Glycopeptide components of influenza viral glycoproteins

VIROLOGY

86.432-442 (1978)

Glycopeptide KIYOTO

Components NAKAMURA

of influenza Viral Glycoproteins RICHARD

AND

W. COMPANS’

Department of Microbiology, University of Alabama Medical Center,

Birmingham, Alabama

3.5294

Accepted January 2, 1978 The oligosaccharide moieties of the WSN strain of influenza virus were analyzed by Pronase digestion of viral glycoproteins followed by gel filtration. Oligosaccharides of intluenxa virions grown in MDBK cells consist of at least two size classes. The larger one contains glucosamine, mannose, galactose, and fucose and is designated type I. In addition, smaller oligosaccharides (type II) contain a high amount of mannose in addition to glucosamine but probably lack galactose and fucose. Type I glycopeptides were found both in HA, and HA2 (two cleavage products of hemagglutinin), whereas type II was found in HAI only. The oligosaccharides of virions grown in MDCK and BHKBl-F cells also consisted of two size classes. The presence of both types of glycopeptides in HA, and the absence of type II in HA, were also observed with virions grown in MDCK cells. The presence of sulfated glycopeptides was demonstrated with virions grown in all of these cell types, and most of the 35S042-label appears to be linked to type I glycopeptides. However, the elution profile of sulfated glycopeptides was slightly different from that of type I glycopeptides, suggesting that type I glycopeptides may be heterogeneous with respect to the extent of sulfation. The molecular weights of type I and type II glycopeptides of virions grown in MDBK cells were estimated to be about 2990 and 1650 to 2260, respectively. However, the sizes of the oligosaccharide components varied with the host cell type. These values, together with previous estimates of the carbohydrate content of the HA glycoprotein, suggest that in the case of virions grown in MDBK cells HA2 contains a single type I oligosaccharide group, whereas HAI contains two type I oligosaccharides plus one or two type II oligosaccharides. INTRODUCTION

The envelope of influenza virus contains two distinct glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The HA glycoprotein may be proteolytically cleaved into products designated HAI and HAz. Previous cell fractionation studies have revealed that HA polypeptides are synthesized in association with rough membranes and migrate through smooth membranes to plasma membranes (Compans, 1973a; Hay, 1974; Klenk et al., 1974; Meier-Ewert and Compans, 1974). During this process, HA polypeptides undergo two types of modifications, glycosylation and sulfation. Glycosylation of HA polypeptides appears to be initiated at rough membranes and completed at smooth membranes (Compans, 1973b; Stanley et al., 1973; Compans and Choppin, 1975; Klenk et al., 1977). Sulfation ’ Author to whom requests for reprints should be addressed.

of HA polypeptides is also initiated at rough membranes and appears to progress further in smooth membranes (Nakamura and Compans, 1977). Previous studies have shown that the carbohydrate moiety of influenza virions contains glucosamine, mannose, galactose, and fucose, and the overall composition of the carbohydrate is similar to that of host cells (Ada and Gottschalk, 1956; Frommhagen et al., 1959). The most remarkable difference between the carbohydrate components of influenza virus and those of most other groups of enveloped viruses is the absence of sialic acid in influenza virus, which is probably due to the action of viral neuraminidase (Klenk et al., 1970a, b; Palese et al., 1974). The carbohydrate analysis of the isolated HAI glycoprotein (Laver, 1971) indicates that it contains all of the four sugars glucosamine, mannose, galactose, and fucose at a molar ratio of 6:4:1:1, respectively. However, little infor-

432 0042-6822/76/0662-0432$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

GLYCOPEPTIDES

OF INFLUENZA

mation has been obtained about the detailed structure of the carbohydrate moiety of influenza viral glycoproteins. In the present report, we have characterized the glycopeptide size classes obtained from influenza WSN virions grown in four cell types. The results indicate the presence of at least two types of oligosaccharides in virions grown in various host cells. We also describe the glycopeptides obtained from the isolated glycoproteins HAI and HA2. Our results when compared with the recent results of Schwarz and co-workers (1977) on the carbohydrates of fowl plague virus indicate that significant differences in glycosylation occur among the hemagglutinin proteins of influenza A viruses. MATERIALS

AND

METHODS

Virus and cells. The &/WSN (HoNI) strain of influenza virus was used for all experiments. Virus stocks were grown in MDBK cells as described by Choppin (1969). The MDBK line of bovine kidney cells and the BHKBl-F line of hamster kidney cells were grown according to described procedures (Holmes and Choppin, 1966; Choppin, 1969). The MDCK line of canine kidney cells was grown in Eagle’s minimal medium (MEM) supplemented with 10% newborn calf serum. Primary cultures of chick embryo fibroblasts (CEF) were prepared from 10 day-old embryos and grown in MEM with 10% newborn calf serum. Growth andpurification of radioactively labeled virus. To prepare radioactively labeled virus, cells were inoculated with stock virus at a multiplicity of 1 to 10 PFU/cell. After an adsorption period of 2 br, MEM with 2% calf serum containing radioactive precursors was added. Radioisotopes were used at the following concentrations: 3Hlabeled sugar precursors, 3 pCi/ml; [14C]glucosamine, 0.5 @X/ml; 35S042-,20 @i/ml; C3H]leucine, 4 pCi/ml; 14C-amino acid mixture, 1 $X/ml. At about 20 hr after infection, the virions were purified from culture fluids by precipitation with polyethylene glycol and banding in a potassium tartrate gradient as described previously (Compans et al., 1970; Landsberger et al., 1971). The virus band was collected, diluted in 0.005 M sodium phosphate (pH 7.2), and pelleted at

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35,000 rpm for 45 min in a Beckman SW 50.1 rotor. The pellet was suspended in either 0.005 M sodium phosphate (pH 7.2) for SDS-polyacrylamide gel electrophoresis or 0.1 M Tris-HCl (pH 8.0) containing 0.01 M CaClz for glycopeptide analysis. Isolation of glycoproteins. Viral glycoproteins were isolated by SDS-polyacrylamide gel electrophoresis. The gels were sliced, and proteins were eluted by incubating each slice with 0.5 ml of 0.1% SDS in 0.01 M sodium phosphate (pH 7.2) for 24 hr at 37”. The radioactivity in a portion of each fraction was determined, and the fractions containing the glycoprotein(s) of interest were combined, lyophylized, and redissolved in 0.5 ml of water. Viral proteins were precipitated with 10 vol of absolute ethanol at -20” for 2 hr utilizing bovine serum albumin (100 pg) as a carrier. The precipitate was sedimented by a centrifugation at 18,000 g for 10 min and dissolved in 0.1 M Tris-HCl (pH 8.0) containing 0.01 M CaCh. Removal of NA from virions by trypsin treatment. Purified virions suspended in 0.05 M Tris-HCl (pH 7.5) containing 0.1 M NaCl and 0.001 M EDTA were mixed with trypsin dissolved in the same buffer at a final concentration of 50 pg/ml and incubated at 37” for 20 min. At the end of the incubation period, the mixture was diluted in 0.005 M sodium phosphate (pH 7.2) and virions were collected by pelleting. Neuraminidase activity of virions was reduced to less than 5% of control after this treatment, while hemagglutinating activity remained unchanged (Schulze, 1970). Pronase digestion and gel filtration of glycopeptides. Either purified virions or isolated viral glycoproteins suspended in 0.1 M Tris-HCl (pH 8.0) containing 0.01 M CaCh were extensively digested with Pronase as described previously (Nakamura and Compans, 1977). The resultant glycopeptides were applied to a column of Bio-Gel P-6 (100-200 mesh, 1.0 x 115 cm) equilibrated with 0.1 M Tris-HCl (pH 8.0), and fractions of 0.7 ml were collected. The molecular weights of glycopeptides were estimated by cochromatography with glycopeptides obtained from Sindbis virions grown in CEF utilizing Bio-Gel P-6

434

NAKAMURA

AND

(200-400 mesh). Sefton and Keegstra (1974) have identified four size classesof glycopeptides in Sindbis virions under these conditions with molecular weights of 3300,2800, 2490, and 1660. The glycopeptides obtained from influenza virions showed similar elution profiles with either the lOO-200- or the 200-400-mesh gel. SDS-polyacrylamide gel electrophoresis. Samples in 1% SDS and 1% /?-mercaptoethanol in 0.005Msodium phosphate (pH 7.2) were boiled for 1 min (Maize1 et al., 1968). For both analytical and preparative purposes, 7.5% acrylamide, 0.12% N,N’methylene bisacrylamide gels containing SDS were used. Processing of gels for determination of radioactivity was done as described previously (Cornpans, 1973b). Chemicals and isotopes. Reagents for polyacrylamide gels were obtained from Canal Industrial Corp. (Rockville, Maryland). Pronase ( Streptomyces grkeus protease), B grade, was obtained from Calbiochem (La Jolla, California). 35S042-;[4,53H]leucine (60 Ci/mmol) and the 14C-amino from were obtained acid mixture Schwarz/Mann (Orangeburg, New York). D-[6-3H]Glucosamine (6.3 Ci/mmol) and D-[l-‘4C]glucosamine (58.2 mCi/mmol) were obtained from ICN Pharmaceuticals (Irvine, California). D-[ l-3H]Mannose ( 13.2 Ci/mmol) and D-[l-3H]galactose (22 Ci/mmol) were obtained from Research Products International Corp. (Elk Grove Village, Illinois). D-[2-3H]mannose (2 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, Illinois).

COMPANS

peptides obtained from vii-ions labeled with [l-‘4C]glucosamine. The elution profile of glycopeptides labeled with either [3H]fucase (Fig. 1A) or [3H]galactose (Fig. 1B) indicated that most of these sugars were associated with the initial major peak of glycopeptides, which is designated peak I. Little or no 3H-label was detected in fractions 51 through 60 containing shoulder(s) of 14C-label. However, in contrast to fucose and galactose, at least two peaks were detectable with [2-3H]mannose (Fig. lc): One peak eluted with the main peak of [‘“Cl glucosamine label, and the other co-eluted with the fractions containing a shoulder of 14C-label. These results indicate that influenza virions grown in MDBK cells contain at least two types of oligosaccharide chains. One contains glucosamine, mannose, galactose, and fucose (type I). The other contains

RESULTS

Glycopeptides of influenza virions. It has been shown that influenza virions contain glucosamine, galactose, fucose, and mannose (Ada and Gottschalk, 1956; Frommhagen et al., 1959; Laver, 1971). To analyze the viral glycopeptides with respect to sugar composition, the incorporation of various sugar precursors into the glycopeptides of influenza virions grown in MDBK cells was investigated. Purified influenza virions labeled with [6-3H]fucose, [ l-3H]galactose, or [2-3H]mannose were digested with Pronase, and the resultant glycopeptides were cochromatographed with glyco-

20

30

40

50 60 70 FRACTION NO

SO

90

FIG. 1. Distribution of sugars into glycopeptides of influenza WSN virions grown in MDBK cells. Influenza virions labeled with (A) [6-3H]fucose, (B) [l-3H]galactose, or (C) [2-3H]mannose were mixed with virions labeled with [l-“C]glucosamine. The mixture was digested with Pronase and chromatographed on Bio-Gel P-6.

GLYCOPEPTIDES

OF INFLUENZA

a high amount of mannose and probably some glucosamine but probably lacks fucase and galactose and is designated type II. However, neither size class appears to be a homogeneous population. The elution profiles of type I glycopeptides differ slightly depending on the labeled sugar precursors used. Furthermore, two shoulders are often resolved with labeled glucosamine in the fractions containing type II glycopeptides. As can be seen in Fig. lC, the shoulder observed at fraction 57 contains more mannose label than observed in fraction 54. The components which eluted around the void volume have been shown to contain host cell-derived mucopolysaccharides (Nakamura et al., 1978). Glycopeptides of individual glycoproteins isolated from influenza virions. When the WSN strain of influenza virus is grown in MDBK cells in the presence of serum, most HA polypeptides are present in virions as the cleavage products HAI and HA2. The NA polypeptides, which are only a minor component of vii-ions, possess an electrophoretic mobility similar to HA,. Schulze (1970) has demonstrated that NA polypeptides can be removed from virions without removal of HA1 or HA2 by mild trypsin treatment. We have utilized this approach to determine the distribution of individual sugar precursor into HAI and HA2 as well as to obtain HAI free of NA using SDS-polyacrylamide gel electrophoresis. Table 1 shows the calculated distribution of each sugar precursor into HAI, HAz, and NA based on the amount of label associated with each glycoprotein peak. It is apparent that the ratio of label incorporated into HAI/HA2 varied with the sugar precursor used: A much higher ratio was obtained with [2-3H]mannose than the other precursors, and the ratio was slightly higher with [6-3H]glucosamine than with [ l-3H]galactose, indicating that the carbohydrate compositions of HA1 and HA2 are distinct. To examine if such differences in carbohydrate composition are reflected in the elution profile of glycopeptides, HA1 and HA2 were purified from trypsin-treated virions labeled with either [6-3H]glucosamine or [2-3H]mannose, and glycopeptides associated with each glycoprotein were an-

435

VIRUS TABLE

1

DISTRIBUTIONOFSUGARPRECURSORSINTO GLYCO~ROTEINSOFINFLUENZAVIRIONSGROWNIN MDBK CELLS" Sugar precursor*

[6-3H]glucosamine [ l-3H]galactose [2-3H]mannose

Percentage of labe1 in glycoprotein NA

HA,

HAs

18.3 8.8

56.3 57.3

25.4 33.9

19.1 66.4

14.5

Ratio, HAT/HA,

-

2.22 1.69 4.56

a The data were obtained as follows: A portion of purified influenza WSN virions labeled with the respective sugar precursor was treated with trypsin under the condition described under Materials and Methods, and both the trypsin-treated and untreated virions were analyzed by SDS-polyacrylamide gel electrophoresis. The ratios of HAI/HA2 were determined from the trypsin-treated sample. The difference in the incorporation between HAI plus NA in the control, and HAI in the trypsin-treated sample was normalized to HA2 and used to estimate the amount of label associated with NA. b In addition to the precursors listed, the distribution of [6-3H]fucose into viral glycoproteins was determined with control virions. The HA, + NA/HA2 ratio was 2.27.

alyzed by gel filtration. The glycopeptides associated with HAI purified from virions labeled with [6-3H]glucosamine showed an elution profile similar to those of whole virions (Fig. 2A). On the other hand, the glycopeptides obtained from HA2 (Fig. 2B) appear to be more homogeneous than those of whole vii-ions, and the profile lacks shoulders in fractions 54 through 60, which were resolved with both HA1 and whole vii-ions. The difference between HA1 and HAp was demonstrated even more clearly using [23H]mannose. As shown in Figs. 2C and 2D, two distinct peaks corresponding to type I and type II glycopeptides were resolved with HA1, whereas only a single peak corresponding to the type I glycopeptide was obtained from HA2. These results indicate that HA1 and HA2 are differentially glycosylated, with HA2 lacking the small mannose-rich glycopeptide species that are found in HA1. Effect of host cell type on influenza virion glycopeptides. There is general agreement that viral glycoproteins are probably glycosylated in large part, if not entirely, by host cell-specific transferases (Compans

436

NAKAMURA

AND I46 dpm

3Hdpm

y?.s*bl,j

I[;/h,,,r

COMPANS

3Hdpm

iYA

.,., *..;*,]

20 30 40 50 60 70 SO 90

4;

;/;id

,

I

;

1

20 30 40 50 60 70 so 90 FRACTION

FRACTION NO

NO

FIG. 2. Comparison of glycopeptides associated with HAI and HA, from influenza virions grown in MDBK cells. Influenza virions labeled with either [6-3H]glucosamine or [2-3H]mannose were purified. After trypsin treatment of the virions under the condition described under Materials and Methods, HAI and HA, were isolated by gel electrophoresis, digested with Pronase, and applied to a column of Bio-Gel P-6. (A) HAI labeled with [6-3H]glucosamine; (B) HA2 labeled with [6-3H]glucosamine; (C) HAI labeled with [2-3H]mannose; (D) HA2 labeled with 12-3Hlmannose. The dashed lines show marker glycopeptides from virions grown in MDBK cells labeled with [‘I-“C]glucosamine.

and Choppin, 1975; Rott and Klenk, 1977). In the case of influenza virus, a difference in the electrophoretic mobility of HA polypeptides has been demonstrated depending on the host cell type (Haslam et al., 1970; Compans et al., 1970; Schulze, 1970), suggesting variability in carbohydrate content. To compare the sizes of glycopeptides of virions grown in different host cell types, influenza virions labeled with [3H]glucosamine were grown in MDCK, BHK21-F, or CEF cells. The estimated molecular weights of HA glycoproteins from these four cell types are given in Table 2. After digestion with Pronase, the resultant glycopeptides were cochromatographed with glycopeptides obtained from MDBK virions labeled with [‘4C]glucosamine (Figs. 3A, 3B, and 3C). The major peak of glycopeptides from MDCK-grown virions was coincident with that of MDBK virions, indicating that the sizes of type I glycopeptides of both virions were similar. However, the type I glycopeptides of MDCK virions are more homogeneous than those of MDBK vii-ions. Components which elute heterogeneously in fractions 52 through 60

TABLE

2

MOLECULARWEIGHTSOF HA GLYCOPROTEINSAND GLYCOPEPTIDESOFINFLUENZAVIRIONSGROWNIN VARIOUS HOSTCELLTYPES Host cell

HA”

Glycopeptides? Type I

MDBK MDCK BHKSl-F CEF

74,509’ 74,ocMl 73,096-73,509 71,500-72,609

2,900 2,900 2,756 2,599

Type II 1,650-2,266 1,656-2,200 1,600-2,606 1,650-2,160

a The molecular weights of HA polypeptides from various cell types were determined by SDSpolyacrylamide gel electrophoresis of virions labeled with [3H]leucine utilizing the proteins of influenza virions grown in MDBK cells labeled with ‘%unina acids as internal markers (Compans et aZ., 1970). ’ Molecular weights of each glycopeptide were determined as described under Materials and Methods, ’ This value has been previously determined (Compans et aZ., 1970).

were also apparent with MDCK virions, which probably correspond to type II glycopeptides of MDBK virions. The type I glycopeptides of BHK21-F vii-ions were more homogeneous and slightly smaller than those of MDBK virions (Fig. 3B).

GLYCOPEPTIDES

OF INFLUENZA

+,dom

20

30

40

50

60

FRACTION

70

NO

80

90

20

so

40

50

60

FRACTION

70

SO 90

NO.

FIG. 3. Comparison of glycopeptides of influenza virions grown in various host cell types. Influenza virions labeled with [1-3H]mannose or [6-3H]glucosamine were prepared from MDCK, BHK%l-F, or CEF cells. After digestion of the virions with Pronase, the resultant glycopeptides were analyzed by gel filtration. (A) MDCK virions labeled with [6-3H]glucosamine; (B) BHKSl-F virions labeled with [6-3H]glucosamine; (C) CEF virions labeled with [6-3H]glucosamine; (D) MDCK virions labeled with [l-3H]mannose; (E) BHKOl-F virions labeled with [1-3H]mannose. (F) CEF virions labeled with [1-3H]mannose. The dashed lines show marker glycopeptides of influenza virions grown in MDBK cells and labeled with [l-‘4C]glucosamine.

BHK21-F-grown virions also appear to contain type II glycopeptides which are similar in size to those of MDBK virions. The most remarkable difference in size of glycopeptides was observed with CEF virions (Fig. 3C). The elution profile demonstrates a peak at fraction 49 and a shoulder in fractions 53 through 65, which suggests the presence of at least two size classes of glycopeptides in CEF virions. Type I glycopeptides of CEF virions are much smaller than those of MDBK virions. To provide further evidence that influenza virions contain both type I and type II glycopeptides irrespective of host cell type, virions were grown in MDCK, BHKBl-F, or CEF cells in the presence of [l-3H]mannose and digested with

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pronase, and the resultant glycopeptides were analyzed by gel filtration. The results illustrated in Figs. 3D, 3E, and 3F show that at least two size classes of glycopeptides labeled with r3H]mannose are obtained from virions grown in MDCK or BHK21-F cells, supporting the conclusion described above. However, two glycopeptide size classes were not resolved clearly in virions grown in CEF cells using mannose label. The smaller glycopeptides of MDCK virions labeled with [3H]mannose reproducibly formed two peaks as shown in Fig. 3D, and those of BHK virions usually showed a shoulder on the descending slope of peak II as can be seen in fraction 58 of Fig. 3E, which may suggest the presence of two or more kinds of type II glycopeptides in these virions. Although it was not possible to determine accurately the recovery of [3H]mannose label into each type of glycopeptide because of incomplete separation of the corresponding peaks, it was apparent that the relative amount of C3H]mannose label recovered in type II glycopeptides of MDCK-grown virions was much lower than in either BHK21-F- or CEF-grown virions, indicating that the distribution of mannose into type I and type II glycopeptides may depend on the host cell type. When [ I-“Hlmannose was used for labeling, a portion of the label incorporated into virions (22.8% for MDCK virions, 33.5% for BHK virions, and 15.5% for CEF virions) eluted heterogeneously in fractions 70 through 90. It appears likely that these counts reflect the metabolic conversion of [l-“Hlmannose into amino acids since we have detected significant amounts of label in NP and M proteins using this precursor (data not, shown). The molecular weight of each type of glycopeptide obtained from virions grown in either of four host cell types was estimated as described under Materials and Methods, and the results are summarized in Table 2. It appears that host cell-dependent size differences are particularly apparent with type I glycopeptides, whereas the size of type II glycopeptides was less variable. The results described above indicate that HA1 of MDBK virions contains both type I and type II glycopeptides, while HA2 con-

438

NAKAMUHA

AND

tains only type I. To examine whether this is also the case with virions grown in other cell types, we analyzed the glycopeptides of glycoproteins isolated from MDCK virions. Since a large fraction of HA polypeptides was present as uncleaved HA in MDCK virions, it was possible to analyze the glycopeptides of HA in addition to NA + HA, and HA2. As shown in Fig. 4, both types of glycopeptides were present in HA and in NA + HA1 and type II glycopeptides were absent from HAz. Incorporation of 35S042- into glycopeptides of influenza virions. The selective incorporation of %Od2- label into influenza viral glycoproteins grown in either MDBK or BHK21-F cells has recently been demonstrated (Compans and Pinter, 1975). Recent studies have also shown that almost all 35S042-label incorporated into the glycoproteins of influenza virions grown in MDBK cells is recovered in association with glycopeptides after extensive digestion of virions with Pronase (Nakamura and Compans, 1977). We have observed sulfation of influenza viral glycoproteins in both MDCK- and CEF-grown virions, and the extent of sulfation appeared to depend on the host cell type (data not shown). We have also compared the distribution of 35S042- label in glycopeptides prepared from virions grown in each of four cell types doubly labeled with [3H]glucosamine and %Od2-. The results shown in Fig. 5 indicate that three 35S042-peaks were obtained independently of host cell type. It has been demonstrated that the peak which elutes near the void volume is primarily due to 35S042-incorporated into host cell-derived mucopolysaccharides, and the peak which elutes in fractions 75 through 79 is free %S04’- (Nakamura and Compans, 1977; Nakamura et al., 1978). The 35S042-label associated with glycoproteins co-elutes with glycopeptides irrespective of host cell type. Furthermore, the results suggest that most 35S042-label is associated with type I glycopeptides. However, in all cell types the ratio of 35S042-to [3H]glucosamine on the ascending slope of the peak corresponding to type I glycopeptides is higher than that on the descending slope. These observations suggest that type I glycopeptides are heterogeneous with respect to the extent of

COMPANS

lOI

i

ci

FRACTION NO

FIG. 4. The glycopeptides of isolated glycoproteins of influenza virions grown in MDCK cells. IntIuensa virus was grown in MDCK cells and labeled with [l3H]mannose. The purified virions were subjected to SDS-polyacrylamide gel electrophoresis without pretreatment of virions with trypsin. Glycoproteins were isolated, digested with Pronase, and applied to a column of Bio-Gel P-6, (A) Uncleaved HA; (B) NA + HAI; (C) HAL?.

sulfation, and the highly sulfated glycopeptides elute before those that are unsulfated or sulfated to a lower extent. DISCUSSION

The oligosaccharide moieties of influenza viral glycoproteins have been analyzed by extensive digestion of either virions or isolated viral glycoproteins with Pronase followed by gel filtration. The results reveal that WSN strain influenza virions contain two distinct types of glycopeptides when grown in any of three cell types, which were designated type I and type II glycopeptides. Type I contains glucosamine, marmose, galactose, and fucose, whereas type II appears to contain only glucosamine and mannose. The presence of glycopeptides similar to these two types has been demonstrated previously in Sindbis virus (Sefton and Keegstra, 1974), immunoglobulin (Johnson and Clamp, 1971), and more recently in fowl plague virus grown in CEF (Schwarz et aZ.,

GLYCOPEPTIDES

FRACTION

OF INFLUENZA

NO.

VIRUS

FRACTION

439

NO.

FIG. 5. The glycopeptides of influenza virions labeled with ?‘3042- and [6-3H]glucosamine. Influenza virions doubly labeled with %302- and [6-3H]glucosamine were prepared from (A) MDBK, (B) MDCK, (C!) BHK21-F, or (D) CEF cells. The purified virions were digested with Pronase and analyzed by gel filtration.

1977). Type I and type II glycopeptides appear to correspond to the A and B types of glycopeptides as defined by Johnson and Clamp (1971). Both the type I and II glycopeptides appear to consist of heterogeneous populations of glycopeptides. It is unlikely that the observed heterogeneity is due entirely to the difference in the number of amino acid residues associated with glycopeptides, since the heterogeneity varied with the host cell type. The elution profiles of type I glycopeptides were slightly different depending on the sugar precursor used for labeling (see Fig. l), and the heterogeneity was more clearly demonstrated by labeling with 35S042-(see Fig. 5), suggesting that the type I glycopeptides are heterogeneous with respect to carbohydrate composition as well as extent of sulfation. The heterogeneity of type II glycopeptides is suggested by the finding that two shoulders which differ in relative amounts of r3H]mannose incorporation are frequently resolved in the fractions corresponding to type II glycopeptides. The extent of heterogeneity of glycopeptides depends on host cell type, as is particularly evident with type I glycopeptides. The type I glycopeptides of MDBK virions appear to be more heterogeneous than those of virions grown in other host cell types (see Fig. 3), although all cell types showed heterogeneity in the extent of glycopeptide sulfation.

The glycopeptides obtained from isolated glycoproteins showed less heterogeneous elution profiles compared with those obtained from virions. This was observed with every kind of glycoprotein tested and was evident particularly with type II glycopeptides. Only one shoulder was detectable in [3H]glucosamine fractions corresponding to type II glycopeptides of HA1, and the type II glycopeptides obtained from either HA, or HA1 + NA isolated from virions labeled with [3H]mannose showed a single homogeneous peak. The reason for this observation remains to be understood. We have found that HAI contains both type I and type II glycopeptides, whereas HA2 contains only type I with WSN virions grown in either of two cell types. Differential glycosylation of the viral glycoproteins has been also demonstrated with fowl plague virus grown in CEF (Schwarz et al., 1977). However, both types of glycopeptides were observed on HA2 and NA, but only type I was found on HAI of fowl plague virus. The mechanism specifying which of the two alternative oligosaccharide types is added to an acceptor site on influenza glycoproteins is of considerable interest. In principle the addition of type I vs type II oligosaccharides could be determined by the structural location of acceptor sites in the hemagglutinin protein or, alternatively, a specific amino acid sequence might deter-

440

NAKAMURA

AND

mine the type of oligosaccharide to be added. The present data taken together with the results of Schwarz and co-workers (1977) do not support the former possibility, since HA2 probably occupies a similar structural location in the hemagglutinin spike of both WSN and fowl plague viruses and yet contains type II glycopeptides only in the latter case. Therefore, we suggest that a specific amino acid sequence determines whether a type I or type II oligosaccharide is added at a given site on the glycoprotein molecule. Analysis of the primary structure of tryptic peptides containing each type of carbohydrate side chain may provide evidence for this possibility. A previous study of the glycoproteins of influenza B virus grown in the HKCC line of hamster cells indicated that the HAI and HA2 glycoproteins of this virus are also differentially glycosylated (Choppin et al., 1975). In this instance HA1 was labeled by either glucosamine or fucose, whereas HA2 contained glucosamine but no detectable fucose. If the glycoproteins of influenza B virions contain oligosaccharides of similar composition to those found in influenza A, these results indicate that HA2 of influenza B virions probably contains type II glycopeptides but lacks the fucose-containing type I glycopeptides. In this regard intluenza B virions differ from either A/WSN or fowl plague viruses, both of which contain fucose in HAz. The glycopeptides associated with NA were not directly examined in the present study. However, it was calculated in Table 1 that the NA of MDBK-grown virions contains approximately 10, 22, and 24% of galactose, glucosamine, and mannose label, respectively, that is incorporated into HA. Similar values were obtained by SDSpolyacrylamide gel electrophoresis of MDBK virions grown in the absence of serum (data not shown). Previous studies have also shown that [3H]fucose is incorporated into NA polypeptides (Nakamura and Compans, 1978). The presence of both galactose and fucose indicates the presence of type I glycopeptides in the NA glycoprotein. The variability in carbohydrate contents of influenza viral glycoproteins according to

COMPANS

host cell type has been suggested previously because of host-dependent differences in the electrophoretic mobilities of viral glycoproteins (Haslam et al., 1970; Compans et al., 1970; Schulze, 1970; Schwarz et al., 1977). This was confirmed in the present study utilizing four different host cell types. A host-dependent size difference was also observed with glycopeptides. This was particularly evident with type I glycopeptides while the size of type II glycopeptides was fairly constant irrespective of host cell type. Furthermore, the difference in size of type I glycopeptides appears to parallel the difference in the apparent molecular weight of the HA glycoprotein. The number of monosaccharide units contained in type I glycopeptides can be estimated as 14 to 16 for MDBK and MDCK virions, 13 to 15 for BHK virions, and 12 to 14 for CEF virions if it is assumed that Pronase-derived glycopeptides contain one to three amino acid residues (Spiro, 1965). Type II glycopeptides appear to be composed of 7 to 12 monosaccharides independent of host cell type. As described above, however, type II glycopeptides of virions grown in MDBK, MDCK, or BHK 21-F cells may contain at least two sizes of glycopeptides of molecular weights 1600 to 1650 and 2000 to 2200 consisting of 7 to 9 and 9 to 12 monosaccharide units, respectively. Host cell-dependent size differences similar to those described above were also observed between the glycopeptides of fowl plague virions grown in MDBK and CEF cells (Schwarz et al., 1977). The molecular weight of HA of virions grown in MDBK cells was estimated as 74,500 to 75,000 (Compans et al., 1970).We have previously estimated that the molecular weight of carbohydrate-free HA polypeptides produced in the presence of tunicamycin is 63,000 (Nakamura and Compans, 1978). It therefore appears that the HA glycoproteins of virions grown in MDBK cells contain 11,500 to 12,006 daltons of carbohydrate. Similar estimates have been made for the carbohydrate content of the hemagglutinin of other influenza virus strains (Laver, 1971; White, 1974; Schwarz et al., 1977). Furthermore, the results shown in Table 1 together with the distribution of sugar precursors into type I

GLYCOPEPTIDES

OF INFLUENZA

and type II glycopeptides suggest that HAI contains about twice as many type I glycopeptides as HA2. On the basis of these estimates, it is suggested that in the case of MDBK virions, HA2 contains only one type 1 glycopeptide and HA1 contains two type I glycopeptides. HA1 also must contain one or two type II glycopeptides on the average, if the above estimates of the total carbohydrate content of HA are correct. Further evidence is needed to determine if all hemagglutinin glycopeptides are equally glycosylated and if the above values are applicable to WSN virions grown in other cell types. The incorporation of r3H]mannose and [3H]glucosamine into type II glycopeptides of MDCK vii-ions was lower than that of virions grown in other cell types, suggesting that fewer type II glycopeptides are present in MDCK virions than in virions grown in other cells. The present results together with those of Schwarz and co-workers (1977) suggest that influenza A virus hemagglutinin subtypes may exhibit significant differences in the types of oligosaccharide side chains which are attached to the HA1 and HA2 portions of the glycoprotein. It will be of interest to examine the possibility that such variation may play a role in determining the immunological properties of this glycoprotein. If the location of oligosaccharide groups is determined by a specific amino acid sequence, a single mutation in the hemagglutinin gene could introduce a new glycosylation site or remove a previously existing glycosylation site in the glycoprotein, possibly causing a major change in the overall shape of the molecule. These possibilities may be examined by further analysis of the location of oligosaccharides on various hemagglutinin subtypes, as well as determination of the role of carbohydrates, in determining the immunological specificity of the glycoprotein. ACKNOWLEDGMENTS This research was supported by Grant No. AI 12660 from The National Institute of Allergy and Infectious Diseases, USPHS. REFERENCES ADA, G. L., and GOTTSCHALK, A. (1956). The component sugars of the influenza virus particle. Biochem.

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Strand Viruses and the Host Cell” (B. W. J. Mahy and R. D. Barry, eds.) Academic Press, New York. In press. PALESE, P., TOBITA, K., UEDA, M., and COMPANS,R. W. (1974). Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61,397-410. Row, R., and KLENK, H.-D. (1977). Structure and assembly of viral envelopes. In “Virus Infection and the Cell Surface” (G. PO& and G. L. Nicolson, eds.), pp. 47-81. North-Holland, Amsterdam. SCHULZE,I. T. (1970). The structure of influenza virus: I, The polypeptides of the virion. Virology 42, 890-904. SCHWARZ,R. T., SCHMIDT, M. F. G., ANWER, U., and KLENK, H.-D. (1977). Carbohydrates of influenza virus: I, Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars. J. Viral. 23,217-226. SEF~ON,B. M., and KEEGSTRA, K. (1974). Glycoproteins of Sindbis virus: Preliminary characterization of the oligosaccharides. J. Virol. 14,522-530. SPIRO, R. G. (1965). The carbohydrate unite of thyroglobulin. J. Bill. C&n. 240, X03-1610. STANLEY, P., GANDHI, S. S., and WHITE, D. 0. (1973). The polypeptides of intluenza virus: VII, Synthesis of the hemagglutinin. Virology 53,92-K%. WHITE, D. 0. (1974). Influenza viral proteins: Identification and synthesis. Curr. Topics Microbial. Zmmunol. 63, 2-48.