Effect of herpes simplex virus type 1 on cellular pools of oligosaccharide-lipid

VIROLOGY

147, l-8

(19%)

Effect of Herpes Simplex Virus Type 1 on Cellular Pools of Oligosaccharide-Lipid TERESA Department

COMPTON’

of Microbiology University

Received

and Program of Tennessee, December

AND

RICHARD

in Cellular, Knoxville,

J. COURTNEY’

Molecular Tennessee

11, 1984; accepted

July

and Developmental 37996-0845

Biology,

17, 1985

Incorporation of [3H]mannose into cellular pools of mannosylphosphoryl dolichol (ManP-Dol), oligosaccharide-lipid, and glycoprotein was measured and compared in herpes simplex virus type 1 (HSV-1)-infected cells and -uninfected cells. While mannose incorporation into the monosaccharide-dolichol fraction was similar in infected or uninfected Vero cells, incorporation into the oligosaccharide-lipid fraction was markedly reduced in HSV-l-infected cells (64% of control levels). In contrast, mannose incorporation into glycoprotein was significantly increased in virus-infected cells versus uninfected cells (194% of control levels). The kinetics of incorporation into the various fractions was examined and it was determined that there was minimal increase in mannose incorporation into oligosaccharide-lipid after 8 hr postinfection in virus-infected cells. This corresponded to the time at which nonglycosylated precursors of the HSV-1 glycoproteins were first detected in association with the nuclear fraction. These data suggest that there is an accelerated turnover of oligosaccharide-lipid in virus-infected Vero cells which is most likely due to 0 1985 Academic Press. Inc. extensive glycoprotein synthesis.

A, the lipid-linked oligosaccharides Mana_ 5GlcNAc2 were found on the cytoplasmic side of RER-derived vesicles while lipidlinked Man6_gGlcNAc2 and Glq-aMangGlcNAc were found facing the lumen (Snider and Rogers, 1984). These authors concluded that Man5GlcNAc2-lipid is synthesized on the cytoplasmic side of the membrane and is then translocated to the luminal side where it serves as a donor in peptide glycosylation (Snider and Rogers, 1984). This translocation proposal provides a mechanism for the export of sugar residues from the cytoplasm during glycoprotein synthesis. Thus, while the basic steps in the assembly of N-glycosidically linked oligosaccharide chains in glycoproteins are well understood, there is considerably less information available about the regulation of the intermediates involved in the biosynthetic pathway. The glycoproteins of enveloped viruses have been studied extensively as model membrane proteins as they utilize the same biosynthetic pathway employed by cellular plasma membrane proteins (for review see

INTRODUCTION

The assembly of glycoproteins with asparagine-linked (N-) oligosaccharides involves the synthesis of a large lipid-linked oligosaccharide precursor and its en bloc transfer to protein in the rough endoplasmic reticulum (RER) (for review see Struck and Lennarz, 1980; Hubbard and Ivatt,1981).Theoligosaccharide,GlcaMangGlcNAcz is assembled on the lipid carrier dolichol pyrophosphate by the stepwise addition of sugar residues. These sugars are either donated by nucleotide- or dolichol-phosphate carriers. The series of reactions that results in the assembly of the oligosaccharide chain are well described; however, it is difficult to account for the presence of nucleotide sugars in the lumen of the RER. Using the lectin concanavalin i Current address: Department of Cell Biology, New York University Medical Center, 550 First Avenue, New York, N. Y. 10016. a Author to whom requests for reprints should be addressed. 1

0042-6822/85 Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved

2

COMPTON AND COURTNEY

Sabatini et ah, 1982). Herpes simplex virus type 1 (HSV-1) codes for four virus-specific glycoproteins designated gB, gC, gD, and gE (Spear, 1976) which are present on the viral envelope (acquired from the inner nuclear membrane) as well as all membranes of the infected cell (Spear and Roizman, 1982; Eisenberg et aZ., 19’79). Studies on the distribution of the HSV-1 glycoproteins have demonstrated that the highmannose precursors, pgBll0 (mol wt llO,OOO), pgClO5 (mol wt 105,000), pgD52 (mol wt 52,000), and pgE65 (mol wt 65,000) (Spear, 1976; Baucke and Spear, 1979; Serafini-Cessi and Campadelli-Fiume, 1981; Wenske et ah, 1982) are the predominant components of the nuclear fraction of infected cells (Compton and Courtney, 1984a). Furthermore, in certain cell lines such as Vero cells (African green monkey kidney cells) incubated at 34”, the nonglycosylated precursors are also detectable in the nuclear fraction in the absence of inhibitors of glycosylation (Compton and Courtney, 1984b). The nonglycosylated precursor of gC, pgC75 (mol wt 75,000), is capable of being post-translationally modified by the addition of high-mannose core sugars if further protein synthesis is inhibited. It was proposed that the accumulation of the nonglycosylated forms may be the result of a limitation of some biosynthetic precursor component such as the dolichol phosphate carrier or the oligosaccharide-lipid intermediate. The results of this study support such an hypothesis as steady-state levels of oligosaccharide-lipid were achieved sooner and were lower (based on mannose incorporation) in infected cells versus uninfected cells. The data presented below suggest that there is an accelerated turnover of oligosaccharidelipid in HSV-l-infected Vero cells which may be due to extensive glycoprotein synthesis within the cells. MATERIALS

AND METHODS

Cell culture and virus. Vero cells were grown in Eagle medium supplemented with 10% fetal bovine serum (Flow Laboratories). The KOS strain of HSV-1 was grown in human embryonic lung fibroblasts

(MRC-5) and all virus titrations were eonducted in Vero cell monolayers (Bone and Courtney, 1974). Infection of cells and cell fractionation. Monolayers of Vero cells cultured in lOOmm or 60-mm dishes were infected at a multiplicity of infection of 10 PFU/cell. After a 1-hr absorption at 34”, the inoculum was removed and maintenance medium containing 2% serum was added. At appropriate times postinfection cells were scraped into the medium and washed twice with phosphate-buffered saline. Cytoplasmic and nuclear fractions were obtained by the modified Penman procedure (Warner et ah, 1963; Penman, 1966; Courtney, 1976). Details of this procedure have been described previously (Compton and Courtney, 1984b). SodizLm dodecyl sedate-~~~ac~~arnide gel e~~troph~esis ~S~S-PA~~~ and imrn~n~lotti~g. Details of the methods used for SDS-PAGE have been previously described (Powell and Courtney, 1975). All slab gels were 7% bis-acrylamide. The proteins resolved by SDS-PAGE were dried and exposed to X-Omat film or transferred to nitrocellulose paper (Schleicher and Schuell, Keene, N. H.) and immunoautoradiography was performed as previously described (Compton and Courtney, 1984a). Hyperimmune monospecific rabbit antiserum to gC was prepared as previously described (Eberle and Courtney, 1980). Assay for the incorporation of [“H]mannose into lipid-linked monosaccharide, oligosaccharide, and glycoprotein, The assay for the incorporation of labeled mannose into dolichol-linked monosaccharide and oligosaecharide as well as glycoprotein was essentially as described by Waechter et al. (1983). Confluent monolayers of Vero cells (60-mm dishes) were either infected with HSV-1 or mock infected. After the 1-hr absorption period the inoculum was removed and maintenance medium was added. Tunicamycin (5 pg/ml) was added to one-half of the dishes at this point and all samples were run in triplicate. Three hours postinfection the medium was removed and replaced with medium containing 5 &i/ml [3~]mannose. At the indicated times, the incorporation periods were terminated by

OLIGOSACCHARIDE-LIPID

AND

the removal of medium and the addition of 1 ml of CH30H. The cells were removed from the dish by scraping and transferred to a conical tube. The cells remaining in the dish were recovered by an additional rinse of 0.5 ml CH30H. The composition of the extraction mixture was adjusted to CHCls:CH30H (2:l) by the addition of 3 ml of CHC&. The mixture was vigorously vortexed and centrifuged at 20009 for 5 min. The lipid extract containing glycolipids was transferred to a separate tube. The cell pellet was reextracted with 1 ml of CHC&: CH30H (2:l) and the lipid extracts were pooled. After washing the extracts with l/ 5 volume of 0.9% NaCl, the aqueous phase was discarded and the lower phase was washed with 1 ml of CHCla:CH30H:0.9% NaCl(3:48:47). The organic phase containing mannosylphosphoryl dolichol (mannosyl-P-dolichol) was transferred to a scintillation vial and the organic solvent was removed by evaporation. Ten milliliters of scintillation fluid was added and the amount of radioactivity in this fraction was measured. The partially delipidated cell residue was then dried under a stream of N2 and resuspended in 3.0 ml of 0.9% NaCl. The cell residue was pelleted and washed with HzO. Mannose-labeled oligosaccharide-lipid was extracted by the addition of 1 ml of CHCls:CH30H:H20 (10:10:3). After centrifugation the remaining residue was reextracted with an equal volume of the same solvent mixture and the extracts were pooled in a counting vial. The extraction solvent was evaporated in a Savant Speed Vat Concentrator and redissolved in 0.1 ml of CHC13:CH30H:H20 (10:10:3) for counting in 10 ml of scintillation cocktail. Radiolabeled proteins remaining in the delipidated residue were solubilized in a solution which was 1% SDS and 0.1% mercaptoethanol followed by heating at 100” for 5 min. This solubilized cell residue was counted in 10 ml of scintillation cocktail for aqueous samples. Paper chromatography. The oligosaccharide-lipid fraction was analyzed by descending paper chromatography on Whatman 3MM paper as previously described (Waechter and Scher, 1978). The solvent mixture used was isobutyric acid:concen-

HSV-INFECTED

CELLS

3

trated NHIOH:HzO (57:4:39). The mannoselabeled product recovered in the CHC&: CHZOH:HzO (10:10:3) fraction had a mobility (Rf = 0.65-0.70) characteristic of dolichol-bound oligosaccharide (Waechter and Scher, 1978). Furthermore, mild acid hydrolysis which cleaves the glycosyl phosphate bond (0.1 N HCl in 80% tetrahydrofuran, 50”, 30 min) produced a labeled compound migrating to the position expected for a free oligosaccharide on the same paper chromatography system (data not shown). RESULTS

Post-translational addition of high-mannose oligosaccharide chains to the gC glycoprotein of HSV-1. Previously we have reported that Vera cells infected with HSV1 and incubated at a reduced temperature (34”) synthesize and accumulate the nonglycosylated forms of the virus-specific glycoproteins in addition to the high-mannose precursors (Compton and Courtney, 1984b). If cycloheximide (CX) was added to cultures at a time when the nonglycosylated precursor of gC, pgC75, was detectable, multiple discrete endoglycosidase H-sensitive bands were observed in the nuclear fraction. It was concluded that high-mannose oligosaccharide chains were being added to pgC75 in a post-translational fashion (Compton and Courtney, 198413). Although as many as four additional bands were observed within 20 min after the addition of CX, there were no additional chains acquired in incubations up to 1 hr nor did the stepwise or “ladder” pattern of bands approach the mobility of the authentic high-mannose precursor, pgC105. Extended incubations up to 6 hr past the addition of CX had the same results (Fig. 1, right panel), therefore the observed four additional bands migrating above pgC75 appears to be a maximum number. In the previous study it was proposed that the number of bands detected was a reflection of the total number of carbohydrate chains added (Compton and Courtney, 1984b). The fact that the maximum number of chains was acquired by 20 min is consistent with the study by Hubbard and Robbins (1980) who reported that

COMPTON

AND

COURTNEY

lmmunoblot:

WC

P&

Ihr +CXI

2

4

Ihl ,+cx1

6

$I=* gB* 05’

0

2

Anti

4

-9C

6

6

-p&105‘P&5

FIG. 1. Immunoblot analysis and [‘Hlmannose incorporation into the post-translationally glycosylated species of the gC glycoprotein of HSV-1. HSV-l-infected Vero cells were incubated at 34” until 14 hr postinfection. One culture was harvested and the remaining cultures were treated with cycloheximide (CX) (50 pg/ml) with one set being incubated with [3H]mannose and another left unlabeled. At 2, 4, or 6 hr after the addition of CX, the cultures were harvested and fractionated into cytoplasmie and nuclear fractions. Unlabeled nuclear fractions were analyzed by SDS-PAGE and immunoblotting with anti-gC (right panel). One sample was treated with tunicamycin (TM) to demonstrate that the ladder-like pattern of bands are not detected in the presence of the N-linked glycosylation inhibitor. [3H]Mannose-labeled nuclear fractions (left panel) were analyzed by SDSPAGE and autoradiography was performed. An HSV-1 whole cell extract (WC) labeled with [3Hlglucosamine is included for comparison.

the entire pool of oligosaccharide-lipid turns over in a variety of cell types within 20 min. It was also reported that no further oligosaccharide-lipid intermediates accumulate under conditions in which protein synthesis is inhibited (Schmitt and Elbein, 1979; Hubbard and Robbins, 1980). In order to confirm that the series of multiple bands contained mannose-rich oligosaccharides, rH]mannose was added to infected cultures concurrent with the CX block (Fig. 1, left panel). Since there was no preincubation with the protein synthesis inhibitor, some authentic high-mannose precursor was labeled by the mannose. In addition, the slower-migrating bands of the ladder pattern are also labeled. Thus on the basis of incorporation of the radiolabeled metabolite, we have extended the data in support of post-translational acquisition of highmannose oligosaccharide chains to the nonglycosylated form of the gC glycoprotein of HSV-1. Inccwporation of [‘Hlmannose into Vero cellfractions. In order to determine if there

was a limitation in biosynthetic components of glycosylation in infected Vero cells incubated at 34”, a differential extraction procedure was employed which separates monosaccharide-linked dolichol from oligosaccharide-linked dolichol and glycoproteins [3H]Mannose (5 &i per ml) was added to infected or uninfected dishes at 3 hr postinfection; a time in the HSV replication cycle when virus-specific glycoprotein synthesis begins (Eisenberg et a& 1979; Compton and Courtney, 1984a). Cultures were harvested at 23 hr postinfection and the results of [3H]mannose incorporation into the various fractions are presented in Table 1. Tunicamycin-treated cultures were used as a control to monitor the extraction process since the inhibitor prevents the formation of the oligosaccharide chain but does not affect the abundance of mannosylP-dolichol (Heiftz et aL, 1979) nor does it affect protein synthesis in Vero cells at the concentration used (data not shown). The data presented in Table 1 indicate that there was no significant difference in in-

OLIGOSACCHARIDE-LIPID

AND TABLE

[3H]M~~~~~~

INCORPORATION

Uninfected Infected Uninfected Infected Uninfected + TM, Infected + TM,

cells, 34” cells, 34” cells, 39” cells, 39” cells 34” cells 34’

5

CELLS

1 INTO VERO CELL FRACTIONS Oligosaccharidelipid

Mannosyl-Pdolichol Sample

HSV-INFECTED

Glycoprotein

CPM”

%b

CPM

%

CPM

%

27,908 25,363 27,109 25,070

100 91 100 93

8509 5310 6976 7009

100 64 100 100

92,821 179,891 5,200 12,725

100 194 100 245

26,344

94

1212

14

8,259

9

25,374

91

1112

13

8,551

9

Note. Vero cells were grown to confluency in 60-mm dishes and infected the absorption period, tunicamycin (5 pg/ml) was added to the indicated [3H]mannose (5 &i/ml) was added to cells which were incubated at 34”. the radiolabel, the cultures were extracted as described under Materials a Values presented represent an average value of five separate experiments b Relative percentage was determined by comparison of values obtained treated cells to those of uninfected cells at each temperature of incubation.

corporation of mannose into mannosyl-Pdolichol regardless of infection, the temperature of incubation, or the addition of tunicamycin. In contrast, there was reduced incorporation (64% of control value) of mannose into the oligosaccharide-lipid fraction of virus-infected cells as compared to uninfected cells which were incubated at 34’; however, in cells incubated at 39” there was no difference in incorporation into this fraction. Furthermore, incorporation of mannose into glycoproteins was dramatically increased in infected cells compared to uninfected cells at either temperature of incubation. The incorporation of mannose into glycoproteins of infected cells is a direct reflection of the virus-specific glycoproteins present since HSV inhibits host cell protein synthesis early in infection. To determine when the reduction in incorporation of [3H]mannose into the oligosaccharide-lipid fraction of virus-infected cells incubated at 34” occurred, infected and mock-infected cells were harvested at 2, 8, 16, and 22 hr after addition of the radiolabel. The differential extraction procedure was carried out at the various time periods to compare the incor-

with HSV-1. Immediately following samples. Three hours postinfection Twenty hours after the addition of and Methods. under identical labeling conditions. from infected cells and tunicamycin-

poration of [3H]mannose into mannosyl-Pdolichol, oligosaccharide-lipid, and glycoproteins in the infected vs uninfected cells (Fig. 2). The kinetics of mannose incorporation into the monosaccharide-dolichol fraction from infected cells paralleled that of uninfected cells. Mannose incorporation into the oligosaccharide-lipid from uninfected cells steadily increased until 16 hr and leveled off somewhat by 22 hr; however, after 8 hr in infected cells, mannose incorporation into this fraction did not increase substantially. HSV-1 nonglycosylated forms are detectable in nuclear fractions from Vero cells approximately 8 hr postinfection (Compton and Courtney, 198413) corresponding to the time that reduced levels of oligosaccharide-lipid were manifested. In contrast, mannose incorporation into glycoproteins from infected cells continued to increase throughout the time course of the experiment indicating extensive glycoprotein synthesis was occurring in virus-infected cells compared to the uninfected cell counterparts. DISCUSSION

The data presented in this study are an extension of our previous work which dem-

COMPTON

1 0

AND

I

I

I

I

6

I2

I6

24

TIME

COURTNEY

6

0

12 TIME

(hours)

16

24

(hours)

16 -

12 -

6-

4-

0

6

I2 TIME

I8

24

(hours)

FIG. 2. Kinetics of [aH]mannose incorporation into mannosyl-P-dolichol, oligosaccharide-lipid and glycoproteins from HSV-l-infected or -uninfected Vero cells. Confluent monolayers of Vero cells were either virus-infected or mock-infected and allowed to absorb for 1 hr. Three hours postinfection the medium was removed and replaced with medium containing 5 &i/ml [“Hlmannose. At 2, 8, 16, and 20 hr after the addition of the radiolabel, the incorporation was terminated and the cells were extracted into the various fractions as described. Mock-infected cells, 0; HSV-infected cells, 0.

onstrated that the nonglycosylated forms of HSV-1 glycoproteins are detectable in the absence of inhibitors of glycosylation. The detection of the nonglycosylated precursors is enhanced by incubation of Vero

cells at a slightly reduced temperature of 34”. Furthermore, the nonglycosylated form of gC, pgC75, is capable of being posttranslationally modified by the addition of high-mannose core sugars (Compton and

OLIGOSACCHARIDE-LIPID

Courtney, 1984b). The data presented in Fig. 1 demonstrate that the multiple discrete bands detected after the addition of CX are metabolically labeled by rH]mannose adding further support to the conclusion that these stepwise patterns of bands represent the nonglycosylated core plus mannose-rich, endo H-sensitive chains. In addition, after extended incubation times in the presence of CX, analysis by immunoblotting revealed that four additional bands was the maximum number detected. Since it has been demonstrated that lipid-linked oligosaccharides do not accumulate when protein synthesis has been inhibited in a variety of cell types (Schmitt and Elbein, 1979; Hubbard and Robbins, 1980), the most likely explanation for the detection of forms containing up to four oligosaccharide chains is a depletion of the remaining pool of oligosaccharidelipid in the nuclear membrane. It would be of interest to reverse the CX block and determine if complete glycosylation of the partially glycosylated forms can occur or whether they represent biosynthetic end products. Furthermore, it is not known whether the partially glycosylated forms in association with the nuclear fraction are capable of being incorporated into virions which acquire their lipid envelope as well as the virus-specific glycoproteins from the nuclear membrane. Under normal conditions, the available pool of completed lipid-linked oligosaccharide appears to exceed the requirements of protein glycosylation (Hubbard and Ivatt, 1981). The rate of turnover is closely tied to the rate at which completed oligosaccharide is transferred to the protein acceptor. A genetic lesion in a mutant cell line of Chinese hamster ovary cells has been described (Tenner et al., 1977; Tenner and Schemer, 1979) in which it was determined that the overall pool size of oligosaccharide-lipid was the same at permissive and nonpermissive temperature and that the actual defect was a reduced rate of transfer of the oligosaccharide chain. The data presented in Table 1 and Fig. 2 argue against a slow transfer of the oligosaccharide chain to pgC75 since the pool size of oligosaccharide-lipid is lower in vi-

AND

HSV-INFECTED

CELLS

7

rus-infected cells compared to uninfected Vero cells. A possible explanation is that Vero cells incubated at 34” cannot accommodate the dramatic increase in demand for oligosaccharide chains as indicated by enhanced mannose incorporation into virally encoded glycoproteins. Indeed, there may be an accelerated transfer of the oligosaccharide chains to glycoproteins suggesting the availability of protein acceptor sites for N-linked glycosylation may influence the cellular pool size of this biosynthetic intermediate. It is also possible to hypothesize that the reason Vero cells cannot accommodate the increased demand for oligosaccharide chains is due to a slow recycling ability or a limitation in one of the regulatory enzymes. The exact effect of the enhancement of this phenotype at a lower temperature of incubation is not known. Pool sizes of oligosaccharide-lipid are the same in uninfected and infected Vero cells incubated at 39” (see Table 1) as well as in MRC-5 cells-a cell line which does not accumulate nonglycosylated forms (unpublished results). Furthermore, the appearance of the nonglycosylated form is particularly prominent in the nuclear fraction which may infer that the inability of Vero cells to accommodate glycosylation may be peculiar to the nuclear membrane and nuclear-associated RER. Herpes simplex virus may prove to be a useful model system to study the regulation of the dolichol pathway and the cells’ capacity to function under the stressful conditions with respect to increased demand for glycoprotein synthesis. ACKNOWLEDGMENTS This work was supported by Public Health Service Grant CA 24564 from the National Institutes of Health. T.C. is a predoctoral fellow supported by National Research Service Award Training Grant T32 AI07123 from the National Institutes of Health. We thank Jackie Pratt for expert assistance in preparation of the manuscript. We also gratefully acknowledge the encouragement and helpful discussions with C. J. Waechter during the course of this work. REFERENCES BAUCKE, R. B., and SPEAR, P. G. (1979). Membrane proteins specified by herpes simplex virus. V. Iden-

COMPTON

AND

tification of an Fc-binding glycoprotein. J. I%&. 32, 779-789. BONE, D. R., and COURTNEY, R. J. (1974). A ts mutant of herpes simplex virus type 1 defective in the synthesis of the major capsid protein. J. Gen. Fiirol. 24, 17-27. COMPTON, T., and COURTNEY, R. J. (1984a). Virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type l-infected cells. J. Viral. 49, 594-597. COMPTON, T., and COURTNEY, R. J. (1984b). Evidence for post-translational glycosylation of a nonglycosylated precursor protein of herpes simplex virus type 1. J. Viral. 52, 630-637. COURTNEY, R. J. (1976). Herpes simplex virus protein synthesis in the presence of 2-deoxy-n-glucose. Vi7dfl{Jy 73, 286-294. EBERLE, R., and COURTNEY, R. J. (1980). Preparation and characterization of specific antisera to individual glycoprotein antigens comprising the major glycoprotein region of herpes simplex virus type 1. J. Viral. 35, 902-917. EISENBERG, R. J., HYDREAN-STERN, C., and COHEN, G. H. (1979). Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of HSV-1. J. Viral. 31, 608620. HEIFETZ, A., KEENAN, R. W., and ELBEIN, A. D. (1979). Mechanism of action of tunicamycin on the UDPGlcNAc:dolchylphosphate transferase. Bkhemistry 18,2186-2192. HUBBARD, S. C., and TVATT, R. J. (1981). Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 50, 555-583. HUBBARD, S. C., and ROBBINS, P. W. (1980). Synthesis of the N-linked oligosaccharides of glycoproteins. Assembly of the lipid-linked precursor oligosaccharide and its relation to protein synthesis in vi?o. J. Biol. Chern. 255, 11782-11793. PENMAN, S. (1966). RNA metabolism in the HeLa cell nucleus. J. Mol. Bid 17, 117-130. POWELL, K. L., and COURTNEY, R. J. (1975). Polypeptides synthesized in herpes simplex virus type 2infected HEp-2 cells. Virology 66, 217-228. SABATINI, D. D., KREBICH, G., MORIMOTO, T., and ADESNIK, M. (1982). Mechanism for the incorporation of proteins in membranes and organelles. J. Cell Biol. 92, l-22.

COURTNEY SCHMITT, J. W., and ELBEIN, A. D. (1979). Inhibition of protein synthesis also inhibits synthesis of lipidlinked oligosaccharides. J. Biol. Chem. 254, 1229b 12294. SEKAFINI-CESSI, F., and CAMPADELLI-FILJME, G. (1981). Studies on benzhydrazone, a specific inhibitor of herpesvirus glycoprotein synthesis. Size distribution of glycopeptides and endo-/-N-acetgl glucosimindase H treatment. Arch. Vid 70, 33-343. SNIDER, D., and ROGERS, C. (1984). Transmembrane movement of oligosaccharide-lipids during glycoprotein synthesis. Cell 36, 753-761. SPEAR, P. G. (1976). Membrane proteins specified by herpes simplex viruses. I. Identification of four glycoprotein precursors and their products in type 1 infected cells. J. Viral. 17, 991-1008. SPEAR, P. G., and ROIZMAN, 8. (1972). Proteins specified by herpes simplex virus, V. Purification and structural proteins of the herpesvirion. J. Viral. 9, 14% 159. STRTJCK, D. K., and LENNARZ, W. J. (1980). The function of saccharide-lipids in synthesis of glycoproteins. Ire “The Biochemistry of Glycoproteins and Proteoglycans” W. J. Lennarz (ed.), pp. 35-73. Plenum, New York. TENNER, A. J., and SCHEFFLER, I. E. (1979). Lipid-saccharide intermediate and glycoprotein biosynthesis in a temperature-sensitive Chinese hamster cell mutant. J. Cell. Physiol. 98, 251-266. TENNER, A. J., ZIEC, J., and SCHEFFLER, I. E. (1977). Glycoprotein synthesis in a temperature-sensitive Chinese hamster cell cycle mutant. J. Cell Physiol. 90, 145-160. WAECHTER, C. J., and &HER, M. G. (19’78). Glucosylphoryl dolichol: Role as a glucosyl donor in the biosynthesis of an oligosaccharide lipid intermediate by calf brain membranes. Arch. Biochem. Bioph ys. 188,385-393. WAECHTER, C. J., SCHMITT, J. W., and CATTF:KALL, W. A. (1983). Glycosylation is required for maintenance of functional sodium channels in neuroblastoma cells. J. Biol. Chem. 258,5117-5123. WARNER, J. R., KNOPF, P. M., and RICH, A. (1963). A multiple ribosomal structure in protein synthesis. Proc. Natl. AC&. Sci. USA 49, 122-129. WENSKE, E. A., BRATTON, M. W., and COIJRTNEY, R. J. (1982). Endo-8-N acetyl glucosaminidase H sensitivity of precursors to herpes simplex virus type-l glycoproteins gB and gC. J Viral. 44, 241-248.