Apr 25, 1989 - 0-linked sugar addition, confirming that 0-linked oligosaccharides ... sugar addition on the transport of G to the cell surface were measured.
Vol. 63, No. 11
JOURNAL OF VIROLOGY, Nov. 1989, p. 4767-4776
0022-538X/89/114767-10$02.00/0 Copyright © 1989, American Society for Microbiology
Structure and Cell Surface Maturation of the Attachment Glycoprotein of Human Respiratory Syncytial Virus in a Cell Line Deficient in 0 Glycosylation GAIL W.
WERTZ,`*
MONTY KRIEGER,2 AND L. ANDREW BALL'
Department of Microbiology, University of Alabama Medical School, Basic Health Sciences Building, Birmingham, Alabama 35294,1 and Department of Biology and the Whitaker College, Massachusetts Institute of Technology, E25-236, Cambridge, Massachusetts 021392 Received 25 April 1989/Accepted 14 July 1989
The synthesis of the extensively 0-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, IdID, which has a defect in protein 0 glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or 0-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant IdlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or 0-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the 0-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of 0-linked sugar addition, confirming that 0-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of 0 glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and 0-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either 0 glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and 0-linked oligosaccharides was severely depressed.
of glycosylation have confirmed that G contains both Nlinked carbohydrate and 0-linked (0-glycanase-sensitive, mucin-type) carbohydrates and have identified a number of partially glycosylated intermediates (3, 5, 6, 16, 30). On the basis of the limited structural information available, the human RS virus G protein appears to represent one of the most highly glycosylated type II integral membrane proteins (i.e., oriented with amino terminus on the cytoplasmic side of the membrane and carboxy terminus on the exoplasmic side) described to date. Human RS virus is the major cause of bronchiolitis and pneumonia in infants. One of the most perplexing features of this disease is the ability of the virus to infect infants in the presence of maternally donated antiviral antibodies and to cause repeated infections in children who have developed neutralizing antibodies to the virus during previous infections. The relatively late maturation of responsiveness to carbohydrates in general in humans (11) has led to the hypothesis that the extensive glycosylation of G may be an important factor in determining the immune response to G. Thus, it is important to determine the extent of glycosylation of G and to understand whether the extensive glycosylation may be affecting the immune response of infants to the G protein. However, because of difficulty in isolating large quantities of the protein for structural studies and because of the lack of specific inhibitors of 0 glycosylation, little is
The attachment protein, G, of human respiratory syncytial (RS) virus is a transmembrane glycoprotein with a combination of structural features unusual among viral surface glycoproteins. The RS virus G protein has extensive 0-linked glycosylation, some N-linked carbohydrate, a high content of the amino acids serine and threonine (30%), and a 10% proline content (18, 22, 30). These structural features are similar to those observed in the cellular mucinous proteins but are not common among viral proteins. Analysis of the nucleotide sequence of the G protein gene predicts a polypeptide of 298 amino acids with a calculated molecular mass of 32 kilodaltons (kDa) (22, 30). The mature G protein has an electrophoretic mobility that corresponds to a size of approximately 90 kDa under reducing conditions, which suggests that as much as 60% of the molecular mass of the mature G protein may be contributed by carbohydrate. The G protein has a single major hydrophobic domain between residues 38 and 66 which is postulated to serve as both signal and transmembrane anchor. Compatible with the extensive 0 glycosylation, the extracellular carboxy-terminal three-quarters of the protein contains 71 serine and threonine residues which are potential sites for 0-linked carbohydrate addition and 4 potential sites for N-linked glycosylation. Studies using endoglycosidases and inhibitors *
Corresponding author. 4767
4768
J. VIROL.
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B
A
SA
SA Gal
Galoctose
-
Gal l-P
Glc 1-P
Glucose
*
UDP-Glc
Glc-Glc6P
UDP- GlcNAc
Gal GlcNAc
MAN
MAN
\MAN/
~~~~~4-Epame,ose
+
GicNAc 6-P
UDPGol
Gal GkNAc
|?| UDP-GoINAc GoINAc 1-P
GoINAc
GolNAc
N-Acetyl
GIcNAc
Gal -SA
GIcNAc
GaINAc-SA
-AS)N-
-SER-
~IGoloctosomsne
Z
_
FIG. 1. Glycosylation defect in IdID cells. (A) In culture conditions with glucose as the sole sugar in the medium, Gal and GalNAc cannot be added to glycoproteins in ldlD cells because their UDP-GalIUDP-GalNAc-4-epimerase deficiency (indicated by x) prevents the synthesis of UDP-Gal and UDP-GalNAc from glucose. The 4-epimerase deficiency can be bypassed by exogenous addition of Gal and GalNAc to the culture medium. Several intermediates in the pathways have been deleted for simplicity. (B) Typical carbohydrate structures for asparagine-linked (N-linked) and serine- or threonine-linked (0-linked) carbohydrates are shown. The positions of Gal and GalNAc are indicated in boldface; GlcNAc, N-acetylglucosamine; Man, mannose; SA, sialic acid. Adapted from reference 19.
known about the exact carbohydrate content, the role of that carbohydrate in the maturation and function of G, or its effect on the immune response to G protein. To examine the role of 0 glycosylation in G protein structure and cell surface maturation, we have used a Chinese hamster ovary (CHO) cell line, ldlD, which has a reversible defect in 0 glycosylation. The ldlD cells are UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase deficient (10) and therefore are unable to synthesize UDPgalactose and UDP-N-acetylgalactosamine under culture conditions where glucose is the sole external source of monosaccharide. These two nucleotide sugars are required to add galactose and N-acetylgalactosamine (GalNAc) to asparagine-linked (N-linked) and serine- or threonine-linked (0-linked) oligosaccharides on glycoproteins (12; see Fig. 1). The structures of typical 0-linked and N-linked oligosaccharides are shown in Fig. 1. GalNAc is the first sugar added in 0-linked biosynthesis, and galactose (Gal) is required for completion of both N- and 0-linked chains (12). The defect in sugar addition in the ldlD cells can be reversed, however, by the exogenous addition of galactose and GalNAc which then can be converted by a salvage pathway to the nucleotide sugars, thus bypassing the epimerase defect (10). Moreover, by addition of either galactose or GalNAc, one may restore N- or 0-linked glycosylation, respectively. The addition of only galactose restores the ability to complete N-linked oligosaccharides, but synthesis of 0-linked oligosaccharides does not occur. Addition of GalNAc, but not galactose, allows synthesis of truncated 0-linked chains and truncated N-linked chains. Addition of both sugars allows normal synthesis of both N- and 0-linked sugars. Thus, the ldlD cells provide a powerful tool for analysis of the role of carbohydrate in structure and maturation of glycoproteins. The ldlD cell line has been used to demonstrate the requirement for 0-linked sugars in normal cell surface expression of the low-density lipoprotein receptor and interleukin-2 receptor (10, 14, 15) and to study the role of 0 glycosylation in secretion of chorionic gonadotropin (19). These cells have allowed us to examine the effect of 0 glycosylation of the G protein on structure and cell surface maturation. MATERIALS AND METHODS
Cells and virus. Wild-type Chinese hamster ovary (CHO) cells and a UDP-Gal/UDP-GalNAc-4-epimerase-deficient
were described previously (10). Stock cultures were maintained in Ham F-12 medium supplemented with 5% fetal calf serum and glutamine (2 mM). The growth of human RS virus (A2 strain) in HEp-2 cells and the growth of vaccinia virus (VV) (WR strain) in HeLa cells and in thymidine kinase negative (tk-) 143B cells have been described previously (1, 24, 25, 30). We previously described the construction and use of a recombinant expression vector for the RS virus G protein by using VV (1). CHO cells, however, are not permissive for VV, whereas they can support the replication of the related cowpox virus (CPV) (8, 23). We therefore created a recombinant between our existing recombinant vector and CPV. The VV/CPV recombinant, VVCG301, was isolated by coinfecting HeLa cells (which are permissive for both VV and CPV) with recombinant VVG301 which contains the entire coding sequence for the RS virus G protein gene and with wild-type CPV (Brighton strain; American Type Culture Collection). Recombinant virus was selected by three alternate cycles of growth on CHO cells where CPV can replicate and on tk- 143B cells, in the presence of bromodeoxyuridine, where VVG301 (with an interrupted tk gene) can replicate. Viruses that could grow on both cell lines were then plaque purified and screened by quick blot hybridization for presence of the RS virus G gene as described previously (25). Restriction enzyme analysis of the viral DNA of the recombinant selected for use (VVCG301) showed that the site of the VV/CPV recombination was near the left end of the VV genome, within the Hindlll C fragment of VV DNA, and close to the position of the CPV gene that is required for replication in CHO cells (23). This recombinant, termed VVCG301, expressed G protein in wild-type CHO cells in abundant quantities, and its synthesis and maturation resembled that previously described by us for G protein expressed from VVG301 in HEp-2 cells (1, 25). Metabolic analysis of G protein. Wild-type CHO or ldlD cells were seeded to dishes or cover slips as required in Ham F-12 medium containing 5% fetal calf serum. Twelve hours after seeding, this medium was removed, the cells were washed twice with HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid) buffered saline (pH 7.6) and reincubated in serum-free medium for 12 to 18 h. Identical results were obtained when cells were starved in serum-free medium for 48 h. Cells were then infected with RS virus or
mutant (ldlD-14) cells
VOL. 63, 1989
recombinant VVCG301 at a multiplicity of infection of 20, and infection was allowed to proceed for 20 or 5 h, respectively, in serum-free medium or in serum-free medium to which Gal (25 ,uM) or GalNAc (400 ,uM) or both sugars had been added to compensate for the metabolic defect in the ldlD cells. It was necessary to use serum-free medium to prevent scavenging of sugar components from serum. In some experiments, tunicamycin was added to inhibit Nlinked sugar addition. The purified beta homolog of tunicamycin which is the least inhibitory to protein synthesis was used at 5 ,ug/ml (2). G protein synthesis was assayed by metabolic labeling with [3H]threonine (100 ixCi/ml; specific activity, 9 Ci/mmol) or by immunoblotting as described below. For metabolic pulse-chase studies, cells were seeded and infected with VVCG301 as described above but incubated in serum-free medium with no exogenous sugar additions for 5 h postinfection. Medium was then withdrawn, and serum-free medium containing [3H]threonine (100 ,uCi/ml) was added for 40 min. At the end of the labeling period, medium containing label was withdrawn and cells were washed twice in ice-cold HEPES buffered saline and reincubated for a chase period in serum-free medium containing five times (240 ,ug/ml) the normal medium concentration of unlabeled threonine and either no exogenous sugar additions or Gal (25 ,M) or GalNAc (400 ,uM) or both sugars. For protein analyses, cells were lysed at 0°C in 0.3 ml of 0.01 M Tris hydrochloride (pH 7.4)-66 mM EDTA-1% Nonidet P-40-0.4% sodium deoxycholate per 35-mm plate, and the nuclei were removed by centrifuging for 3 min in an Eppendorf centrifuge. Labeled proteins in the resultant cytoplasmic extract were analyzed by immunoprecipitation, or unlabeled proteins were analyzed by Western blot (immunoblot) analysis as described below. Protein analysis. Proteins synthesized in infected cells were labeled with [3H]threonine and analyzed by electrophoresis on sodium dodecyl sulfate (SDS)-10% polyacrylamide gels following immunoprecipitation with either a G-proteinspecific monoclonal antibody, L-9 (kindly provided by E. Walsh), and anti-mouse Immunobeads (Bio-Rad Laboratories) or anti-RS virus horse serum (Flow Laboratories) and IgG Sorb (The Enzyme Center, Inc.). Endoglycosidase H (endo H) digestions were carried out on immunoprecipitated proteins as described previously (4). Digestion was for 18 h at 37°C. The samples were then electrophoresed on SDSpolyacrylamide gels as described above. Alternatively, proteins were detected by immunoblotting. Unlabeled proteins were separated by electrophoresis in SDS-10% polyacrylamide gels and transferred to nitrocellulose by using an ABN Transblot apparatus under the manufacturer's recommended conditions. The sheet to be immunostained was then incubated in 1% bovine serum albumin for 1 h at 37°C, rinsed in phosphate-buffered saline (PBS) containing Tween 20 (0.05%), incubated for 90 min at 37°C with monoclonal antibody L-9, rinsed five times, and incubated for 90 min at 37°C with peroxidase-conjugated rabbit anti-mouse immunoglobulin G, and the antibody-specific proteins were visualized by reaction of the peroxidase conjugate with 4-chloro1-naphthol (Sigma Chemical Co.). Indirect immunofluorescence. For surface staining, CHO or ldlD cells grown on cover slips under conditions described above (plated in medium with 5% sera, plating medium removed, cells washed and reincubated in serum-free medium for 12 to 18 h) were mock infected or infected with recombinant VVCG301 or wild-type VV at a multiplicity of infection of 10 PFU per cell. Cells were incubated for 5 h at 37°C under desired conditions of exogenous sugar addition in
RS VIRUS G PROTEIN GLYCOSYLATION
4769
serum-free medium. At 5 h postinfection, medium was removed, cells were washed twice with PBS, and 10 RI of a 1:50 dilution of monoclonal antibody L-9 or IC2 (both specific for the RS virus G protein) was added to each cover slip for 15 min at 20°C. The first antibody was removed by washing twice with PBS, and 10 RI of phycoerytherinconjugated goat anti-mouse immunoglobulin (Southern Biotechnology Associates, Inc., Birmingham, Ala.) was added for 20 min at 20°C. The conjugate was removed by washing cells three times in PBS, the cells were fixed by the addition of 1% Formalin, and the cover slips were mounted on glass slides for examination. Flow cytometry. Wild-type CHO or ldlD cells plated and infected exactly as described above under designated conditions of exogenous sugar addition were harvested at 5 h postinfection by washing and incubation in 0.2% EDTA for 20 min at 37°C. Cells (5 x 105) were washed once in PBS containing 1% bovine serum albumin, pelleted, suspended in 20 RI of a 1:50 dilution of monoclonal antibody L-9, and incubated for 20 min at 4°C. Cells were then washed with PBS containing 1% bovine serum albumin, repelleted, and incubated in 20 RI of phycoerytherin-conjugated goat antimouse antibody (Southern Biotechnology Associates). For one-color staining, cells were fixed with 1% paraformaldehyde in PBS. For two-color staining, cells were treated as described above except the first antibody was rabbit anti-VV antibody and the second antibody was goat-antirabbit immunoglobulin conjugated to fluorescein isothiocyanate (Southern Biotechnology Associates). Cells were then fixed with 1% paraformaldehyde. Double fluorescent labeling allowed us to monitor G expression as a function of cells infected with and expressing the genes of the recombinant VV vector. Samples were stored at 4°C until analysis by flow cytometry.
RESULTS To examine the role of 0-linked oligosaccharides in the structure and cell surface expression of human RS virus G protein, we infected wild-type CHO or mutant ldlD cells with the recombinant VV/CPV expression vector, VVCG301, which contains a cDNA with the complete coding sequence for the G protein gene and which replicates efficiently in CHO cells (see Materials and Methods). The contributions of N- or 0-linked or both types of sugar addition on the synthesis of the human RS virus G protein were examined by comparing synthesis of G protein in wild-type CHO cells with that in the mutant ldlD cells in the absence of any added sugars or in the presence of exogenously added Gal or GalNAc. In addition, the form of G synthesized in the ldlD cells in the presence of tunicamycin to prevent any N-linked chain addition was also examined. These analyses were done by three methods: (i) immunoblotting of extracts of infected cells was used to examine steady-state levels of G, (ii) metabolic labeling and endo H sensitivity were used to examine synthesis and intracellular transport, and (iii) metabolic pulse-chase analyses were used to examine precursorproduct relationships. Figure 2 shows the results of an experiment examining steady-state levels of the forms of G synthesized under various conditions of sugar addition. In the wild-type parent CHO cell line infected with recombinant vector VVCG301, three forms of G protein were observed by Western blotting of cytoplasmic extracts (Fig. 2): (i) the mature form of G with an electrophoretic mobility of approximately 88 to 90 kDa, (ii) an intermediate form with a mobility of approximately 45 kDa, and (iii) the nonglyco-
4770
WERTZ ET AL. VVc
CHO
Cells Tunic.
m
IdiD
0 +
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+
0
Sugar G-
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TM
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0 0 a
.
.
expression from VVcG301, however, this vector was used in the subsequent studies. In IdID cells, the form of G synthesized varied according to the composition of the medium with regard to sugar addition (Fig. 2). In IdID cells grown in serum-free medium without addition of exogenous sugars, a species of G was observed having an estimated molecular mass of 45 kDa. Under these conditions in ldlD cells, only incomplete N-
Cells
Recombinant lnfectecd
G
J. VIROL.
0
+
Gal Gal NA GNAc I:NAc
* U*
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68
-
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29 FIG. 2. Effects of sugar additions on hu man RS virus G protein structure in wild-type CHO or IdID cells. Wild-type or IdID cells were incubated for 12 h in serum-free jmedium prior to mock infection or infection with recombinant VV,CG301. Mock- or vectorinfected cells were then incubated in serrum-free medium which contained the following: no additions (0), tunicamycin at 5 ,ug/ml (Tunic), galactose at 25 ,uM (Gal), N-acetylEgalactosamine at 400 F.M (GNAc), or both galactose and N-acetylgalh actosamine (Gal GNAc). At 5.5 h postinfection, cells were harvested, proteins were separated by electrophoresis on an SDS-10% poly acrylamide gel, and Gspecific proteins were visualized by Weste blotting using mono'rn clonal antibody L-9. kd, Kilodaltons.
sylated polypeptide backbone of G wthich we identified in previous work by in vitro translation of purified G mRNA (30). The molecular mass of this precuirsor polypeptide (Gp), estimated from its electrophoretic miobility in SDS-polyacrylamide gels, is approximately 306 kDa, although the nucleotide sequence of G mRNA pre(dicts a polypeptide of 32 kDa. The G protein sequence hzas a 10% content of proline; it is possible that this high proline content may account for the anomalous mobility oif G on gels, or, alternatively, some other as yet undetermiined modification of G may be responsible. It should be emp hasized at this point, however, that all molecular mass dleterminations by gel electrophoresis of proteins with a high content of proline or carbohydrate are, at best, approximat ;ions. The apparent molecular mass of the mature form of G was decreased by approximately 8 to 9 k;Da in wild-type cells treated with tunicamycin to inhibit N-1linked sugar addition, demonstrating a contribution to the IG structure by some N-linked side chains as observed pre' viously (5, 6, 16, 30). The addition of tunicamycin also increatsed the amount of the nonglycosylated precursor present in wild-type CHO cells and resulted in the absence of the 45-k Da intermediate (Fig. 2). The three forms of G observed in wild-type CHO cells infected with recombinant vector VV( CG301 resemble those observed previously in experiments usiing other types of host cells (1, 25). In addition to using the re-combinant vector for expression of G, we also carried out ti ie experiments shown in Fig. 2 by using RS virus to infect thke wild-type CHO and the ldlD cells. Analysis of G expressioin by Western blotting from RS virus-infected cells gave resuilts qualitatively identical to those obtained with the rec ombinant expression vector (data not shown). Because of thte superior levels of G
linked oligosaccharides can be synthesized. In ldlD cells in serum-free medium with tunicamycin added to inhibit N. linked oligosaccharide addition, conditions where no sugars should be added, a major band of G with an estimated mass of 36 kDa was observed. This observation confirms previous data from in vitro translation of purified G mRNA that this species represents the nonglycosylated polypeptide backbone or precursor (Gp) of G (30). If galactose is added to serum-free medium in the absence of an inhibitor of N-linked oligosaccharide biosynthesis, N-linked sugars should be able to mature to the complex form, but no 0-linked sugars can be added because of the inability of ldlD cells to add GalNAc. Under these conditions, the 45-kDa form was observed, as well as a heterodisperse population of G with a mobility increased over that of the 45-kDa form. When GalNAc alone was added to the medium of ldlD cells, conditions where incomplete N-linked and incomplete 0linked sugars can be added, two forms of G were observed: the 45-kDa species, as well as a species of approximately 74 to 78 kDa. If GalNAc was added alone in the presence of tunicamycin, so that incomplete 0-linked chains but no N-linked sugars could be attached, a single species of approximately 67 to 68 kDa was observed (data not shown). Addition of Gal plus GalNAc to the medium, which should allow both N- and 0-linked glycosylation, resulted in synthesis of the mature 88- to 90-kDa form as well as the 45-kDa form. These species had the same electrophoretic mobility as the mature form of G and the 45-kDa form observed in wild-type CHO cells. Similarly, when tunicamycin was added to IdlD cells to which both Gal and GalNAc were added, the mobility of G was reduced to approximately 82 kDa, indicative of the absence of any N-linked chains, as seen in wild-type CHO cells. A summary of the conclusions drawn from the data presented in Fig. 2 and Fig. 3 is presented in Table 1 for reference. These experiments showed that the 45-kDa form of G was a form that contained only N-linked sugars. To test whether the 45-kDa species contained immature forms of the Nlinked side chains, endo H digestion was done with immunoprecipitated, radiolabeled G species synthesized in ldlD or wild-type CHO cells under each of the conditions of inhibition or exogenous sugar addition described in the legend to Fig. 2. Endo H is known to cleave between the two proximal N-acetylglucosamine residues of the large high-mannosetype oligosaccharides. Complex-type N-linked carbohydrates are resistant to this enzyme, and it has been shown that the extent of oligosaccharide processing correlates with endo H sensitivity (12, 13, 21, 26). Because the oligosaccharide modifications that result in endo H resistance occur in the Golgi (13), the acquisition of endo H resistance can also be used as a measure of transport to this compartment. The data presented in Fig. 3 show that the majority of the 45-kDa form of G made in either wild-type CHO or ldlD cells was sensitive to endo H digestion. Endo H digestion converted the 45-kDa species to a form approximately 2 kDa larger than the nonglycosylated precursor of G. In ldlD cells in the absence of any sugar additions, the 45-kDa form of G was predominantly, but not completely, endo H sensitive. It
RS VIRUS G PROTEIN GLYCOSYLATION
VOL. 63, 1989
G
4771
-97 kd
-
*
GTMl
*I*
- 68
G45
-
Gp
-
- 43 A& -o
FIG. 3. Effect of endo H on G protein synthesized in CHO and IdlD cells. The various structural forms of G synthesized in wild-type CHO or IdlD cells in the absence or presence of exogenously added sugars were analyzed for sensitivity to digestion by endo H. Conditions of infection and inhibitor or sugar additions were exactly as described in the legend to Fig. 2 except that proteins were labeled with [3H]threonine (100 ,uCi/ml) from 3.5 to 5.5 h postinfection. Samples were immunoprecipitated, one-half of each sample was digested with endo H as previously described, and samples were analyzed by electrophoresis on a 10%o polyacrylamide gel. M, Mock-infected cultures; Vc, wild-type CPV; kd, kilodaltons.
is likely that this 45-kDa band contains an 0-linked deficient 45-kDa endo H-sensitive precursor of G synthesized in the endoplasmic reticulum and an 0-linked deficient endo Hresistant form which may contain trimmed but immature complex-type N-linked sugars which are truncated (agalacto-, asialo- forms) because of the unavailability of galactose. This is demonstrated more clearly by the pulsechase experiments shown in Fig. 4B. In the case of G synthesized in the presence of galactose (which should allow completion of complex N-linked sugar synthesis), two forms of G were observed: (i) an endo H-sensitive 45-kDa form which presumably represents the high-mannose-type Nlinked chain precursor initially synthesized in the endoplasmic reticulum and (ii) a doublet of endo H-resistant protein with a molecular mass greater than 45 kDa which must represent G which has passed into the Golgi and been processed to a complex form. Two forms of G were synthesized when GalNAc, which allows addition of 0-linked side chains, was added to ldlD TABLE 1. RS virus G protein synthesis in IdID cells Medium addition
Oligosaccharide(s)
G protein
Inhibitor
attached
product
None None
Tunica
Gal GalNAc GalNAc
None Tunic None
None N-linked, high-mannose immature complex N-linked mature complex 0-linked N-linked immature complex, 0-linked
Gal + GalNAc Gal + GaINAc
Tunic None
Sugar
None
a Tunic, Tunicamycin.
0-linked N-linked mature complex,
0-linked
Gp
G45K G45K+
G68K
G74-78K
G82K G90K
cells. The predominant species was a form of G with a molecular mass approximately 10 to 14 kDa smaller than that of the mature 90-kDa form. There was also some 45-kDa species present. The high-molecular-mass form of G was completely endo H resistant, whereas the 45-kDa form was sensitive. When Gal and GalNAc were both added to the medium of ldlD cells, the major form of G synthesized was the mature 88- to 90-kDa form which was completely resistant to endo H digestion. Some 45-kDa species which was endo H sensitive was also present (Fig. 3). Metabolic pathways of G maturation. To determine the role of these intermediates and the contribution of N- and 0linked sugars to the formation of the mature 90-kDa form of G, metabolic pulse-chase experiments were carried out. The ldlD cells were exposed to a 40-min pulse-label with [3H]threonine in the absence of any exogenously added sugars. The label was then removed, and a 45-min chase period followed in media with an excess of unlabeled threonine and in the absence of added sugars or with the addition of exogenous galactose or GalNAc or both sugars. Essentially the same results were obtained with shorter pulselabeling times (20 min). In wild-type CHO cells at the end of a 40-min labeling period, most of the label was found in the 45-kDa doublet species of G with a small amount in the nonglycosylated precursor species and a significant amount in mature G (Fig. 4A). During the 45-min chase period, most of the precursor 45-kDa doublet matured to the fully glycosylated 90-kDa form of G. In ldlD cells labeled for 40 min in the absence of added sugars, most of the label entered the 45-kDa species, which appeared as a doublet. During the chase period, in the absence of added sugars, the top band of the 45-kDa doublet remained; however, the bottom band of the doublet disappeared. Additionally, as shown in Fig. 4B, whereas the 45-kDa species synthesized during the pulse was predominantly endo H sensitive, the 45-kDa species acquired partial
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Cell Type
CHO
_
Vector
M VVcG
M
Chase Sugars
0
0
0
O
+
0
0
Cell Type
dXD VVcG 0
+
0
+A Gai
+ + Gal NA G
Vector
M
Sugars
0
VVcG301 0 0
Chase
0
0
Endo H
G
+
M 0
+-
+
_
_
0
VVcG301 0 G31 GNAc
0
+
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+
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-
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-
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-
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FIG. 4. Effects of sugar additions on the posttranslational processing of G protein. (A) Wild-type CHO or IdiD cells were incubated in serum-free medium for 12 h prior to infection or mock infection with recombinant VV,G301 in serum-free medium. At 4 h postinfection, cells were pulse-labeled for 40 min with [3H]threonine. Following the labeling period, isotope was removed and cells were washed twice in ice-cold HEPES buffered saline, harvested immediately (Chase = 0) or incubated for a 45-min period at 37°C (Chase = +) in serum-free medium having a 5 x concentration of unlabeled threonine and containing the following: no additions (0), galactose at 25 ,uM (Gal), Nacetylgalactosamine at 400 ,uM (GNAc), or both galactose (25 ,uM) and N-acetylgalactosamine at 400 FM (Gal GNAc). M, Mock infected. At the appropriate time, cells were harvested, cytoplasmic extracts were prepared, and proteins were immunoprecipitated with monoclonal antibody L-9 and analyzed by electrophoresis in a SDS-10% polyacrylamide gel. (B) Following immunoprecipitation, each sample described above was divided and equal portions incubated with or without endo H for 18 h at 37°C. Samples were then analyzed by SDS-polyacrylamide gel electrophoresis. Vc, Wild-type CPV.
endo H resistance during the chase period, indicating its passage to the Golgi for processing. Only when Gal or GalNAc was added exogenously did the material present in
the 45-kDa band mature into the high-molecular-mass endo H-resistant form of G (Fig. 4B). With the addition of Gal, the 45-kDa band matured to a higher-molecular-mass form which was shown to be endo H resistant (Fig. 4B). This is consistent with the ability of the N-linked sugars to mature to the complex form when Gal is restored to the medium and is consistent with transport to the Golgi. Addition of GalNAc, which permitted addition of 0-linked side chains during the chase period, was associated with maturation of the 45-kDa species into high-molecular-mass material with a mobility of approximately 74 to 78 kDa, slightly faster than that of the 82-kDa form of G synthesized in the presence of tunicamycin. This is consistent with the synthesis of both incomplete N-linked and incomplete 0-linked side chains. The mature 90-kDa form of G was observed only after a chase period in which both Gal and GalNAc were present to restore complete N and 0-linked oligosaccharide biosynthesis. It is interesting that although intermediates in the pathway of 0-linked glycosylation did not accumulate during the chase period in CHO cells, such intermediates were detected as a heterodisperse population of molecules in ldlD cells (Fig. 4A and B). This can probably be attributed to the fact that under the chase conditions, activated Gal and GalNAc were limiting in concentration and this restricted the rate of O glycosylation. It is possible that intermediates in 0 glycosylation could be isolated from ldlD cells under these conditions and used to determine whether oligosaccharide addition to different sites occurs in a defined sequence. The second type of metabolic pulse-chase study focused exclusively on 0-linked biosynthesis by the use of pulselabeling experiments in the presence of tunicamycin to inhibit any N-linked glycosylation. The pulse-labeling was followed by a chase period in the absence or presence of
exogenously added sugars in order to determine the contribution of 0-linked sugars to the maturation of G and to investigate whether, following synthesis of a nonglycosylated precursor, this precursor could then mature by addition of 0-linked sugars. Pulse-labeling of G in wild-type CHO cells with no inhibitors added again showed entry of label primarily into GP and the 45-kDa doublet precursor forms, which during a chase period matured to the 88- to 90-kDa mature form of G (Fig. 5). In contrast, pulse-labeling in wild-type CHO cells in the presence of tunicamycin showed entry of label into the nonglycosylated precursor of G (with some label progressing to the size of the mature G lacking N-linked sugars). No label was observed to accumulate in the 45-kDa form, which is further confirmation of its identity as a N-linked intermediate. During the chase period in wild-type CHO cells, the nonglycosylated precursor (GP) matured to the approximately 82-kDa form of G lacking N-linked sugars (GTM). Only the nonglycosylated precursor form of G was synthesized during pulse-labeling of ldlD cells in the presence of tunicamycin (Fig. 5). Interestingly, under pulse-labeling conditions this form of G appeared as a doublet. The reasons for a doublet are unknown at this time. One possibility, although unlikely, is that the doublet may be associated with a proteolytic cleavage near the membrane-spanning region which has been implicated in creating a shed form of G (7). The precise nature of this doublet, however, will require further investigation. During a 45-min chase in the absence of exogenously added sugars, there was no change in the mobility of the precursor form, indicating that maturation to higher-molecular-mass forms required addition of N- or 0-linked sugars. When GalNAc was added to permit 0linked chain addition, the material in the nonglycosylated precursor band chased into a form that was fractionally smaller than the fully 0-glycosylated (but non-N-glycosylated) form observed in CHO cells in the presence of
RS VIRUS G PROTEIN GLYCOSYLATION
VOL. 63, 1989
_ IdlD
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- 29 FIG. 5. Effects of iN-acetylgalactosamine addition on metabolism of human RS viru s G protein in the absence of N-linked sugar synthesis. Wild-type c)r IdlD cells were incubated in serum-free medium for 12 h prior to infection or mock infection with recombinant vector VVCG301. At 4 h postinfection, cells were pulse-labeled for 40 min with [3H]thrreonine in the presence (+) or absence (0) of tunicamycin (5 ,ug/ml) 1to inhibit N-linked sugar addition. At the end of the pulse, the radic)label was removed and cells were washed twice with ice-cold buffFered saline and harvested immediately (Pulse nr int;UVU4tU = umin UMldU e-hneiPC1 UU dlt /1 Afnrhnei =- U) fl) ori1int-iih!ntp,d lUFl fnr n -Ali+J-111111 U t11n1sC (Chase = 45 min) in serum-free medium containing excess unlabeled threonine and the following: no additions (0), N-acetylgalactosamine at 400 ,uM (GNAc), or both galactose and N-acetylgalactosamine (Gal GNAc). M, Mock infected. Cells were harvested, cytoplasmic extracts were prepared, and proteins were immunoprecipitated with monoclonal antibody L9 and analyzed by electrophoresis on a 10% polyacrylamide gel.
tunicamycin (Fig. 5). However, when the chase was carried out in the presence of both Gal and GalNAc the nonglycosylated precursor matured into a form of G that possessed the same mobility as G synthesized in wild-type CHO cells in the presence of tunicamycin (i.e., lacking in N-linked sugars) (Fig. 5). These results demonstrate that addition of 0-linked sugars alone accounts for the majority of the molecular mass increase in the maturation of nonglycosylated precursor to the mature 88- to 90-kDa form of G. These data also confirm the previous finding that 0 glycosylation can occur posttranslationally (15) and that maturation from the nonglycosylated precursor form (Gp) to the exclusively 0-linked form of G (G82K) can occur posttranslationally in the absence of N-linked side chain addition. Cell surface expression. The cell surface expression of G in wild-type CHO or ldlD cells under designated conditions of sugar addition was examined both by indirect immunofluorescence and by flow cytometry. The former technique determined whether G was expressed on the surface and allowed investigation of the distribution of G on the cell surface, and the latter technique allowed estimation of levels of cell surface expression. As shown by the data in Fig. 6, the G protein is expressed on the surface of ldlD cells both in the absence and in the presence of exogenously added sugars. The level of surface fluorescence in ldlD cells with both sugars added exogenously to restore both N- and
4773
0-linked glycosylation was indistinguishable from that in wild-type CHO cells (data not shown). Addition of sugars, either Gal or GalNAc, to the ldlD cells increased the level of fluorescence above that in cells without exogenously added sugars. Levels of surface fluorescence in cells treated with tunicamycin such that no sugar addition occurred were severely reduced but reproducibly detectable. Analysis of cell surface expression by flow cytometry confirmed the results obtained by indirect immunofluorescence. In cells where no sugar addition occurred (panel A), less than half
the cells monitored expressed G at the surface and the mean
level of fluorescence intensity was eight- to ninefold lower than under conditions of full sugar addition. Examination of panel B clearly showed that cell surface expression of G occurs in ldlD cells in the absence of any 0 glycosylation. Approximately 80% of cells expressed G on their surface in the absence of 0 glycosylation, and the mean level of expression was approximately 50% of that seen under conditions of full sugar addition (Fig. 6B and E). The cause of the reduced level of expression in the absence of 0 glycosylation is unknown at this time, but it may be related to the reduced stability observed for several other normally 0glycosylated membrane proteins when they were synthesized in ldlD cells under similar conditions (10). When tunicamycin was added to inhibit N-linked sugar addition in either ldlD cells in the presence of GalNAc or in wild-type CHO cells, G was expressed on the cell surface (data not shown). Thus, these data demonstrate that whereas absence of any sugar addition severely inhibits cell surface expression, the presence of either 0-linked or N-linked (even immature N-linked) sugars correlates with efficient cell suriatureN-ink face expression.
DISCUSSION The role of 0-linked glycosylation in protein maturation, structure, and function has been difficult to assay because of lack of inhibitors specific for this process. The isolation of a mutant CHO cell line (ldlD) with a deficiency in the UDPGalIUDP-GalNAc-4-epimerase has provided a powerful approach for analysis of 0 glycosylation (10). The properties of this cell line coupled with the use of tunicamycin, an inhibitor of N-linked glycosylation have allowed us to investigate the role of N- and 0-linked glycosylation in the structure and maturation of the extensively glycosylated attachment protein G of human RS virus. In wild-type CHO cells infected with the recombinant that expressed G protein, three major forms of G protein were observed: (i) the nonglycosylated precursor (predicted molecular mass, 32 kDa), (ii) a 45-kDa doublet intermediate, and (iii) the 88- to 90-kDa mature form of G. Treatment of cells with tunicamycin to inhibit N-linked synthesis resulted in synthesis of a fourth species of G having an electrophoretic mobility indicating that it was approximately 8 to 9 kDa smaller than the mature form. These forms of G have been observed by us and others in a variety of host cells infected with RS virus (3, 5, 16, 25, 30). Previous studies of G in virus-infected cells, using inhibitors and enzymatic digestion, have suggested that the 45-kDa form is predominantly an N-linked intermediate (3, 5, 16, 17). The suggestion has been put forth by one group that the mature G may consist of a dimer of two 45-kDa subunits (3). However, it is known that a glycosylated form of G with an apparent molecular mass of 82 kDa is synthesized in the absence of N-linked synthesis (tunicamycin), and digestion with O-glycanase has indicated that this form contains 0-linked sugars (16), indi-
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Fluorescence * FIG. 6. Analysis of cell surface expression of G protein in ldlD cells. Cells were incubated in serum-free medium for 16 h prior to infection or mock infection with recombinant VVCG301. Mock- or vector-infected ldlD cells were then incubated in serum-free medium which contained no sugar addition and tunicamycin at 5 pLg/ml (A); no additions (B); galactose (Gal) added at 25 ,uM (C); N-acetylgalactosamine (GalNAc) added at 400 ,uM (D); Gal plus GalNAc added at 25 ,uM and 400 ,uM, respectively (E); and uninfected control in serum-free medium (F). At 5 h postinfection, cells in each group were analyzed for surface expression of G protein by either indirect immunofluorescence or flow cytometry as described in Materials and Methods. 4774
VOL. 63, 1989
cating that it is unlikely that the mature form of G consists of dimers of the 45-kDa form. We have used the reversible glycosylation defects of ldlD cells to examine the structures of intermediates and the role of 0-linked glycosylation in the maturation of the G protein. In ldlD cells with no exogenous sugars added, a condition in which only incomplete N-linked carbohydrate can be added and no 0-linked carbohydrate is added, the primary species of G observed is the 45-kDa intermediate. This species is identified as the polypeptide backbone plus immature N-linked sugars by the observation that (i) its synthesis is completely sensitive to tunicamycin, with only the nonglycosylated form being observed in the presence of this drug, and (ii) the 45-kDa species is sensitive to digestion with endoglycosidase H (5; Fig. 3). Treatment with endo H yields a product with mobility just slightly slower than that of the nonglycosylated precursor (Fig. 3 and 4B), presumably because of the fact that endo H cleavage takes place between the two proximal N-acetylglucosamine residues of the Nlinked chain. The higher-molecular-mass forms of G were observed in ldlD cells only when 0 glycosylation was permitted. Exogenous addition of GalNAc resulted in a 78-kDa species of G, while addition of both Gal and GalNAc resulted in synthesis of the mature 90-kDa form of G protein. Metabolic labeling, pulse-chase studies carried out in both wild-type CHO and mutant ldlD cells indicated that the maturation of G involves polypeptide synthesis with attachment of high-mannose forms of N-linked sugars in the rough endoplasmic reticulum and subsequent Golgi-associated conversion of the N-linked chains to the complex form. Addition of 0-linked sugars resulted in appearance of the mature form of G. The current data do not allow us to determine when the initiation of 0-linked sugar addition occurs. However, the data do show that 0 glycosylation can occur posttranslationally, as previously shown for the LDL receptor (15) and a herpesvirus glycoprotein (9), and that maturation to the high-molecular-mass form of G does not occur in the absence of GalNAc (Fig. 2 and 4), probably because of the GalNAc requirement for the synthesis of 0-linked sugars. Future experiments will address the possibility that any of the GalNAc dependence in the maturation of G in ldlD cells to the mature form may be due to other types of 0-linked residues, for example chondroitin sulfate. Additionally, in the presence of the N-glycosylation inhibitor tunicamycin, G protein was observed to mature directly from a nonglycosylated protein (precursor) form with an apparent mass of 36 kDa (Gp) to an 82-kDa form (addition of GalNAc plus galactose) because of GalNAc-dependent 0linked glycosylation (Fig. 5). These data indicate that the mature G protein is not a dimer of two 45-kDa exclusively N-glycosylated species and that most of the shift in electrophoretic mobility is a consequence of GalNAc-dependent 0 glycosylation. This 0-glycosylation-dependent maturation can occur in the absence of N glycosylation. Transport of newly synthesized G protein to the surface of ldlD cells could be detected readily under conditions in which either N glycosylation or 0 glycosylation was completely blocked; however, very little surface expression was observed when both types of glycosylation were simultaneously blocked (tunicamycin treatment in the absence of exogenous GalNAc). The low level of surface expression detected in the absence of any glycosylation agrees with the observation that a small amount of nonglycosylated G can be detected in virions (16). The mean level of cell surface expression of RS virus G protein in the absence of 0 glycosylation was approximately 50% of that of the fully
RS VIRUS G PROTEIN GLYCOSYLATION
4775
glycosylated protein. This reduction may be related to the reduced stability of three other normally 0-glycosylated membrane proteins-the LDL receptor (10, 15), decayaccelerating factor (P. Reddy, I. Caras, and M. Krieger, manuscript in preparation), and the major envelope glycoprotein of Epstein-Barr virus (K. Kozarsky, M. Silberklang, and M. Krieger, unpublished data)-when synthesized without 0-linked sugars in ldlD cells. However, the reduction in the surface expression of G in the absence of 0 glycosylation was far less than that for these other proteins. This difference may be a consequence of the normal shedding of cell
surface G protein into the extracellular fluid (7), whereas these other proteins do not normally undergo substantial shedding. The effect of 0 glycosylation on cell surface expression of the interleukin-2 receptor has also been investigated in ldlD cells (14). In the case of the 0-linked carbohydrate-deficient interleukin-2 receptor, cell surface expression was decreased, but the reason for the decrease in expression was due to missorting of the receptor which occurred in or beyond the trans-Golgi apparatus (14). The ability to produce G protein in eucaryotic cells that are deficient in 0-linked sugars offers us the opportunity to examine the role of the extensive glycosylation of G in its function in viral attachment and in the immune response to G. We are now in a position to produce RS virions in ldlD cells under conditions where no 0 glycosylation occurs and examine the specific infectivity of these virions. Previous work using enzymatic removal of 0-linked sugars from virions indicated that 0 glycosylation was important for infectivity (16). Most importantly, the ldlD cells offer the opportunity to analyze the effect of 0-glycosylation on the immune response to the RS virus G protein. It appears that the immune response to the G protein differs from that to the other surface glycoprotein of RS virus, the fusion protein, which has only N-linked carbohydrate (20, 27-29). Analysis of immunoglobulin G (IgG) subclass antibody responses provides information on recognition as predominately a protein or carbohydrate antigen. Following RS virus infection in humans, the IgG1/IgG2 ratio to F was fourfold higher than that to G, with G having an IgG1/IgG2 ratio of 1 (27). It has been hypothesized that the extensive carbohydrate of the G protein may affect the immune response to this protein and that the nature of the response to G may play an important role in the repeated infections observed in children with RS virus disease. The opportunity to produce G with and without 0-linked sugars will allow us to examine the ability of these different forms of G to elicit a protective immune response (24) and to study the specificity of antibodies formed during infection. ACKNOWLEDGMENTS This work was supported by Public Health Service grants R37 A112464 and Al 20181 (to G.W.W.) and R37 Al 18270 (to L.A.B.) from the National Institute of Allergy and Infectious Diseases. The investigation also received support from the World Health Organization Programme on Vaccine Development. We thank D. Lichtenstein and K. Barry for assistance with flow cytometry. LITERATURE CITED 1. Ball, L. A., K. Young, K. Anderson, P. Collins, and G. W. Wertz. 1986. Expression of the major glycoprotein G of human respiratory syncytial virus from recombinant vaccinia virus vectors. Proc. Natl. Acad. Sci. USA 83:246-250. 2. Duksin, D., and W. C. Mahoney. 1982. Relationship of the structure and biological activity of the natural homologues of tunicamycin. J. Biol. Chem. 257:3105-3109.
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3. Fernie, B., G. Dapolito, P. Cote, and J. Gerin. 1985. Kinetics of synthesis of respiratory syncytial virus glycoproteins. J. Gen. Virol. 66:1983-1990. 4. Florkiewicz, R. Z., A. Smith, J. E. Bergmann, and J. K. Rose. 1983. Isolation of stable mouse cell lines that express cell surface and secreted forms of the vesicular stomatitis virus glycoprotein. J. Cell Biol. 97:1381-1388. 5. Gruber, C., and S. Levine. 1985. Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins. J. Gen. Virol. 66:417-432. 6. Gruber, C., and S. Levine. 1985. Respiratory syncytial virus polypeptides. V. The kinetics of glycoprotein synthesis. J. Gen. Virol. 66:1241-1247. 7. Hendricks, D. A., K. McIntosh, and J. L. Patterson. 1988. Further characterization of the soluble form of the G glycoprotein of respiratory syncytial virus. J. Virol. 62:2228-2233. 8. Hruby, D. E., D. L. Lynn, R. C. Condit, and J. R. Kates. 1980. Cellular differences in the molecular mechanisms of vaccinia virus host range restriction. J. Gen. Virol. 47:485-488. 9. Johnson, D. C., and P. G. Spear. 1983. 0-linked oligosaccharides are acquired by herpes simplex glycoproteins in the Golgi apparatus. Cell 32:987-997. 10. Kingsley, D. M., K. F. Kozarsky, L. Hobbie, and M. Krieger. 1986. Reversible defects in 0-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant. Cell 44:749-759. 11. Klein, J. O., D. W. Tecle, J. Sloyer, Jr., J. Ploussard, V. Howie, P. Makela, and P. Karma. 1982. Use of pneumococcal vaccine for prevention of recurrent episodes of otitis media, p. 305. In J. B. Robbins, J. C. Hill, and J. C. Sadoff (ed.), Seminars in infectious diseases, vol. 4. Bacterial vaccines. Thieme-Stratton, Inc., New York. 12. Kornfeld, R., and S. Kornfeld. 1980. Structure of glycoproteins and their oligosaccharide units, p. 1-32. In W. J. Lenarz (ed.), The biochemistry of glycoproteins and proteoglycans. Plenum Publishing Corp., New York. 13. Kornfeld, R., and S. Kornfeld. 1985. Assembly of asparaginelinked oligosaccharides. Am. Rev. Biochem. 54:631-664. 14. Kozarsky, K. F., S. M. Call, S. K. Dower, and M. Krieger. 1988. Abnormal intracellular sorting of 0-linked carbohydrate-deficient interleukin-2 receptors. Mol. Cell. Biol. 8:3357-3363. 15. Kozarsky, K., D. Kingsley, and M. Krieger. 1988. Use of a mutant cell line to study the kinetics and function of 0-linked glycosylation of low density lipoprotein receptors. Proc. Natl. Acad. Sci. USA 85:4335-4339. 16. Lambert, D. M. 1988. Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoprotein. Virology 164:458-466. 17. Lambert, D. M., and M. W. Pons. 1983. Respiratory syncytial virus glycoproteins. Virology 130:204-214. 18. Levine, S., R. Klaiber-Franco, and P. R. Paradiso. 1987. Dem-
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onstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J. Gen. Virol. 68:2521-2524. Matzuk, M. M., M. Krieger, C. L. Corless, and I. Boime. 1988. Effects of preventing 0-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 84:6354-6358. Murphy, B. A., B. S. Graham, G. A. Prince, E. E. Walsh, R. M. Chanock, D. T. Karzon, and P. F. Wright. 1986. Serum and nasal-wash immunoglobulin G and A antibody response of infants and children to respiratory syncytial virus F and G glycoproteins following primary infection. J. Clin. Microbiol. 23:1009-1014. Robbins, P., S. Hubbard, S. Turco, and D. Wirth. 1977. Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoprotein. Cell 12:893-900. Satake, M., J. Coligan, N. Elango, E. Norrby, and S. Venkatesan. 1985. Respiratory syncytial virus envelope glycoprotein (G) has a novel structure. Nucleic Acids Res. 13:7795-7812. Spehner, D., S. GilMard, R. Drillien, and A. Kirn. 1988. A cowpox virus gene required for multiplication in Chinese hamster ovary cells. J. Virol. 62:1297-1304. Stott, E. J., L. A. Ball, K. Anderson, K. Young, A. M. Q. King, and G. W. Wertz. 1987. Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus. J. Virol. 61:3855-3861. Stott, E. J., L. A. Ball, K. K. Young, J. Furze, and G. W. Wertz. 1986. Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge. J. Virol. 60:607-613. Tarentino, A. L., and F. Maley. 1974. Purification and properties of an endo-,B-N-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem. 249:811-817. Wagner, D. K., P. Mullenaer, F. Henderson, M. Snyder, C. Reimer, E. Walsh, L. Anderson, D. L. Nelson, and B. R. Murphy. 1989. Serum immunoglobulin G antibody subclass response to respiratory syncytial virus F and G glycoproteins after first, second, and third infections. J. Clin. Microbiol. 27:589592. Wagner, D. K., D. L. Nelson, E. E. Walsh, C. B. Reimer, F. W. Henderson, and B. R. Murphy. 1987. Differential immunoglobulin G subclass antibody titers to respiratory syncytial virus F and G glycoprotein in adults. J. Clin. Microbiol. 25:748-750. Ward, K. A., P. R. Lambden, M. M. Ogilvie, and P. J. Watt. 1983. Antibodies to respiratory syncytial virus polypeptides and their significance in human infection. J. Gen. Virol. 64:18671876. Wertz, G. W., P. L. Collins, Y. Huang, C. Gruber, S. Levine, and L. A. Ball. 1985. Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proc. Natl. Acad. Sci. USA 82:40754079.
