Vol. 28, No. 1
JOURNAL OF VIROLOGY, Oct. 1978, p. 368-374 0022-538X/78/0028-0368$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Assembly of Viral Membranes: Maturation of the Vesicular Stomatitis Virus Glycoprotein in the Presence of Tunicamycin T. G. MORRISON,* C. 0. McQUAIN, AND D. SIMPSON Department of Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Received for publication 11 April 1978
The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375-385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.
The vesicular stomatitis virus (VSV) particle is surrounded by a membrane which contains on its external surface one viral encoded protein, the glycoprotein (G) (3, 4, 8, 22, 25, 26, 34, 36). The immediate precursors of mature virions are regions of host-cell plasma membrane which contain glycoprotein as well as the rest of the virion components (26). The glycoprotein arrives at the external surface of the host-cell plasma membrane (15) by a complex series of steps which begin even as the protein is synthesized. First, the nascent polypeptide chain associates with the membrane of the rough endoplasmic reticulum (2, 7, 9, 10, 21, 23). Second, the protein is transported from the cytoplasmic side to the lumenal side of the endoplasmic reticulum (13, 24, 28, 32, 33). The glycoprotein then migrates to smooth membranes and finally to the plasma membrane of infected cells (11, 14, 17, 35). The total transit time from the rough endoplasmic reticulum to the plasma membrane has been estimated at 20 to 25 min (1, 6, 15). Glycosylation of the G protein begins in the rough endoplasmic reticulum with the addition of the core oligosaccharides (12, 27) and proceeds as the protein migrates to the smooth membrane fraction of infected cells (12, 15). Tunicamycin is a glucosamine-containing antibiotic which interferes with the formation of N-acetylglucosamine-lipid intermediates that serve as sugar donors for the synthesis of the core region of oligosaccharide chains of glycoproteins (30, 31). Thus, glycoproteins synthesized in the presence of tunicamycin fail to be glycosylated (19, 20, 31). Leavitt et al. (19) have shown that VSV-infected Chinese hamster ovary (CHO) cells 368
treated with tunicamycin synthesize the G protein in an unglycosylated form and do not release mature virus particles. Furthermore, the glycoprotein does not even reach the plasma membrane of tunicamycin-treated cells (20). Thus, a block in the glycosylation of the VSV glycoprotein appears to inhibit some step in the morphogenesis of the virus particle. We report here that in tunicamycin-treated VSV-infected cells, the glycoprotein attached to membranes normally and was transported across membranes to the lumen of the endoplasmic reticulum but did not move to low-density membranes. MATERIALS AND METHODS Cells and virus. The cells used were CHO cells. VSV, Indiana serotype, was grown and purified as described previously (29).
Preparation of cytoplasmic extracts. CHO cells (4.0 x 106) growing at 37°C were infected with purified B particles of VSV at a multiplicity of 5 PFU/cell as described previously (23). Tunicamycin was added at 1.5 h postinfection (0.5 tig/ml). At 4.5 h postinfection, cells were harvested by centrifugation, washed once in modified Eagle minimal essential medium (MEM) minus methionine (supplemented with nonessential amino acids and dialyzed fetal calf serum), and resuspended in 0.5 ml of supplemented MEM minus methionine. [35S]methionine (0.125 mCi/ml; Amersham Corp.; 1,170 Ci/mmol) was added to the infected cells, and incubation was continued for 15 min at 37°C. The infected cells were then rapidly mixed with ice-cold MEM, harvested by centrifugation, washed once in cold 10% sucrose, and disrupted with a tight-fitting Dounce homogenizer. Just prior to cell disruption, 4 x 107 unlabeled, uninfected CHO cells which had been washed in 10% sucrose were added to the infected cells. Nuclei were removed by centrifugation and
VOL. 28, 1978
MATURATION OF VSV GLYCOPROTEIN
washed once in 10% sucrose. The resulting supernatant fluid was combined with the cytoplasmic extract. Preparation of infected-cell membranes by sedimentation. The cytoplasmic extracts were layered over preformed discontinuous sucrose gradients containing a layer of 80% (wt/vol) sucrose (5 ml) and a layer of 20% (wt/vol) sucrose (8 ml). The gradients were centrifuged for 3 h in a Beckman SW27.1 rotor at 24,000 rpm (90,000 x g) at 5°C. Visible membranous material was seen at the interface between the 80% and 20% sucrose layers. Preparation of infected-cell membranes by flotation. Membranes prepared by sedimentation were made 80% with respect to sucrose, placed in the bottom of a Beckman SW27.1 centrifuge tube, and overlayered with 65% sucrose (8 ml) and 20% sucrose (2 ml). The gradient was centrifuged to equilibrium (24,000 rpm, 90,000 x g, 18 h, 5°C). Visible membranous material floats to the interface of the 65% and 20% sucrose layers. Polyacrylamide gel electrophoresis. Polypep-
A
369
tides were resolved on 10% polyacrylamide slab gels (14 by 12 by 0.15 cm) prepared and run as outlined by Laemmli (17). The polyacrylamide gels were then fixed and stained with Coomassie brilliant blue as described by Clinkscales et al. (5), dried, and subjected to autoradiography (Kodak X-ray film, X-Omat). RESULTS
Effect of tunicamycin on the membrane association of the glycoprotein. To determine whether the glycoprotein made in the presence of tunicamycin is membrane associated, membranes were isolated from cytoplasmic extracts of infected cells treated with the antibiotic. VSV-infected cells which had been incubated with tunicamycin were radioactively labeled with [3S]methionine. Infected cells to which no drug was added were labeled in parallel. As has
C
B
D
A" I
OW-0
"'m
OR
N-
-j
G
I
-N
FIG. 1. Membrane association of the unglycosylated G protein. Membranes prepared by sedimentation (channels A and B) or flotation (channels C and D) as described in Materials and Methods were acetone precipitated, and the precipitate was resuspended in gel sample buffer (1% sodium dodecyl sulfate, 0.125 M Tris-hydrochloride, pH 6.8, 0.7 M f3-mercaptoethanol, and 20% glycerol), boiled, and subjected to polyacrylamnide gel electrophoresis on 10% Laemmli gels (1 7). The figure shows autoradiograms of the fixed, dried gels. Channels A and C: no tunicamycin added. Channels B and D: tunicamycin (0.5 pg/ml) added. Autoradiograms shown in channels A and B were exposed to X-ray film for 48 h. Equal amounts of radioactivity were applied to A and B. Autoradiograms shown in channels C and D were exposed to X-ray film for 24 h. Equal amounts of radioactivity were applied to C and D.
370
J. VIROL.
MORRISON, McQUAIN, AND SIMPSON
been reported by Leavitt et al. (19), incorporation of [35S]methionine into trichloroacetic acidprecipitable material in extracts derived from tunicamycin-treated cells was reduced to 40% of the incorporation detected in extracts derived from untreated cells (Table 1). Membranous material in these cytoplasmic extracts was first isolated by sedimentation in discontinuous sucrose gradients as described in Materials and Methods. By this procedure, 22% of the total protein content of both extracts sedimented into the membrane fractions of the gradient, and approximately the same fraction of the total trichloroacetic acid-precipitable radioactivity in both cell extracts sedimented with membranous material (44% of the radioactivity present in extracts derived from tunicamycintreated cells and 38% of the radioactivity present in extracts derived from untreated cells, Table 1). Therefore, tunicamycin had no effect on the distribution of radioactivity in infected cells. Channels A and B in Fig. 1 show autoradiograms of polyacrylamide gels containing the VSV proteins present in the membranous material derived from both treated and untreated infected cells. Both membrane preparations contained the G protein as well as the N, NS, M, and L proteins. Non-membrane-associated material found in the top of the discontinuous gradients contained no G protein (not shown). Unglycosylated glycoprotein migrates on sodium dodecyl sulfate-containing polyacrylamide gels with an apparent molecular weight of 63,000 (16, 23), whereas the fully glycosylated form migrates with an apparent molecular weight of 69,000. Consistent with the report of Leavitt et al. (19), the G protein made in the presence of tunicamycin had an apparent molecular weight of 63,000. To eliminate the possibility that the G protein found in this membrane fraction resulted from cosedimentation of membranes and aggregates of the G protein, membranes were isolated by a
A B
G N
NS----
M
C
D
_
-- _ _ 'krV ''
Jrw
_
t
| 4W
_
+
_
+
FIG. 2. Trypsin sensitivity of the G protein. Cytoplasmic extracts of infected cells were digested with trypsin (Worthington Biochemicals Corp., 50 pg/ml) for 30 min at 25°C (channels B and D). Control extracts were incubated in the absence of trypsin (channels A and C). Soybean trypsin inhibitor (Boehringer/Mannheim, 200 pg/ml) was added, and the extracts were acetone precipitated. The precipitate was resuspended in gel sample buffer containing 0.1% Triton and 0.1% deoxycholate and was subjected to electrophoresis on 10% polyacrylamide gels. Extracts derived from untreated cells are shown in channels A and B; extracts derived from tunicamycin-treated cells are shown in channels C and D.
TABLE 1. Fraction of radioactivity present in the membrane fraction of infected cells after tunicamycin treatmenta Without tunicamycin treatment
With tunicamycin treatment
Fraction Total protein (jug)
Radioactivity (cpm)
Total protein (,ug)
Radioactivity (cpm)
2.6 x 106 Cytoplasmic extract. 2,600 1.0 x 106 3,200 Membranes isolated by sedi580 (22%)b mentation ...... 9.9 x 105 (38%)C 4.4 x 105 (44%)C 710 (22%)b Membranes isolated by flotation ....... 280 (11%) 6.4 x 105 (25%) 370 (12%) 3.1 x 105 (31%) Infected celLs were radioactively labeled for 15 min with [3S]methionine at 4.5 h after initiation of the infection. Cells treated with tunicamycin contained the antibiotic from 1.5 h after the initiation of infection. Cytoplasmic extracts were prepared and membranes were isolated from cell extracts as described in Materials and Methods. Total protein concentration was determined by use of a Bio-Rad protein assay kit. b Percentage of total protein. c Percentage of total radioactivity. a
MATURATION OF VSV GLYCOPROTEIN
VOL. 28, 1978
second procedure which involved flotation of membranes in a discontinuous sucrose gradient. Table 1 shows that the fraction of the total cytoplasmic trichloroacetic acid-precipitable radioactive material recovered in membrane fractions of extracts derived from treated and untreated cells was similar (31% and 25%, respectively). Channels C and D in Fig. 1 show autoradiograms of polyacrylamide gels containing VSV proteins present in the membranous material isolated by flotation. Both membrane preparations contained primarily the G protein and M protein, in approximately the same proportions. Thus, the results of both membrane purification procedures suggest that tunicamycin does not prevent membrane association of the G protein. Transport of the glycoprotein across membranes in the presence of tunicamycin. We have previously presented evidence that the
371
glycoprotein is transported across intracellular membranes to the lumen of the endoplasmic reticulum (24). This conclusion was indicated by the fact that newly made glycoprotein is largely resistant to digestion with trypsin. However, trypsin does reduce the apparent size of the glycoprotein by 3,000 daltons, suggesting that the glycoprotein is transmembranal and that approximately 3,000 daltons of the polypeptide is on the cytoplasmic side of the membrane. Cell extracts derived from tunicamycintreated, infected cells were digested with trypsin. Extracts derived from untreated cells were also digested in parallel. After digestion, the products were resolved on polyacrylamide gels (Fig. 2). Trypsin treatment of cell extracts derived from tunicamycin-treated, infected cells as well as untreated cells reduced the apparent size of the G proteins by 3,000 daltons (Fig. 2, channels C and D). After trypsin digestion, 80% of both the
10min
65 60 45
65 60 45 40 %Sucrose % Sucrose FIG. 3. Density of membranes containing pulse-labeled VSV proteins. At 4.5 h postinfection, infected cells were pulse-labeled with f3S]methionine for 2, 5, 10, and 15 min. Incorporation of radioactivity was linear during the 15-min label. Incorporation was terminated by mixing the infected cells with a large volume of frozen 1 0% sucrose. Cytoplasmic extracts of these infected cells were prepared as described in Materials and Methods, and membranes were purified from the cytoplasmic extracts by flotation. The membranes were then made 80% (wt/vol) with respect to sucrose (p = 1.298 g/cm3 at 20°C), overlayered with 2 ml of 65% sucrose (p = 1.243g/cm3), 2 ml of 60% sucrose (p = 1.225g/cm3), 4 ml of 45% sucrose (p = 1.171 g/cm3), 2 ml of 40% sucrose (p = 1.151 g/cm3), and 2 ml of 30% sucrose (p = 1.112 g/cm3), and centrifuged to equilibrium (24,000 rpm or 90,000 x g in a Spinco SW27.1 rotor for 18 h at 5°C). Gradients were collected from the bottom. The figure shows the pattern of trichloroacetic acid-precipitable material across the gradient after centrifugation.
372
J. VIROL.
MORRISON, McQUAIN, AND SIMPSON
glycosylated and unglycosylated glycoprotein was recovered in the cleaved form. Movement of the glycoprotein to lowdensity membranes. Numerous studies of the morphogenesis of VSV have presented evidence that the VSV glycoprotein initially associates with rough endoplasmic reticulum (7, 10, 11, 14, 15, 18, 21), the site of its synthesis (2, 9, 23). With time, the nascent glycoprotein moves to smooth membranes and finally to the cell surface (1, 11, 14, 15, 18). The time required for this transit has been estimated to be about 25 min (1, 11, 15). In agreement with these results, we have found that membranes prepared from extracts derived from cells radioactively labeled for 2 and 5 min contained VSV protein only in high-density membranes (Fig. 3, panels A and B). However, after a labeling period of 10 min, some VSV protein could be detected in lowerdensity membranes, and after 15 min of labeling a significant amount of VSV protein could be found in low-density membranes (Fig. 3, panels C and D), suggesting that the G protein had moved to smooth membrane fractions of infected cells. Both low- and high-density membranes contain both G and M proteins (24). To determine whether the VSV glycoprotein made in the presence of tunicamycin is capable of moving from high-density to low-density membranes, infected cells, untreated and pretreated with tunicamycin, were labeled with [35S]methionine for 15 min. Membranes were isolated by flotation and then centrifuged to equilibrium in discontinuous sucrose gradients constructed to resolve high- and low-density membranes. Figure 4 shows the pattern of trichloroacetic acid-precipitable material across the gradients. Panel B shows the pattern typical of membranes derived from untreated cells: radioactivity was found in both high- and lowdensity membranes. Panel A shows the pattern obtained from membranes isolated from tunicamycin-treated cells. The membrane-associated material remained in high-density membranes. Both G and M proteins were present in this material (Fig. 1, channel D). DISCUSSION The G protein is unique among the VSV proteins in that it is glycosylated (34). The precise role of the carbohydrate in viral morphogenesis is unclear. We have sought to determine what effect a failure to glycosylate the glycoprotein might have on viral morphogenesis. These studies utilized the antibiotic tunicamycin. Treatment of VSV-infected cells with tunicamycin seems to result in the synthesis of an unglycosylated glycoprotein (19). There are two
co1 M
E4B
U
3 2
70
40 130 X Sucrose
85145
FIG. 4. Density of membranes containing VSV proteins pulse-labeled in the presence of tunicamycin. Infected cells were treated with tunicamycin (0.5 pg/ml) from 1.5 to 4.5 h postinfection. At 4.5 h postinfection, the cells were pulse-labeled with [5S]methionine for 15 min. VSV-infected cells untreated with antibiotic were labeled in parallel. Membranes were isolated by flotation from these infected cells as described in Materials and Methods. Purified membranes were made 70%o with respect to sucrose (p = 1.263 g/cm3 at 20°C) and reisolated by flotation in gradients containing 2 ml of 65% sucrose, 4 ml of 45% sucrose, 4 ml of 40%o sucrose, and 2 ml of 30%lo sucrose (24,000 rpm or 90,000 x g, Spinco SW27.1 rotor, 18 h, 5°C). The figure shows the pattern of trichloroacetic acid-precipitable material across the gradient after centrifugation. (A) Membranes isolated from cells treated with tunicamycin. (B) Membranes isolated from untreated cells. Equal amounts of radioactivity were present in each gradient.
lines of evidence which support this conclusion. First, VSV-infected cells which have been treated with tunicamycin synthesize a glycoprotein which has a molecular weight of 63,000 (19), a size similar to that of the unglycosylated G protein made in membrane-free cell-free protein-synthesizing systems (16,23). Second, Leavitt et al. (19) have reported that in the presence
MATURATION OF VSV GLYCOPROTEIN
VOL. 28, 1978
of tunicamycin glucosamine is not incorporated into the glycoprotein. We have found that unglycosylated G protein is fully membrane associated, as is the glycosylated form of the G protein (18, 35). Using two different procedures for membrane purification, we found the unglycosylated G protein in the membrane fraction of cell extracts to the same extent as the glycosylated glycoprotein. Furthermore, no G protein was found in the soluble fraction of either treated or untreated cells. We have also found that, just as in the case of extracts derived from untreated cells, trypsin digestion of cell extracts derived from tunicamycin-treated infected cells reduced the apparent size of the G protein by 3,000 daltons. This result suggests that the unglycosylated G protein is also transported to the lumen of the endoplasmic reticulum in much the same manner as the fully glycosylated G protein: a small portion of the molecule remains on the cytoplasmic face of the membrane. Finally, in extracts derived from tunicamycintreated cells, no labeled VSV protein could be found in low-density membranes. This result suggests that the unglycosylated G protein is unable to migrate from the rough endoplasmic reticulum. Failure to migrate to smooth membranes may be attributed to the failure to be glycosylated: sequential glycosylation could be responsible for the migration of the protein. Alternatively, the absence of carbohydrate on the molecule may alter the conformation of the protein such that it is unable to participate in the normal mechanisms responsible for the migration of proteins through membranes (20). ACKNOWLEDGMENTS We thank Donald J. Tipper for tunicamycin, M. A. Bratt and C. Madansky for critical reading of the manuscript and helpful discussion, and P. Theriault for preparation of the manuscript. This work was supported by National Science Foundation grant PCM 76-23290 and by Public Health Service grant NIH 1 R01 AI 13847-OlAl from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Atkinson, P., S. Moyer, and D. Summers. 1976. Assembly of vesicular stomatitis virus glycoprotein and matrix protein into Hela cell plasma membranes. J. Mol. Biol. 102:613-632. 2. Both, G., S. Moyer, and A. Bannerjee. 1975. Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells. J. Virol. 15:1012-1019. 3. Cartwright, B., C. J. Smale, and F. Brown. 1969. Surface structure of vesicular stomatitis virus. J. Gen. Virol. 5:1-10. 4. Cartwright, B., P. Talbot, and F. Brown. 1970. The proteins of biologically active subunits of vesicular stomatitis virus. J. Gen. Virol. 7:267-272. 5. Clinkscales, W. C., M. A. Bratt, and T. G. Morrison.
6.
7. 8. 9.
10.
11. 12. 13.
373
1977. Synthesis of Newcastle disease virus polypeptides in a wheat germ cell-free system. J. Virol. 22:97-101. Cohen, G., P. Atkinson, and D. Suimmers. 1971. Interactions of vesicular stomatitis virus structural proteins with Hela plasma membrane. Nature (London) New Biol. 231:121-124. David, A. 1977. Assembly of the vesicular stomatitis virus envelope: transfer of viral polypeptides from polysomes to cellular membranes. Virology 76:98-108. Eger, R., R. Compans, and P. Rifkin. 1975. The organization of the proteins of vesicular stomatitis virions: labeling with pyridoxal phosphate. Virology 66:610-615. Grubman, M., S. Moyer, A. Bannerjee, and E. Ehrenfeld. 1975. Sub-cellular localization of vesicular stomatitis virus messenger RNAs. Biochem. Biophys. Res. Commun. 62:531-538. Grubman, M. J., J. A. Weinstein, and P. A. Shafritz. 1977. Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein mRNA into membrane bound polyribosome complexes. J. Cell Biol. 74:43-57. Hunt, L., and P. Summers. 1976. Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus. J. Virol. 20:637-645. Hunt, L. A., and P. F. Summers. 1976. Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells. J. Virol. 20:646-657. Katz, F., J. E. Rothman, V. R. Lingappa, G. Blobel, and H. F. Lodish. 1977. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc. Natl. Acad. Sci. U.S.A.
74:3278-3282. 14. Knipe, D., D. Baltimore, and H. Lodish. 1977. Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus. J. Virol. 21:1128-1139. 15. Knipe, D., H. Lodish, and D. Baltimore. 1977. Localization of two cellular forms of the vesicular stomatitis viral glycoprotein. J. Virol. 21:1121-1127. 16. Knipe, D., J. R. Rose, and H. F. Lodish. 1975. Translation of individual species of vesicular stomatitis viral
mRNA. J. Virol. 15:1004-1011. 17. Laemmli, U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 18. Lafay, F. 1974. Envelope proteins of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups III and V. J. Virol. 14:1220-1228. 19. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J. Virol. 21:375-385. 20. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. J. Biol. Chem. 252:9018-9025. 21. Lodish, H. F., and S. Froshauer. 1977. Binding of viral glycoprotein mRNAs to endoplasmic reticulum membranes is disrupted by puromycin. J. Cell Biol. 74:358-365. 22. Moore, N., J. Kelley, and R. Wagner. 1974. Envelope proteins of vesicular stomatitis virions: accessibility to iodination. Virology 61:292-296. 23. Morrison, T., and H. Lodish. 1975. Site of synthesis of membrane and nonmembrane proteins of vesicular stomatitis virus. J. Biol. Chem. 250:6955-6962. 24. Morrison, T. G., and C. 0. McQuain. 1978. Assembly of viral membranes: the nature of the association of vesicular stomatitis virus proteins to membranes. J. Virol. 26:115-125. 25. Mudd, J. 1974. Glycoprotein fragment associated with vesicular stomatitis virus after proteolytic digestion. Virology 62:573-577. 26. Nakai, T., and A. F. Howatson. 1968. The fine structure of vesicular stomatitis virus. Virology 35:268-281.
MORRISON, McQUAIN, AND SIMPSON
J. VIROL.
27. Robertson, J. S., J. R. Etchison, and D. F. Summers. 1976. Glycosylation sites of vesicular stomatitis virus glycoprotein. J. Virol. 19:871-878. 28. Rothman, J. E., and H. F. Lodish. 1977. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature (London) 269:775-780. 29. Stampfer, M., A. Huang, and D. Baltimore. 1969. Ribonucleic acid synthesis of vesicular stomatitis virus. I. Species of ribonucleic acid found in Chinese hamster ovary cells infected with plaque-forming and defective particles. J. Virol. 4:154-161. 30. Takatsuki, A., K. Arima, and G. Tamura. 1971. Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. J. Antibiot. 24:215-223. 31. Takatsuki, A., K. Kohro, and G. Tamura. 1975. Inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin. Agric. Biol. Chem. 39:2089-2091. 32. Toneguzzo, F., and H. P. Ghosh. 1977. Synthesis and
glycosylation in vitro of glycoprotein of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 74: 1516-1520. Toneguzzo, F., and H. P. Ghosh. 1978. In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes. Proc. Natl. Acad. Sci. U.S.A. 75:715-719. Wagner, R. R. 1975. Reproduction of rhabdoviruses, p. 1-93. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Press, New York. Wagner, R., M. Kiley, R. Synder, and C. Schnaitman. 1972. Cytoplasmic compartmentalization of the proteins and ribonucleic acid species of vesicular stomatitis virus. J. Virol. 9:672-683. Wagner, R. R., T. C. Schnaitman, R. M. Snyder, and C. A. Schnaitman. 1969. Protein composition of the structural components of vesicular stomatitis virus. J. Virol. 3:611-618.
374
33.
34.
35.
36.
JOURNAL OF VIROLOGY, Oct. 1978, p. 368-374 0022-538X/78/0028-0368$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Assembly of Viral Membranes: Maturation of the Vesicular Stomatitis Virus Glycoprotein in the Presence of Tunicamycin T. G. MORRISON,* C. 0. McQUAIN, AND D. SIMPSON Department of Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Received for publication 11 April 1978
The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375-385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.
The vesicular stomatitis virus (VSV) particle is surrounded by a membrane which contains on its external surface one viral encoded protein, the glycoprotein (G) (3, 4, 8, 22, 25, 26, 34, 36). The immediate precursors of mature virions are regions of host-cell plasma membrane which contain glycoprotein as well as the rest of the virion components (26). The glycoprotein arrives at the external surface of the host-cell plasma membrane (15) by a complex series of steps which begin even as the protein is synthesized. First, the nascent polypeptide chain associates with the membrane of the rough endoplasmic reticulum (2, 7, 9, 10, 21, 23). Second, the protein is transported from the cytoplasmic side to the lumenal side of the endoplasmic reticulum (13, 24, 28, 32, 33). The glycoprotein then migrates to smooth membranes and finally to the plasma membrane of infected cells (11, 14, 17, 35). The total transit time from the rough endoplasmic reticulum to the plasma membrane has been estimated at 20 to 25 min (1, 6, 15). Glycosylation of the G protein begins in the rough endoplasmic reticulum with the addition of the core oligosaccharides (12, 27) and proceeds as the protein migrates to the smooth membrane fraction of infected cells (12, 15). Tunicamycin is a glucosamine-containing antibiotic which interferes with the formation of N-acetylglucosamine-lipid intermediates that serve as sugar donors for the synthesis of the core region of oligosaccharide chains of glycoproteins (30, 31). Thus, glycoproteins synthesized in the presence of tunicamycin fail to be glycosylated (19, 20, 31). Leavitt et al. (19) have shown that VSV-infected Chinese hamster ovary (CHO) cells 368
treated with tunicamycin synthesize the G protein in an unglycosylated form and do not release mature virus particles. Furthermore, the glycoprotein does not even reach the plasma membrane of tunicamycin-treated cells (20). Thus, a block in the glycosylation of the VSV glycoprotein appears to inhibit some step in the morphogenesis of the virus particle. We report here that in tunicamycin-treated VSV-infected cells, the glycoprotein attached to membranes normally and was transported across membranes to the lumen of the endoplasmic reticulum but did not move to low-density membranes. MATERIALS AND METHODS Cells and virus. The cells used were CHO cells. VSV, Indiana serotype, was grown and purified as described previously (29).
Preparation of cytoplasmic extracts. CHO cells (4.0 x 106) growing at 37°C were infected with purified B particles of VSV at a multiplicity of 5 PFU/cell as described previously (23). Tunicamycin was added at 1.5 h postinfection (0.5 tig/ml). At 4.5 h postinfection, cells were harvested by centrifugation, washed once in modified Eagle minimal essential medium (MEM) minus methionine (supplemented with nonessential amino acids and dialyzed fetal calf serum), and resuspended in 0.5 ml of supplemented MEM minus methionine. [35S]methionine (0.125 mCi/ml; Amersham Corp.; 1,170 Ci/mmol) was added to the infected cells, and incubation was continued for 15 min at 37°C. The infected cells were then rapidly mixed with ice-cold MEM, harvested by centrifugation, washed once in cold 10% sucrose, and disrupted with a tight-fitting Dounce homogenizer. Just prior to cell disruption, 4 x 107 unlabeled, uninfected CHO cells which had been washed in 10% sucrose were added to the infected cells. Nuclei were removed by centrifugation and
VOL. 28, 1978
MATURATION OF VSV GLYCOPROTEIN
washed once in 10% sucrose. The resulting supernatant fluid was combined with the cytoplasmic extract. Preparation of infected-cell membranes by sedimentation. The cytoplasmic extracts were layered over preformed discontinuous sucrose gradients containing a layer of 80% (wt/vol) sucrose (5 ml) and a layer of 20% (wt/vol) sucrose (8 ml). The gradients were centrifuged for 3 h in a Beckman SW27.1 rotor at 24,000 rpm (90,000 x g) at 5°C. Visible membranous material was seen at the interface between the 80% and 20% sucrose layers. Preparation of infected-cell membranes by flotation. Membranes prepared by sedimentation were made 80% with respect to sucrose, placed in the bottom of a Beckman SW27.1 centrifuge tube, and overlayered with 65% sucrose (8 ml) and 20% sucrose (2 ml). The gradient was centrifuged to equilibrium (24,000 rpm, 90,000 x g, 18 h, 5°C). Visible membranous material floats to the interface of the 65% and 20% sucrose layers. Polyacrylamide gel electrophoresis. Polypep-
A
369
tides were resolved on 10% polyacrylamide slab gels (14 by 12 by 0.15 cm) prepared and run as outlined by Laemmli (17). The polyacrylamide gels were then fixed and stained with Coomassie brilliant blue as described by Clinkscales et al. (5), dried, and subjected to autoradiography (Kodak X-ray film, X-Omat). RESULTS
Effect of tunicamycin on the membrane association of the glycoprotein. To determine whether the glycoprotein made in the presence of tunicamycin is membrane associated, membranes were isolated from cytoplasmic extracts of infected cells treated with the antibiotic. VSV-infected cells which had been incubated with tunicamycin were radioactively labeled with [3S]methionine. Infected cells to which no drug was added were labeled in parallel. As has
C
B
D
A" I
OW-0
"'m
OR
N-
-j
G
I
-N
FIG. 1. Membrane association of the unglycosylated G protein. Membranes prepared by sedimentation (channels A and B) or flotation (channels C and D) as described in Materials and Methods were acetone precipitated, and the precipitate was resuspended in gel sample buffer (1% sodium dodecyl sulfate, 0.125 M Tris-hydrochloride, pH 6.8, 0.7 M f3-mercaptoethanol, and 20% glycerol), boiled, and subjected to polyacrylamnide gel electrophoresis on 10% Laemmli gels (1 7). The figure shows autoradiograms of the fixed, dried gels. Channels A and C: no tunicamycin added. Channels B and D: tunicamycin (0.5 pg/ml) added. Autoradiograms shown in channels A and B were exposed to X-ray film for 48 h. Equal amounts of radioactivity were applied to A and B. Autoradiograms shown in channels C and D were exposed to X-ray film for 24 h. Equal amounts of radioactivity were applied to C and D.
370
J. VIROL.
MORRISON, McQUAIN, AND SIMPSON
been reported by Leavitt et al. (19), incorporation of [35S]methionine into trichloroacetic acidprecipitable material in extracts derived from tunicamycin-treated cells was reduced to 40% of the incorporation detected in extracts derived from untreated cells (Table 1). Membranous material in these cytoplasmic extracts was first isolated by sedimentation in discontinuous sucrose gradients as described in Materials and Methods. By this procedure, 22% of the total protein content of both extracts sedimented into the membrane fractions of the gradient, and approximately the same fraction of the total trichloroacetic acid-precipitable radioactivity in both cell extracts sedimented with membranous material (44% of the radioactivity present in extracts derived from tunicamycintreated cells and 38% of the radioactivity present in extracts derived from untreated cells, Table 1). Therefore, tunicamycin had no effect on the distribution of radioactivity in infected cells. Channels A and B in Fig. 1 show autoradiograms of polyacrylamide gels containing the VSV proteins present in the membranous material derived from both treated and untreated infected cells. Both membrane preparations contained the G protein as well as the N, NS, M, and L proteins. Non-membrane-associated material found in the top of the discontinuous gradients contained no G protein (not shown). Unglycosylated glycoprotein migrates on sodium dodecyl sulfate-containing polyacrylamide gels with an apparent molecular weight of 63,000 (16, 23), whereas the fully glycosylated form migrates with an apparent molecular weight of 69,000. Consistent with the report of Leavitt et al. (19), the G protein made in the presence of tunicamycin had an apparent molecular weight of 63,000. To eliminate the possibility that the G protein found in this membrane fraction resulted from cosedimentation of membranes and aggregates of the G protein, membranes were isolated by a
A B
G N
NS----
M
C
D
_
-- _ _ 'krV ''
Jrw
_
t
| 4W
_
+
_
+
FIG. 2. Trypsin sensitivity of the G protein. Cytoplasmic extracts of infected cells were digested with trypsin (Worthington Biochemicals Corp., 50 pg/ml) for 30 min at 25°C (channels B and D). Control extracts were incubated in the absence of trypsin (channels A and C). Soybean trypsin inhibitor (Boehringer/Mannheim, 200 pg/ml) was added, and the extracts were acetone precipitated. The precipitate was resuspended in gel sample buffer containing 0.1% Triton and 0.1% deoxycholate and was subjected to electrophoresis on 10% polyacrylamide gels. Extracts derived from untreated cells are shown in channels A and B; extracts derived from tunicamycin-treated cells are shown in channels C and D.
TABLE 1. Fraction of radioactivity present in the membrane fraction of infected cells after tunicamycin treatmenta Without tunicamycin treatment
With tunicamycin treatment
Fraction Total protein (jug)
Radioactivity (cpm)
Total protein (,ug)
Radioactivity (cpm)
2.6 x 106 Cytoplasmic extract. 2,600 1.0 x 106 3,200 Membranes isolated by sedi580 (22%)b mentation ...... 9.9 x 105 (38%)C 4.4 x 105 (44%)C 710 (22%)b Membranes isolated by flotation ....... 280 (11%) 6.4 x 105 (25%) 370 (12%) 3.1 x 105 (31%) Infected celLs were radioactively labeled for 15 min with [3S]methionine at 4.5 h after initiation of the infection. Cells treated with tunicamycin contained the antibiotic from 1.5 h after the initiation of infection. Cytoplasmic extracts were prepared and membranes were isolated from cell extracts as described in Materials and Methods. Total protein concentration was determined by use of a Bio-Rad protein assay kit. b Percentage of total protein. c Percentage of total radioactivity. a
MATURATION OF VSV GLYCOPROTEIN
VOL. 28, 1978
second procedure which involved flotation of membranes in a discontinuous sucrose gradient. Table 1 shows that the fraction of the total cytoplasmic trichloroacetic acid-precipitable radioactive material recovered in membrane fractions of extracts derived from treated and untreated cells was similar (31% and 25%, respectively). Channels C and D in Fig. 1 show autoradiograms of polyacrylamide gels containing VSV proteins present in the membranous material isolated by flotation. Both membrane preparations contained primarily the G protein and M protein, in approximately the same proportions. Thus, the results of both membrane purification procedures suggest that tunicamycin does not prevent membrane association of the G protein. Transport of the glycoprotein across membranes in the presence of tunicamycin. We have previously presented evidence that the
371
glycoprotein is transported across intracellular membranes to the lumen of the endoplasmic reticulum (24). This conclusion was indicated by the fact that newly made glycoprotein is largely resistant to digestion with trypsin. However, trypsin does reduce the apparent size of the glycoprotein by 3,000 daltons, suggesting that the glycoprotein is transmembranal and that approximately 3,000 daltons of the polypeptide is on the cytoplasmic side of the membrane. Cell extracts derived from tunicamycintreated, infected cells were digested with trypsin. Extracts derived from untreated cells were also digested in parallel. After digestion, the products were resolved on polyacrylamide gels (Fig. 2). Trypsin treatment of cell extracts derived from tunicamycin-treated, infected cells as well as untreated cells reduced the apparent size of the G proteins by 3,000 daltons (Fig. 2, channels C and D). After trypsin digestion, 80% of both the
10min
65 60 45
65 60 45 40 %Sucrose % Sucrose FIG. 3. Density of membranes containing pulse-labeled VSV proteins. At 4.5 h postinfection, infected cells were pulse-labeled with f3S]methionine for 2, 5, 10, and 15 min. Incorporation of radioactivity was linear during the 15-min label. Incorporation was terminated by mixing the infected cells with a large volume of frozen 1 0% sucrose. Cytoplasmic extracts of these infected cells were prepared as described in Materials and Methods, and membranes were purified from the cytoplasmic extracts by flotation. The membranes were then made 80% (wt/vol) with respect to sucrose (p = 1.298 g/cm3 at 20°C), overlayered with 2 ml of 65% sucrose (p = 1.243g/cm3), 2 ml of 60% sucrose (p = 1.225g/cm3), 4 ml of 45% sucrose (p = 1.171 g/cm3), 2 ml of 40% sucrose (p = 1.151 g/cm3), and 2 ml of 30% sucrose (p = 1.112 g/cm3), and centrifuged to equilibrium (24,000 rpm or 90,000 x g in a Spinco SW27.1 rotor for 18 h at 5°C). Gradients were collected from the bottom. The figure shows the pattern of trichloroacetic acid-precipitable material across the gradient after centrifugation.
372
J. VIROL.
MORRISON, McQUAIN, AND SIMPSON
glycosylated and unglycosylated glycoprotein was recovered in the cleaved form. Movement of the glycoprotein to lowdensity membranes. Numerous studies of the morphogenesis of VSV have presented evidence that the VSV glycoprotein initially associates with rough endoplasmic reticulum (7, 10, 11, 14, 15, 18, 21), the site of its synthesis (2, 9, 23). With time, the nascent glycoprotein moves to smooth membranes and finally to the cell surface (1, 11, 14, 15, 18). The time required for this transit has been estimated to be about 25 min (1, 11, 15). In agreement with these results, we have found that membranes prepared from extracts derived from cells radioactively labeled for 2 and 5 min contained VSV protein only in high-density membranes (Fig. 3, panels A and B). However, after a labeling period of 10 min, some VSV protein could be detected in lowerdensity membranes, and after 15 min of labeling a significant amount of VSV protein could be found in low-density membranes (Fig. 3, panels C and D), suggesting that the G protein had moved to smooth membrane fractions of infected cells. Both low- and high-density membranes contain both G and M proteins (24). To determine whether the VSV glycoprotein made in the presence of tunicamycin is capable of moving from high-density to low-density membranes, infected cells, untreated and pretreated with tunicamycin, were labeled with [35S]methionine for 15 min. Membranes were isolated by flotation and then centrifuged to equilibrium in discontinuous sucrose gradients constructed to resolve high- and low-density membranes. Figure 4 shows the pattern of trichloroacetic acid-precipitable material across the gradients. Panel B shows the pattern typical of membranes derived from untreated cells: radioactivity was found in both high- and lowdensity membranes. Panel A shows the pattern obtained from membranes isolated from tunicamycin-treated cells. The membrane-associated material remained in high-density membranes. Both G and M proteins were present in this material (Fig. 1, channel D). DISCUSSION The G protein is unique among the VSV proteins in that it is glycosylated (34). The precise role of the carbohydrate in viral morphogenesis is unclear. We have sought to determine what effect a failure to glycosylate the glycoprotein might have on viral morphogenesis. These studies utilized the antibiotic tunicamycin. Treatment of VSV-infected cells with tunicamycin seems to result in the synthesis of an unglycosylated glycoprotein (19). There are two
co1 M
E4B
U
3 2
70
40 130 X Sucrose
85145
FIG. 4. Density of membranes containing VSV proteins pulse-labeled in the presence of tunicamycin. Infected cells were treated with tunicamycin (0.5 pg/ml) from 1.5 to 4.5 h postinfection. At 4.5 h postinfection, the cells were pulse-labeled with [5S]methionine for 15 min. VSV-infected cells untreated with antibiotic were labeled in parallel. Membranes were isolated by flotation from these infected cells as described in Materials and Methods. Purified membranes were made 70%o with respect to sucrose (p = 1.263 g/cm3 at 20°C) and reisolated by flotation in gradients containing 2 ml of 65% sucrose, 4 ml of 45% sucrose, 4 ml of 40%o sucrose, and 2 ml of 30%lo sucrose (24,000 rpm or 90,000 x g, Spinco SW27.1 rotor, 18 h, 5°C). The figure shows the pattern of trichloroacetic acid-precipitable material across the gradient after centrifugation. (A) Membranes isolated from cells treated with tunicamycin. (B) Membranes isolated from untreated cells. Equal amounts of radioactivity were present in each gradient.
lines of evidence which support this conclusion. First, VSV-infected cells which have been treated with tunicamycin synthesize a glycoprotein which has a molecular weight of 63,000 (19), a size similar to that of the unglycosylated G protein made in membrane-free cell-free protein-synthesizing systems (16,23). Second, Leavitt et al. (19) have reported that in the presence
MATURATION OF VSV GLYCOPROTEIN
VOL. 28, 1978
of tunicamycin glucosamine is not incorporated into the glycoprotein. We have found that unglycosylated G protein is fully membrane associated, as is the glycosylated form of the G protein (18, 35). Using two different procedures for membrane purification, we found the unglycosylated G protein in the membrane fraction of cell extracts to the same extent as the glycosylated glycoprotein. Furthermore, no G protein was found in the soluble fraction of either treated or untreated cells. We have also found that, just as in the case of extracts derived from untreated cells, trypsin digestion of cell extracts derived from tunicamycin-treated infected cells reduced the apparent size of the G protein by 3,000 daltons. This result suggests that the unglycosylated G protein is also transported to the lumen of the endoplasmic reticulum in much the same manner as the fully glycosylated G protein: a small portion of the molecule remains on the cytoplasmic face of the membrane. Finally, in extracts derived from tunicamycintreated cells, no labeled VSV protein could be found in low-density membranes. This result suggests that the unglycosylated G protein is unable to migrate from the rough endoplasmic reticulum. Failure to migrate to smooth membranes may be attributed to the failure to be glycosylated: sequential glycosylation could be responsible for the migration of the protein. Alternatively, the absence of carbohydrate on the molecule may alter the conformation of the protein such that it is unable to participate in the normal mechanisms responsible for the migration of proteins through membranes (20). ACKNOWLEDGMENTS We thank Donald J. Tipper for tunicamycin, M. A. Bratt and C. Madansky for critical reading of the manuscript and helpful discussion, and P. Theriault for preparation of the manuscript. This work was supported by National Science Foundation grant PCM 76-23290 and by Public Health Service grant NIH 1 R01 AI 13847-OlAl from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Atkinson, P., S. Moyer, and D. Summers. 1976. Assembly of vesicular stomatitis virus glycoprotein and matrix protein into Hela cell plasma membranes. J. Mol. Biol. 102:613-632. 2. Both, G., S. Moyer, and A. Bannerjee. 1975. Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells. J. Virol. 15:1012-1019. 3. Cartwright, B., C. J. Smale, and F. Brown. 1969. Surface structure of vesicular stomatitis virus. J. Gen. Virol. 5:1-10. 4. Cartwright, B., P. Talbot, and F. Brown. 1970. The proteins of biologically active subunits of vesicular stomatitis virus. J. Gen. Virol. 7:267-272. 5. Clinkscales, W. C., M. A. Bratt, and T. G. Morrison.
6.
7. 8. 9.
10.
11. 12. 13.
373
1977. Synthesis of Newcastle disease virus polypeptides in a wheat germ cell-free system. J. Virol. 22:97-101. Cohen, G., P. Atkinson, and D. Suimmers. 1971. Interactions of vesicular stomatitis virus structural proteins with Hela plasma membrane. Nature (London) New Biol. 231:121-124. David, A. 1977. Assembly of the vesicular stomatitis virus envelope: transfer of viral polypeptides from polysomes to cellular membranes. Virology 76:98-108. Eger, R., R. Compans, and P. Rifkin. 1975. The organization of the proteins of vesicular stomatitis virions: labeling with pyridoxal phosphate. Virology 66:610-615. Grubman, M., S. Moyer, A. Bannerjee, and E. Ehrenfeld. 1975. Sub-cellular localization of vesicular stomatitis virus messenger RNAs. Biochem. Biophys. Res. Commun. 62:531-538. Grubman, M. J., J. A. Weinstein, and P. A. Shafritz. 1977. Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein mRNA into membrane bound polyribosome complexes. J. Cell Biol. 74:43-57. Hunt, L., and P. Summers. 1976. Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus. J. Virol. 20:637-645. Hunt, L. A., and P. F. Summers. 1976. Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells. J. Virol. 20:646-657. Katz, F., J. E. Rothman, V. R. Lingappa, G. Blobel, and H. F. Lodish. 1977. Membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein. Proc. Natl. Acad. Sci. U.S.A.
74:3278-3282. 14. Knipe, D., D. Baltimore, and H. Lodish. 1977. Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus. J. Virol. 21:1128-1139. 15. Knipe, D., H. Lodish, and D. Baltimore. 1977. Localization of two cellular forms of the vesicular stomatitis viral glycoprotein. J. Virol. 21:1121-1127. 16. Knipe, D., J. R. Rose, and H. F. Lodish. 1975. Translation of individual species of vesicular stomatitis viral
mRNA. J. Virol. 15:1004-1011. 17. Laemmli, U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 18. Lafay, F. 1974. Envelope proteins of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups III and V. J. Virol. 14:1220-1228. 19. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J. Virol. 21:375-385. 20. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. J. Biol. Chem. 252:9018-9025. 21. Lodish, H. F., and S. Froshauer. 1977. Binding of viral glycoprotein mRNAs to endoplasmic reticulum membranes is disrupted by puromycin. J. Cell Biol. 74:358-365. 22. Moore, N., J. Kelley, and R. Wagner. 1974. Envelope proteins of vesicular stomatitis virions: accessibility to iodination. Virology 61:292-296. 23. Morrison, T., and H. Lodish. 1975. Site of synthesis of membrane and nonmembrane proteins of vesicular stomatitis virus. J. Biol. Chem. 250:6955-6962. 24. Morrison, T. G., and C. 0. McQuain. 1978. Assembly of viral membranes: the nature of the association of vesicular stomatitis virus proteins to membranes. J. Virol. 26:115-125. 25. Mudd, J. 1974. Glycoprotein fragment associated with vesicular stomatitis virus after proteolytic digestion. Virology 62:573-577. 26. Nakai, T., and A. F. Howatson. 1968. The fine structure of vesicular stomatitis virus. Virology 35:268-281.
MORRISON, McQUAIN, AND SIMPSON
J. VIROL.
27. Robertson, J. S., J. R. Etchison, and D. F. Summers. 1976. Glycosylation sites of vesicular stomatitis virus glycoprotein. J. Virol. 19:871-878. 28. Rothman, J. E., and H. F. Lodish. 1977. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature (London) 269:775-780. 29. Stampfer, M., A. Huang, and D. Baltimore. 1969. Ribonucleic acid synthesis of vesicular stomatitis virus. I. Species of ribonucleic acid found in Chinese hamster ovary cells infected with plaque-forming and defective particles. J. Virol. 4:154-161. 30. Takatsuki, A., K. Arima, and G. Tamura. 1971. Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. J. Antibiot. 24:215-223. 31. Takatsuki, A., K. Kohro, and G. Tamura. 1975. Inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin. Agric. Biol. Chem. 39:2089-2091. 32. Toneguzzo, F., and H. P. Ghosh. 1977. Synthesis and
glycosylation in vitro of glycoprotein of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 74: 1516-1520. Toneguzzo, F., and H. P. Ghosh. 1978. In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes. Proc. Natl. Acad. Sci. U.S.A. 75:715-719. Wagner, R. R. 1975. Reproduction of rhabdoviruses, p. 1-93. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Press, New York. Wagner, R., M. Kiley, R. Synder, and C. Schnaitman. 1972. Cytoplasmic compartmentalization of the proteins and ribonucleic acid species of vesicular stomatitis virus. J. Virol. 9:672-683. Wagner, R. R., T. C. Schnaitman, R. M. Snyder, and C. A. Schnaitman. 1969. Protein composition of the structural components of vesicular stomatitis virus. J. Virol. 3:611-618.
374
33.
34.
35.
36.
