus Temperature pfu,m,m Plaquing efficiency of assay 39/32 'C

Cocal wild-type 32 "C 2 x 1O'O 0.75 39 "C 1.5 X 10"

ts y l 32 "C 39 "C

6 X 10" 1.5 X 9 X 105

MATERIALS AND METHODS*

RESULTS

Analysis of Virus Particles-Plaque assays were performed on wild-type VSV Cocal and ts y l virus stocks at the permissive (32 "C) and nonpermissive (39 "C) temperatures to con- firm the ts nature of the mutant. The size of the plaques a t 39 "C forts y l was much smaller than those at 32 "C, whereas VSV Cocal wild-type plaques were of the same size at both temperatures. As shown in Table I, in the case of ts y l virus, the plaquing efficiency ratio between 32 and 39 "C was of the order of as compared to 0.75 in the case of VSV Cocal wild-type virus.

Analysis of [35S]methionine-labeled virus particles produced at 32 and 39 "C by sucrose density gradient centrifugation showed that the proportion of noninfectious ts y l particles produced at 39 "C was 35% of the infectious particles produced at 32 "C. It should be noted that, although the reduction in particle formation a t nonpermissive temperature was 65%, there was more than 99.9% decrease in infectivity. Analysis of the bouyant density showed that the noninfectious ts y l particles had a density of 1.08 g/ml compared to a value of 1.09 g/ml obtained for infectious ts y l or Cocal wild-type virus particles.

The proteins present in the [35S]methionine-labeled virus particles were analyzed on SDS-polyacrylamide gels (Fig. 1). The results show that the L, N, and M proteins of the VSV Cocal wild-type and ts y l virus particles have similar mobil- ities. The G protein from infectious ts y l virus particles (produced at 32 "C) has a lower apparent molecular weight of 68,000 (lane b) compared to the apparent molecular weight of G protein (71,000) present in VSV Cocal wild-type virus (lanes a and c). However, the noninfectious ts y l virus particles released a t 39 "C totally lacked G protein (lane d).

Thermal Inactivation Profiles of Wikl-type Virus and ts Mutants-The heat inactivation kinetics of VSV Cocal mutant ts y l and Cocal wild-type virus were compared with VSV Indiana ts 045 and VSV Indiana wild-type virus (Fig. 2) as well as VSV Cocal ts mutants representing complementation groups a, 0, and 6 (data not shown) to differentiate those mutants with thermal labile defects in envelope proteins from mutants defective in nucleocapsid proteins. The heat inactivation of ts 045 virus shown here confirmed previous findings of Lafay and Berkaloff (26). The thermal inactivation of ts

* Portions of this paper (including "Materials and Methods" and Figs. 2 and 6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 85M-4104, cite the authors, and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

a b c d

L " Lf t 2

II

FIG. 1. Autoradiogram of [S5S]methionine-labeled viron proteins. VSV Cocal wild-type and ts y l virus particles labeled at 32 "C (lanes a and b ) or at 39 "C (lanes c and d ) were purified from the medium containing the released virus particles by centrifugation through a 30% sucrose cushion (40,000 rpm for 3 h). Viral proteins were analyzed on an SDS-10% polyacrylamide gel (lanes a and b) or an SDS-5-12.575 polyacrylamide gel (lanes c and d). Lanes a and c represent VSV Cocal wild-type viral proteins of particles released at 32 and 39 "C, respectively, and lanes b and d represent proteins of ts y l particles released at 32 and 39 "C, respectively.

y l and ts 045 viruses demonstrated in Fig. 2A was shown to be due to a defective membrane protein since their infectious transcribing nucleoprotein complexes were not inactivated more than the transcribing nucleoproteins of their respective parental wild-type viruses (Fig. 2B). These results provide biological evidence for a defective membrane protein, proba- bly the G protein, in the VSV Cocal ts y l mutant virus and establish a correlation between the ts y l mutant and ts 045 virus which belongs to complementation group V of the VSV Indiana serotype.

Glycoproteins Synthesized at Permissive and Nonpermissive Temperatures-Cells infected with wild-type VSV Cocal or ts y l viruses were labeled with [35S]methionine,, and the viral coded proteins synthesized were analyzed on an SDS-polyacrylamide gel (Fig. 3). The G protein synthesized in wild- type virus-infected cells a t 32 and 39 "C had an apparent molecular weight of 71,000. In contrast, the G protein synthesized by ts y l virus-infected cells a t 32 and 39 "C had apparent molecular weights of 68,000 and 64,000, respectively. The ts y l virus-infected cells at 39°C also showed the presence of a small amount of G protein of M, 68,000. This decrease in the size of the G protein in the ts virus-infected cells at both the permissive and nonpermissive temperatures suggested the two following possibilities. The mutant glycoprotein might have a polypeptide chain of decreased size. Alternatively, the pattern of glycosylation of the mutant G protein at both temperatures could be different from that observed in the case of the wild- type G protein.

That the nonglycosylated polypeptides synthesized by the wild-type and the mutant viruses at both 32 and 39 "C were

Role of Glycosylation in G Protein Transport 8938

a b c

L

N

M

d e f g h i i k

FIG. 3. Autoradiogram of [S5S]methionine-labeled proteins synthesized by VSV Cocal wild-type- or ts 71 virus-infected cells. A 5-12.5% polyacrylamide gel containing 0.1% SDS was used. Lune a contains VSV Indiana viral proteins; hnes b-e represent proteins synthesized in the absence of tunicamycin by cells infected with wild-type VSV Cocal at 32 and 39 "C and with mutant ts y l at 32 and 39 "C, respectively. Lunes f-i represent proteins synthesized in the presence of tunicamycin (4 pg/ml) by cells infected with VSV Cocal wild-type at 32 and 39 "C and with ts yl virus at 32 and 39 "C, respectively. Lanes j and k contain in vitro translation products synthesized in a wheat germ extract programed with mRNA isolated from VSV Cocal wild-type- and ts y l virus-infected cells, respectively.

the same size was shown by using the glycosylation inhibitor tunicamycin. The G protein of VSV Indiana serotype synthesized in the presence of tunicamycin showed a decreased molecular weight (27). As shown in Fig. 3, G proteins synthesized by both the wild-type virus and the mutant virus a t either temperature in the presence of tunicamycin had the same molecular weight of 64,000. This indicates that the size of the nonglycosylated G protein is the same in both the wild- type and the mutant viruses.

We have previously shown that translation of the G mRNA in a wheat germ protein-synthesizing system resulted in the synthesis of a nonglycosylated protein (23) which contained a signal peptide at the NHZ terminus (28). The signal peptide is absent in the mature glycosylated G protein and is proteo- lytically removed from the in vitro synthesized protein in the presence of microsomal membranes (28, 29). When mRNAs isolated from VSV Cocal wild-type- and ts y l virus-infected cells were translated in vitro in a wheat germ protein-synthesizing system, the nonglycosylated G proteins synthesized were identical in size in both cases (about M , 66,000). The in vitro synthesized nonglycosylated G protein showed a higher molecular weight than the nonglycosylated protein synthesized in vivo in the presence of tunicamycin (Fig. 3). This

agreed with our previous observation that the nonglycosylated G protein synthesized in vivo in the presence of tunicamycin lacked its NHz-terminal signal peptide extension (30).

The partial NHp-terminal sequence of G proteins synthesized in vivo a t 32 and 39 "C by ts y l virus-infected cells was determined using [3H]phenylalanine-labeled G proteins. A comparison of the positions of phenylalanine in G proteins synthesized by the mutant virus at the permissive and nonpermissive temperatures with the previously published NHz- terminal sequence of the in vitro synthesized nonglycosylated G protein and the in vivo synthesized mature G protein of the wild-type Cocal virus (25) showed that the mutant G protein possesses the same NH2-terminal sequence as the wild-type G protein (Fig. 4). The results further demonstrated the proper processing of the 17-amino acid-long signal peptide of G protein (25) in the mutant ts y l virus a t both permissive and nonpermissive temperatures.

Lack of Glycosylutwn of ts y l G Protein at Nonpermissive Temperature-The fact that the G protein synthesized by the ts y l mutant virus at the permissive and nonpermissive temperature has a lower molecular weight than the mature G protein of wild-type Cocal VSV, although they contain polypeptides of the same size and an NH2-terminal sequence, suggests that the ts y l G protein synthesized at 32 "C (ts yl G 32 "C) and at 39 "C (ts y l G 39 "C) are glycosylated to different extents compared with wild-type Cocal G protein (Cocal G).

The G protein of VSV Indiana contains two complex oligosaccharide moieties attached to asparagine residues of the polypeptide chain (31, 32). The oligosaccharide moiety contains glucosamine and mannose present as a core with branched chain containing the terminal sugars, galactose, and sialic acid residues (31). In the initial step of the glycosylation, an en bloc transfer of a high mannose- and glucose-containing oligosaccharide occurs from a dolichol lipid carrier to the asparagine residue (2). In the subsequent steps, the mannose residues are trimmed and the terminal sugars are added in the Golgi apparatus (2,33,34). When cells infected with VSV Cocal wild-type or ts y l virus were labeled with [3H]mannose at 32 or 39 "C, Cocal wild-type G protein was labeled at both temperatures. In contrast, ts y l G protein produced a t 32 "C only contained mannose residues (Fig. 5). The absence of any mannose label in ts y l G 39 "C suggests that at 39 "C ts y l G protein is not glycosylated. This was further confirmed by labeling with [3H]glucosamine and [3H]galactose (data not shown). Both of these sugar residues were again absent in ts y l G protein a t 39 "C. The presence of galactose in ts y l G 32 "C shows that at 32 "C the processing of the core oligosaccharide and the transfer of the terminal sugars onto the G protein occur in ts y l virus-infected cells. This was also confirmed by neuraminidase digestion of the G protein when a reduction in the size of ts y l G protein synthesized at 32 "C was observed (data not presented). It appears, therefore, that the G protein synthesized at the nonpermissive temperature by ts y l virus-infected cells lacks glycosyl residues.

ts y l G Protein Synthesized at Permissive Temperature Contains One Oligosaccharide Unit-The fact that ts yl G 32 "C contains terminal sugars but its molecular weight is reduced by about 3000, which corresponds to one oligosaccharide moiety of VSV Indiana G protein, suggests that it contains one less carbohydrate chain than the number present in wild-type Cocal G protein. Since the number of glycosyl chains attached to VSV Cocal G protein was not known, we analyzed the mannose-containing glycopeptides present in wild-type VSV Cocal G and ts y l G 32 "C and compared these with VSV Indiana G protein, using a high performance liquid


1 5 10 15 1 20 25 a . COCAL G1 METLYSPHELEULEULEULEUPHEILE - LEUPROLEU - - - - - PHE - ILE - PHEPRO -

b. COCAL G

C . h l G 32°C

d . h l G 39°C

1 5 LYSPHESERILEVALPHEPROGLN

1 - PHE - - - PHE - - 1 - PHE - - - PHE - -

FIG. 4. A comparison of the partial NH2-terminal sequences of the in vitro synthesized precursor GI and intracellular glycoprotein of VSV Cocal wild-type published earlier (25) with those of ts 71 intracellular dvcoDrotein svnthesized at the Dermissive and nonpermissive temperatures. The arrow at position 17 indicates the signal peptide cleavage site.

chromatographic system (Fig. 6). As expected the G protein of VSV Indiana serotype showed the presence of two glycopeptides labeled with ['H]mannose. Wild-type VSV Cocal G protein also showed two [3HH]mannose-containing glycopeptides; however, their elution pattern was different. In contrast, the ts y l G protein synthesized at 32 "C showed the presence of a single [3H]mannose-containing peptide. It may be noted that the elution pattern of the glycopeptide obtained from ts y l G 32 "C was different from the elution pattern of the two glycopeptides of the wild-type VSV Cocal G.

Expression of G Protein at the Cell Surface Requires Glyco- sylation-In order to determine if the nonglycosylated G protein of ts y l virus is transported from its site of synthesis (endoplasmic reticulum) to the cell surface, we used indirect immunofluorescent staining of infected cells. Results shown in Fig. 7 show that cells infected with ts y l virus at 32 "C showed surface fluorescence. In contrast, cells infected with ts y l virus at 39 "C did not show any fluorescence, indicating that the nonglycosylated G protein does not reach the cell surface. In order to establish that the failure of the unglyco- sylated G to reach the cell surface at 39 "C was due to a lack of glycosyl residues rather than to an altered conformation, we also checked the cell surface fluorescence of cells infected with ts y l virus at 32 "C in the presence of tunicamycin. No surface fluorescence was observed with tunicamycin-treated cells (Fig. 7C) showing that glycosylation of ts y l G protein is essential for its transport to the cell surface. Similar results were also obtained with tunicamycin-treated wild-type VSV Cocal-infected cells (data not shown). When microsomal frac- tions from ts y l virus-infected cells incubated at 39 "C were treated with trypsin, the nonglycosylated G protein was found to be protected from proteolytic digestion. The protected nonglycosylated G protein had, however, a lower molecular weight (data not shown). This agrees with our previous observation that nonglycosylated G protein of VSV Indiana serotype is inserted into endoplasmic reticulum membrane and the signal sequence is properly processed (30). Thus, in the absence of glycosylation, G protein is sequestered in the microsomes and cannot be transported to the cell surface.

DISCUSSION

Previous studies using mutants of VSV Indiana belonging to complementation group V have demonstrated the presence of two subclasses. In one group, mutants such as ts 045, ts M501, and ts L513 at the nonpermissive temperative synthe- size G protein which is blocked in the transport process from rough endoplasmic reticulum to the Golgi apparatus. The defective G protein of these viruses is processed at the permissive temperature with glycosylation at both sites providing high mannose core oligosaccharide which is endoglycosidase H-sensitive (8, 10, 11). Processing of the core oligosaccharide (2,33), addition of terminal sugar residues (34), and covalent linkage of fatty acids (35,36) occuring in the Golgi apparatus

a b c d e

L

G

N

M

FIG. 5. Autoradiogram of [*H]mannose-labeled proteins separated on a SDS- 10% polyacrylamide gel. Lune a represents [35S]methionine-labeled Cocal viral protein; lanes b-e represents ['HI mannose-labeled protein synthesized by cells infected with VSV Cocal wild-type at 32 "C, ts yl virus a t 32 "C, ts y l virus a t 39 "C, and VSV Cocal wild-type at 39 "C, respectively.

are, however, blocked at the nonpermissive temperature. The single mutant ts L511 belonging to the other class at the nonpermissive temperature allows the G protein to proceed from the rough endoplasmic reticulum to the Golgi apparatus. The glycoprotein of ts L511 virus is fully processed, including the addition of terminal sugars. The attachment of fatty acid is, however, blocked, and only a part of the nonacylated glycoprotein moves to the cell surface (11, 12).

The mutant ts y l virus described in this study is quite distinct from both of these subclasses. At the permissive temperature, unlike the two classes of G mutants described above, ts y l glycosylates only one of the two potential carbohydrate attachment sites. The glycosylation is complete at

8940 Role of Glycosylation in G Protein Transport

FIG. 7. Indirect immunofluorescent staining of cells infected with VSV Cocal wild-type or ts y l virus at 32 and 39 OC. A and E, cells infected with ts yl virus a t 32 "C; B and F, cells infected with ts y l virus at 39 "C; C and G, cells infected with ts y l virus at 32 "C in the presence of 6 pg/ml tunicamycin; D and H, cells infected with wild-type VSV Cocal. A-D show immunofluores- cence, and E-H show phase-contrast photomicrographs.


this site as shown by the fact that, as in wild-type G protein, pulse-labeled ts y l G protein synthesized at 32 "C became endoglycosidase H-resistant after a chase period of about 40- 50 min.3 The incorporation of galactose and the susceptibility of ts y l G 32 "C to neuraminidase (data not shown) further show that the terminal sugars are properly added and that the mutant glycoprotein is transported via the Golgi apparatus.

At the nonpermissive temperature, the production of infectious particles of ts y l virus is drastically reduced to about 0.001%. As in the case of the other G protein mutants (8), noninfectious particles lacking only the G protein are synthesized to the extent of 35%. In contrast to the other mutant glycoproteins, ts y l G protein synthesized at 39 "C is completely nonglycosylated and blocked in its intracellular transport to the cell surface. NHn-terminal sequence determination shows that the signal peptide is removed from the nonglycosylated protein and therefore, it must have been inserted into rough endoplasmic reticulum for cleavage to occur. This is in agreement with our previous observation that glycosylation is not required for insertion into rough endoplasmic reticulum and cleavage of signal peptide (30).

Mutants of both classes described previously failed to attach fatty acid onto the G protein at the nonpermissive temperature (11, 12). In the case of ts L511 virus, it was inferred that the lack of fatty acid acylation of G protein resulted in blocking of virus maturation and budding (12). In the case of ts y l virus, the functional G protein synthesized at 32 "C lacked covalently linked fatty acid (palmitic acid). In fact, the wild-type Cocal G protein also lacked fatty acid (37). There- fore, the maturation and budding of VSV may not need, in all cases, the presence of fatty acid in the G protein. Indeed, the G proteins of three strains of VSV New Jersey, namely Concan, Missouri, and Hazelhurst, also lacked fatty acid, whereas two other vesiculoviruses, Chandipura and Piry, contained fatty acid in G protein (37). The absence of fatty acid in G protein of another strain, VSV New Jersey Ogden, was also reported (32). It appears therefore that the requirement for covalently linked fatty acid for biological activity of G protein, namely viral maturation and budding, may not be a general phenomenon (37).

A comparison of the elution profiles of the [3H]mannose- labeled glycopeptides of G proteins of Cocal and ts y l viruses produced at 32 "C shows that the mutant G protein which produced only one tryptic glycopeptide has an altered peptide sequence. The results suggest that the G protein of ts y l virus may have more than one mutation affecting the glycosylation. Although the altered glycopeptides have not been sequenced, one may speculate that a mutation either changes an acceptor asparagine residue or induces a conformational change which restricts glycosylation at that particular site at both temperatures. Yet another mutation could affect the second glycosylation site such that at the nonpermissive temperature the addition of high mannose core oligosaccharide onto the acceptor asparagine residue is blocked (41).

Studies using the glycosylation inhibitor tunicamycin have been made to determine the requirement of glycosylation for transport of glycoproteins. Leavitt et al. (1 7) have shown that in the absence of glycosylation the envelope glycoproteins of both Sindbis virus and the San Juan strain of VSV fail to migrate to the cell surface with a concomitant inhibition of 99% of infectious virus production. In subsequent studies (14), they reported that the G protein of the Orsay strain of VSV was less stringent in its requirement for glycosylation. Thus, at a reduced temperature of 30 "C, tunicamycin blocked

G. Kotwal, unpublished observation,

glycosylation completely, but the nonglycosylated G protein was transported to and expressed on the cell surface, resulting in the production of infectious virion particles containing nonglycosylated G protein (14). It was further shown that mutational changes within the G protein of the Orsay strain of VSV also altered the requirement for carbohydrate in G protein transport and virus morphogenesis (15). Recently, the amino acid sequences for G proteins of the San Juan and Orsay strains of VSV were deduced from the cDNA sequences (38, 39). A comparison of the sequences between the two G proteins shows that aspartic acid replaces tyrosine at position 172 in the Orsay strain, 6 residues from the first glycosylation site at aspargine 178. Aspartic acid at position 172 would increase the hydrophilic nature of Orsay G protein and the folding of the peptide chain around the first glycosylation site, which may lessen the requirement for carbohydrate as suggested previously (14) for maintaining the proper conformation (39).

The studies of ts y l virus reported here have shown that glycosylation of only one of the two sites of G protein is sufficient for normal transport to the cell surface and morphogenesis of infectious virions. Blocking of glycosylation at both sites prevents the migration of G protein to the cell surface. Our results corroborate and extend the earlier obser- vations on the role of glycosylation (15, 17) but avoid the use of glycosylation inhibitors. Although the results with tunicamycin suggest that glycosylation may play a role in maintaining protein conformation for proper transport, our results suggest that glycosylation may have a more direct effect on the transport process which may also involve recognition by other components in this process. Recently, it has also been shown that a single glycosyl residue is sufficient for transport of G protein to plasma membranes of cells expressing G protein from cloned cDNA (42).

Acknowledgments-We thank Martin Butcher for the immunoflu- orescence studies, Dr. C. Nurse and M. Holmes for their help with the fluorescence microscope, and Dale Tomlinson for typing the manuscript.

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8942 Role of Glycosylation in G Protein Transport

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Supp lemen ta ry Ma te r i a l t o

Virus Envelope Glycoprotein: A New Class of Mutant Defect ive R o l e o f G l y c o s y l a t i o n i n T r a n s p o r t o f V e s i c u l a r S t o m a t i t i s

i n G l y c o s y l e t i o n and Transport of G P r o t e i n

G i r i s h 1. Katwal, R. Hark L. Buller, William H. Wunner, Craig R . P r i n g l e and Hars P. Ghosh

MATERIALS AND UETHDDS

H a t e r i a l s

R a d i o a c t i v e l a b e l l e d amino a c i d s , sugars end pa lmi t ic ac id were ob ta ined from New England Nuclear Corporation. Tunicamycin was from E l i L i l l y 6 Co. L td . , I nd ianapo l i s , IN; sequence grade chemicals , used for automated Edman degradat ion were purchased f rom Beckman Inc., and t h c s o l v e n t s u s e d were obtained from Burdick and Jackson L a b o r a t o r i e s . The HPLC e o l v e n t s were obtained f rom Caledon Laborator ies and Burdick and Jackson Laborator ies .

Ce l l s and V i ruses

The Ind iana (Toron to ) s e ro type of VSV was obtained f rom Dr. L. Prevec, HcHaster Univers i ty , Canada; the o r ig ins o f the !IsV Cocal wild-type end the t B Y 1 v i r u s and t empera tu re s ens i t i ve mu tan t s have been desc r ibed p rev ious ly ( 1 3 ) . I n d i a n a La 045 was a g i f t from Dr. A. Flamand (Orsay, France).

L Cel l s were grown i n s u s p e n s i o n i n J o k l i k ' s m o d i f i e d minimum essential medium (MEHI c o n t a i n i n g 5% new b o r n c a l f serum (kats), and L c e l l s u s e d f o r l abe l l i ng were grow t o near c o n f l u e n c e i n I 0 0 m p l a t e s i n HEM c o n t a i n i n g 5% fe t a l ca l f s e rum (FCS) . Ch ick embryo f ib rob la s t s were p repa red as p r e v i o u s l y desc r ibed 119) . P l aque -pur i f i ed v i ruses were grown i n L c e l l s and p u r i f i e d a3 d e s c r i b e d b e f o r e (20). BHK"ZIICI3 c e l l s were grown in Eagle 's medium supplemented with IO% c a l f s e r u .

H e a t I n a c t i v a t i o n o f V i r u s and Transc r ib ing Nuc leopro te in (TNP) Complex

7 . 4 , 90 rnH N a C l , and 1 mN EDTA end hea ted t o 49 .5 2 D.Z0C, t h e t e m p e r a t u r e a t w h i c h t h e g r e a t e s t i n a c t i v a t i o n d i f f e r e n c e s o c c u r r e d b e t w e e n m u t a n t and w i l d - t y p e v i r u s . I n f e c t i o u s TNP complex was i s o l a t e d from p u r i f i e d v i r i o n s a s d e s c r i b e d (21) and hea ted to 49.5OC i n NTE b u f f e r and 3.5 mM d i t h i o t h r e i t o l (Dm). I n f e c t i v i t y o f t h e TNP complex was a s s a y e d a f t e r d i l u t i n g t h e h e a c e d f r a c t i o n w i t h PBS c n t a i n i n g 3.4 mM KCI, 3.5 dl DTT, 1% (v /v) c a l f serum, 5% ( " 1 " ) g l y c e r o l , M l i n o l e i c a c i d , and 800 ugiml DEAE d e x t r a n and adsorbing o n t o BHxZllC13 c e l l s weshed with PBS. A f t e r a d s o r p t i o n f o r 60 m i " a t 37OC, t h e a s s a y p l a t e s were washed twice with 2 m l of PBS c o n t a i n i n g 3.4 mH KC1 and ove r l ayed w i th aga r be fo re i ncuba t ing at 3 1 O G to produce plagues.

V i r u s s t o c k s were d i l u t e d i n NTE b u f f e r c o n t a i n i n g 20 mM Tris-HCI, pH

S y n t h e s i s o f I n t r a c e l l u l a r V i r u s S p e c i f i c P r o t e i n s

Confluent L cells (100 mm p l a t e s ) a d a p t e d t o 39.5'C were i n f e c t e d f o r 45 minutes with VSV Coca1 wild-type or t a Y 1 v i r u s e s a t 32OC or 39OC. The excess medium was removed, and the infected cel ls were washed twice with phosphate b u f f e r e d s a l i n e (PBS), before B medium conta in ing 2% d i a l y z e d f e t a l c a l f s e r a (DFCS) end 2 uglml of Aetimomycin D waa added. A t t h end of 4 . 5 hours t he medim was replaced with HXH conta in ing 2% DFCS and [95Sl L-methionine ( 4 0 uCi/ml) end incubated a t the respec t ive t empere turee for 60 minutes . Label l ing o f p ro te ins in the p resence o f tun icamycin was e n s e n t i a l l y t h e same 88 descr ibed above wi th the except ion ths t cells were p r e t r e a t e d and maintained throughout the experiment in medium conta in ing tun ic-yc in a t 4 uglol. The c e l l s were then washed twice with PES, scraped o f f t h e p l a t e , l y s e d i n 4OOyl of a b u f f e r c o n t a i n i n g 50 mH T r i s HC1, pH 7 . 1 , 150 dl N e C 1 , 1% T r i t o n x-100, 0.1% SDS, I% Na deoxycholate (RIPA b u f f e r ) and 100 Ke l l i Kre in

of ant i -Coca1 virus a n t i a e r m and SO u 1 of B 10% (v/v> svspenaion of P r o t e i n i n a c t i v a t o r u n i t B of Trasylol. The l y s a t e was i m u n o p r e c i p i t a t e d with IO U l

A-Sepharaee beads (Pharmacia). The imnunoprec ip i te ted rad io labe l led p ro te ins were so lub i l i zed i n sample b u f f e r (0.625 H T r i s HC1, pH 6 . 0 , 0.625 H 2-mereeptoethano1, 32 SDS, 10% g l y c e r o l and 0.012 bromphenol blue) heated to

polyacry lamide grad ien t ge ls conta in ing 0.1% SDS and i d e n t i f i e d b y 100°C f o r 3 min. The v i r u s - s p e c i f i c p r o t e i n s were separated on 5 t o 1 2 . 5 %

autoradiography ( 2 3 . 2 4 ) .

30.

31.

32. 33. 34.

35.

36. 37.

38. 39. 40. 41.

42.

Irving, R. A., Hofmann, T., and Ghosh, H. P. (1982) FEBS Lett.

Etchison, J. R., Robertson, J. S., and Summers, D. F. (1977)

Gallione, C. J., and Rose, J. K. (1983) J . Virol. 46,162-169 Tabas, I., and Kornfeld, S. (1978) J. Biol. Chem. 253, 7779-7786 Bretz, R., Bretz, H., and Palade, G. E. (1980) J. Cell Biol. 84,

140,257-259

Virology 78,375-392

87-1 nl Schmidt, M. F. G., and Schlesinger, M. J. (1980) J. Biol. Chem.

Schmidt. M. F. G. (1982) Virobm 116.327-338

-. "_ 255,3334-3339

Kotwal, G. J., and Ghosh, H. P. li984) i. Bwl. Chem. 259,4699-

Rose, J. K., and Gallione, C. J. (1981) J . Virol. 39, 519-528 Gallione, C. J., and Rose, J. K. (1985) J. Virol. 54, 374-382 Nakamura, K., and Compans, R. W. (1978) Virology 84,303-319 Wunner, W. H., Dietzschold, B., Smith, C. L., Lafon, M., and

Machamer, C. E., Florkiewicz, R. Z., and Rose, J. K. (1985) Mol.

4701

Golub, E. (1985) Virology 140, 1-12

Cell. Biol. 5, 3074-3083

I n Vi t ro T rans l a t ion Us ing Wheat Germ E x t r a c t

i n f e c t e d L cells incubated 8t 32OC by e x t r a c t i n g w i t h phenolICHC1 ISDS

and Chosh, 1975). The i s o l a t e d RNA Was used t o d i r ec t VSV-spec i f i c p ro t e in fo l lowed by p r e c i p i t a t i o n w i t h L i C l I e t h e n o l as d e s c r i b e d e a r l i e r ? T o n e g u z r o

s y n t h e s i s i n what germ e x t r a c t s 88 d e s c r i b e d p r e v i o u s l y (25).

VSV Cocal wild-type and tsY 1 v i r u s - s p e c i f i c RRNAs were prepared from

Nh2-Terminal Analysis I s o l a t i o n o f R a d i o a c t i v e I n t r a c e l l u l a r V i r u s S p e c i f i c P r o t e i n s f o r

T h e v i r e l g f o t e i n s were l a b e l l e d w i t h I 3 H 1 amino a c i d s e s s e n t i a l l y a s desc r ibed fo r 1 SI m e t h i o n i n e l a b e l l i n g , w i t h t h e e x c e p t i o n t h a t a f t e r e l e c t r o p h o r e a i s u n f i x e d wet gels were exposed tO X-ray f i lm. The gels were a l i g n e d and r ad ioac t ive bands co r re spond ing t o C p r o t e i n s were excised f rom t h e g e l . The p r o t e i n was e l u t e d i n a e o l u t i o n c o n t a i n i n g IO mu T r i a pH 8.0, I m k 2-mercaptnethanol, 0.01% SDS, 1 dl phenylmethylsul fonyl f luoride and 2 m g l m l

d i a l y z e d a g a i n s t 0.01% SDS and I mM 2-rnercaptoethanol for a f u r t h e r 24 h r a t lysoyzme by s h a k i n g a t room t empera tu re fo r 24 h r s . The e l u t e d p r o t e i n s were

4'C. The e l u t e d sample was l y o p h i l i z e d and suspended i n 0.5 m l o f s t e r i l e water and checked fo r pu r i ty by r e -e l ec t rophores i s o f a small a l i q u o t .

Automated Sequencing

Beckman 890 c sequencer as d e s c r i b e d b e f o r e (25).

L a b e l l i n g o f I n f e c t e d Cells wi th ['HI Sugars

M i c r o s e q u e n c i n g o f t h e r a d i o l a b e l l e d p r o t e i n s vas c a r r i e d o u t i n a

L cells w e r e c u l t u r e d i n 100 mm P e t r i d i s h e s u n t i l n e a r l y c o n f l u e n t ( 6 x

v i r u s a t a m . 0 . i . of 5 0 and incubated a t 32'C or 39OC. A f t e r A5 m i " , t h e lo6 c e l l a ) . The c e l l s were t h e n i n f e c t e d w i t h VSV Cocal wild-type or ts Y I

m l of MEN was added and the c e l l s were i ncuba ted fo r a f u r t h e r 3.75 hours . ""adsorbed v i r u s wag removed and the c e l l s washed wi th prewarmed PBS, b e f o r e 5

The medium was then removed and t h e cells were washed th ree s imes w i th 5 m l o f w r m PES. The c e l l 6 w e r e t h e n l a b e l l e d w i t h f 3 H 1 mannose, H I g a l a c t a s e or I H] glucosamine ( 2 5 u c i l m l ) i n MEN f o r 60 mi". A t t h e end o f t h e l a b e l l i n g pe r iod t he c e l l s were washed, harvested and d i s r u p t e d , and t h e l a b e l l e d p r o t e i n s were analyzed on IO% o r 5-12.52 gradient SDS-polyacrylamide ge l s and iden t i f i ed by au to rad iog raphy (23.24).

T r y p t i c P e p t i d e A n a l y s i s of 1 3 H l Mannoae-Label led Clycoproteins by HPLC

9

v i r u s and l a b e l l e d w i t h [ H] mannose a6 desc r ibed above w i th t he excep t ion L c e l l s were i n f e c t e q w i t h VSV I n d i a n a or VSV Cocal wild-type or t 6 Y 1

g l y c o p r o t e i n s were s e p a r a t e d on a 10% SDS-polyacrylamide gel. The p r o t e i n t h a t t h e amount o f r a d i o a c t i v i t y u s e d was 50 UCiIml. I3HI Hannose-labelled

band cor responding to VSG was exc ised f rom a wet gel and e l u t e d i n t h e

d iges t ed w i th TPCK-treated t r y p s i n ( p r o t e i n t o enzyme r a t i o , 2 : l ) i n a b u f f e r presence o f IO mM Tris-HC1 pH 8.0, 1 mn Z-mercaptoethsnol and 0.01% SDS and

c o n t a i n i n g 50 mH Tris-HCI pH 8.0 and 10 mH CaCIZ. ,The incubation was c a r r i e d o u t a t 37 C f o r 6 hy. The t r y p t i c d i g e s t was a p p l l e d t o an Al tex U l t r a sphe re

mM sodium a c e t a t e , pH 5 .05 , c o n t a i n i n g 5% t e t r a h y d r o f u r a n ) and was e lu t ed w i th ODs column (0.46 x 22 cm) p r e v i o v s l y e q u i l i b r a t e d w i t h 100% o f s o l v e n t A ( 4 . 2 5

a c e t o n i t r i l e ) as f o i l o v s : 0-25% of B in 15 minutes , 25-50% of B i n a f u r t h e r B l i n e a r g r a d i e n t o f t h e s o l v e n t A t o t h e s o l v e n t B (10% t e t r a h y d r o f u r a n i n

o f 1.2 ml /min . F rac t ions o f 1.2 ml w e r e c o l l e c t e d , d r i e d i n an Oven a t 13OoC 35 minutes, and 50-100% of B i n IO m i n u t e s a t room temperature and a f l o w r a t e

and coun ted fo r r ad ioac t iv i ty .

Inmunof luorescence

L c e l l s were on c o v e r s l i p s t o 80% eonfluency and infected with VSV coral wild-type or t s y I m u t a n t v i r u s a t an m . 0 . i . o f 5-10. A f t e r 4 5 min a t 32'c t h e i n o c u l m was removed and t h e c o v e r s l i p s were incubated e t 32OC Or 39Oc. A f t e r 4 h c e l l s were washed twice w i th PBS and t h e n f i x e d w i t h

0.5% bovine serum albumin in PBS. A 1:20 d i l u t i o n o f r a b b i t a n t i - c o r a l formaldehyde in PBS Lor 10 min st 4'C. The c o v e r s l i p s were washed with PBS,

c o v e r s l i p . A f t e r 1 h r t h e c o v e r e l i p s were waehed 3 t imes wi th PBS Conta in ing a n t i s e r a i n 0 .5% bovine serum a l b u m i n i n PBS was t h e n l a y e r e d on t h e

albumin and l aye red w i th a 1:20 di lu t ion of FITC-conjugated goa t an t i - rabbi t ~ g c serum ( c a r p e l L a b o r a t o r i e s ) . A f t e r 30 min t h e c o v e r s l i p s were washed 3 times wi th p ~ s c o n t a i n i n g a l b m i n , a i r d r i e d and mounted onto microscope

equ ipped w i th phase con t r a s t op t i c s . F I ~ ~ ~ ~ s ~ ~ c ~ was observed with a ze i s s f l uo rescence mic roscope

Role of Clycosylation in G Protein Transport

1 6 3 L L " I 3 I

0 10 m 30 Minutes of inactivation

r e s p e c t r v e t s mutants . A. I n d i a n a w i l d - t y p e v i r u s A-A, and t s 045 v i r u s

complexes pur i f ied f r w VSV-Indiana wild-type m"., and t s 045 0-0, A-A; Coca1 wild- type virus m"., and t sYl v i r u s 0-0. 8. TNP

VSV-Coca1 wi ld- type A-A, and t 8 Y L v i r u s A-A. V i r u s e s and 'IN? complexes Mre h e a t e d t o 49.5 0.2OC a n d a s s a y e d f o r r e s i d u a l i n f e c t i v i t y a f t e r v a r i o u s times of i n a c t i v a t i o n 88 d e s c r i b e d i n t h e t e x t and p l o t t e d a8 the minus common logar i thm of s u r v i v i n g t i t e r (T) o v e r t h e i n i t i a l t i t e r (T ).

w2. H e a t i n a c t i v a t i o n k i n e t i c s of VSV w i l d - t y p e s t r a i n s and

I IN0 I .I c0 1 I 197,

P "

8943

1 F R "0 m "0. I". "0

glycopro te ins o f VSV Indiana , Coca1 and t s 1 v i r u s by HPLC. Left panel shows Fig.. Separa t ion of t ryp t ic pept ide6 of 13H1 mannose l a b e l l e d

g lycopept ides ob ta ined f r w VSV Indiana G protein; middle panel shows glycopeptides from VSV Coca1 wild-type G p r o t e i n and the r i gh t pane l shows glycopeptides from ts TI G pro te ins syn thes i zed a t 32'C. n e major peaks

peat8 ( sugges t ive of i n c w p l e t e c l e a v a g e p r o d u c t s ) by arrow heada. The corresponding to the g lycopept ides are i n d i c a t e d by long srrov~ and the minor

recovery of t h e 1 3 H 1 r a d i o a c t i v i t y from t h e column wan about 952. The peak represent ing undiges ted G p r o t e i n c o n t a i n s 11-12.52 of t o t a l r a d i o a c t i v i t y .

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