Host cell- and virus strain-dependent differences in oligosaccharides of hemagglutinin glycoproteins of influenza A viruses

VIROLOGY

95, 8-23

(19’79)

Host Cell- and Virus Strain-Dependent Differences in Oligosaccharides of Hemagglutinin Glycoproteins of Influenza A Viruses KIYOTO Department

NAKAMURA

of Microbiology,

University

AND RICHARD of Alabama

Accepted

Medical

December

27,

W. COMPANS Center,

Birmingham,

Alabama $529.$

1978

The effect of the host cell and virus strain on the glycosylation of influenza A virus hemagglutinin glycoproteins was investigated by analysis of Pronase-digested glycopeptides labeled with radioactive sugar precursors. When influenza A/WSN virus was grown in MDBK cells, HA, contained both complex galactose-containing (type I) and mannose-rich (type II) glycopeptides, but HAI lacked type II glycopeptides. In contrast, type I and type II glycopeptides were found in HA, as well as HA, isolated from virus grown in primary chick embryo fibroblast cells, indicating that whether type I or type II oligosaccharide chains are attached to HA polypeptides is determined in part by the host cell type. However, analyses of glycopeptides from various influenza A strains representative of all human antigenic subtypes grown in a single host cell type, MDCK cells, revealed that the oligosaccharide types associated with HA polypeptides also depend on the virus strain. Both type I and type II glycopeptides were found in all strains tested, and there was no significant difference in the sizes of these glycopeptides among the strains. However, strain-dependent differences were observed in the relative amounts of type I and type II glycopeptides in vu-ions as well as isolated glycoproteins HA, or HA,. Such differences were evident among influenza A strains of the same as well as distinct HA subtypes, which suggests that changes in primary structure of the HA polypeptides induced by either antigenic drift or major antigenic shifts have caused the variation in oligosaccharide chains associated with these polypeptides.

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). Laver (1971) has analyzed the carbohydrate composition of HA, isolated from influenza A/BEL strain grown in allantoic membranes of embryonated hen’s eggs, and reported that it contains all four sugars, glucosamine, mannose, galactose, and fucose, at a molar ratio of 6:4:1:1. In contrast, the carbohydrate analysis of HA glycoproteins isolated from A/PR8 virus grown in MDCK cells has shown that glucosamine, mannose, galactose, and fucose are present in the glycoproteins at a molar ratio of 5:ll: 6:2 (Collins and Knight, 1978). Recently, it has been demonstrated that influenza viral glycoproteins contain at least two size

INTRODUCTION

Two distinct glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are located on the surface of influenza A virions. HA glycoproteins may be present in virions as two cleavage products, HA, and HA,. Major changes in antigenicity of HA and NA of influenza A viruses have occurred periodically in nature, which have resulted in the appearance of new strains with surface antigens that are totally unrelated to previously isolated strains. Thus influenza A strains which belong to different subtypes contain glycoproteins with completely distinct antigenic properties. In addition, many influenza A strains have been isolated within each subtype, and they contain glycoproteins with related but distinguishable antigenic properties. It has been previously shown that the 0042~6822/79/070008-16$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

8

CARBOHYDRATES

OF INFLUENZA

classes of carbohydrate chains (Schwarz and Compans, et al., 197’7; Nakamura 1978b; Collins and Knight, 1978). The larger one consists of glucosamine, mannose, galactose, and fucose, and was designated type I glycopeptides. The smaller one (designated type II) contains a high amount of mannose in addition to glucosamine, but lacks galactose and fucose. Collins and Knight (1978) have examined the sensitivity of these glycopeptides to various exoglycosidases, and found that at least some of the galactose and fucose residues are present in terminal positions of type I oligosaccharide chains, and some mannose residues in type II oligosaccharide are sensitive to a-mannosidase. The results of Schwarz and co-workers (1977) and our laboratory (Nakamura and Compans, 1978b) indicate that influenza viral glycoproteins of various strains are differentially glycosylated. In the former case (fowl plague virus grown in primary chick embryo fibroblast cells), both type I and type II glycopeptides were found on NA and HA,, but only type I was detected on HA,. In contrast, in the latter case (WSN virus grown in either MDBK or MDCK cells), HA, contained both type I and type II glycopeptides, whereas HA, lacked type II glycopeptides. These different observations led us to investigate whether the oligosaccharide types that are attached to a particular viral glycoprotein are specified by the host cell or the virus strain, or both. Evidence will be presented in this paper which indicates that the types of carbohydrate side chains associated with HA polypeptides can vary depending on host cell type as well as virus strain. Further, we will show that the distribution of oligosaccharide chains associated with HA glycoproteins varies among influenza virus strains of the same as well as different antigenic subtypes, and the mechanisms by which different influenza strains control the glycosylation of their HA polypeptides will be discussed. MATERIALS

AND

METHODS

Viruses and cells. The following influenza A viruses were used: A/WSN (H,N,),

A VIRUSES

9

A/I’R/8/34 (HoN1), A/FM/l/47 (H,N,), A/ USSR/90/77 (H,N,), A/FW/1/50 (H,N,), A/Jap/305/57 (H,N,), A/RI/5+/57 (H,N,), A/ Victoria/3/75 (H3N2), A/Aichi/2/68 (H,N,), and Alswine/1976/31 (H,,N,). AlPR/8/34 (PR8), A/FM/l/47 (FMl), and A/Aichi/ 2168 (Aichi/Z) were obtained from the Research Resources Branch of the National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. A/USSR/90/77 (USSR), A/FW/1/50 (FWl), ANictoria/3/75 (Victoria), and A/swine/1976/31 (swine) were obtained from Dr. A. P. Kendal, Center for Disease Control, Atlanta, Georgia. A/Jap/305/57 (Japl305) and A/RI/ 5+/57 (RI/5+) were obtained from Dr. N. J. Dimmock, Australian National University, and Dr. P. W. Choppin, Rockefeller University, respectively. Unless otherwise noted, viruses grown in the allantoic cavity of hen’s eggs were used for infection of cells. The MDBK line of bovine kidney cells was grown according to procedures described previously (Choppin, 1969). The MDCK line of canine kidney cells and primary cultures of chick embryo fibroblast (CEF) cells were grown as described previously (Nakamura and Compans, 1978b). Growth and purification of radioactively labeled viruses. To grow radioactively labeled virus, monolayers of either MDBK, MDCK, or CEF cells on lo-cm plastic petri dishes were infected with stock virus at a multiplicity of about 10 PFU/cell. After an adsorption period of 2 hr, unadsorbed inoculum was removed and Eagle’s minimal medium with 2% newborn calf serum containing 3 &i/ml of 3H-labeled sugar precursor or 0.5 &i/ml of [‘*C]glucosamine was added. At about 24 hr after infection, virions were purified from culture fluids by precipitation with polyethylene glycol followed by banding in a potassium tartrate gradient as described previously (Compans et al., 1970; Landsberger et al., 1971). The virus band was collected, pelleted at 35,000 rpm for 45 min in a Beckman SW50.1 rotor, and suspended in 0.005 1M sodium phosphate (pH 7.2) for SDS-polyacrylamide gel electrophoresis or 0.1 M Tris-HCl (PH 8.0) containing 0.01 A4 CaCI, for glycopeptide analysis.

10

NAKAMURAANDCOMPANS

Isolation of HA, and HA,. HA, and HA, at 37” for 24 hr. The reaction was terwere isolated by SDS-polyacrylamide gel minated by boiling for 2 min. electrophoresis of purified virions treated SDS-polyacrylamide gel electrophowith trypsin according to the procedures resis. This was carried out with 7.5% gels described previously (Nakamura and Com- according to the procedures described prepans, 1978b). Trypsin treatment was neces- viously (Maize1 et al ., 1968; Nakamura sary for isolation of the HA, and HA, and Compans, 1978b), and gels were procglycoproteins since in any influenza A essed for determination of radioactivity strains used, except for PR8 and WSN as described previously (Cornpans, 1973). viruses, no cleavage of HA polypeptides Chemicals and isotopes. Pronase (Strepwas detectable in purified virions when tomyces griseus protease), B grade, was they were grown in MDCK cells. Even obtained from Calbiochem, La Jolla, Caliwith PR8 and WSN viruses, cleavage oc- fornia. Endo-/I-N-acetylglucosaminidase ‘H curred to a much lower extent in MDCK from Streptomyces griseus was obtained cells than in either MDBK or CEF cells. from Seikagaku Kogyo Co., Tokyo, Japan. In addition, NA glycoproteins of viruses Chemicals for polyacrylamide gels were obof the H,N, serotype have electrophoretic tained from Canal Industrial Corp., Rockmobilities similar to that of HA, (Comville, Maryland. D-[l-3H]Glucosamine (6.3 pans et al., 1970), and NA has been Ci/mmol) and D-[l-14C]glucosamine (58.2 shown to be selectively removed from mCi/mmol) were obtained from ICN Pharvirions of the WSN strain without removal maceuticals, Irvine, California. ~-[l-~Hlof HA, and HA, by trypsin treatment, Galactose (22 Ci/mmol) was obtained from under the conditions in which HA is cleaved Research Products International Corp., into HA, and HA, (Schulze, 1970; NakaElk Grove Village, Illinois. D-[2-3H]Manmura and Compans, 1978b). Similar results nose (2 Ci/mmol) was obtained from Amer(not shown) have been obtained with PR8 sham Corp., Arlington Heights, Illinois. virus. 14C-Labeled amino acid mixture (‘“C-aa) Gel Jiltration of glycopeptides. Either was obtained from SchwarzlMann, Orangepurified virions or isolated viral glyco- burg, New York. proteins suspended in 0.1 M Tris-HCl (pH 8.0) containing 0.01 M CaCl, were exRESULTS tensively digested with Pronase under the Host cell-dependent glycosylation of inconditions described previously (Nakamura and Compans, 1977). The resultant glyco- fluenza viral gl ycoproteins . Previous results have indicated that the glycoproteins peptides were analyzed by gel filtration of influenza A viruses contain both complex on a column of Bio-Gel P-6 (lOO/ZOO mesh, 1.0 x 115 cm) equilibrated with 0.1 M Tris(type I) and mannose-rich (type II) oligosacchar-ides, and that the distribution of these HCl @H 8.0) as described previously oligosaccharide types between the HA, (Nakamura and Compans, 1977). varies with the Treatment of glycopeptides with endo- and HA, glycoproteins /3-N-acetylglucosaminidase H (endo-H) . virus strain-host cell system used (Schwarz and Compans, The fractions containing glycopeptides to be et al., 1977; Nakamura 197813; Collins and Knight, 1978). With fowl tested, which were obtained after Pronase digestion of viral glycoproteins followed by plague virus grown in CEF cells, both type I and type II glycopeptides are gel filtration on Bio-Gel P-6, were pooled, lyophilized, and dissolved in deionized found on HA, as well as NA, but only type I is detectable on HA, (Schwarz et al., water. They were desalted by gel f&ration on a column of Bio-Gel P-2 (50/100 1977). In contrast, our previous results with the WSN strain grown in either MDBK or mesh, 1.5 x 25 cm) and then lyophilized. The glycopeptides were dissolved in 250 MDCK cells have indicated that HA, con~1 of 0.05 M citrate-phosphate buffer (pH tains both type I and type II glycopeptides, whereas HA, lacks type II glycopeptides 6.5) and incubated with 0.01 U of endo-H

CARBOHYDRATES

OF INFLUENZA

A VIRUSES

11

obtained from HA, and HA,, these glycoproteins were isolated from virions grown DISTRIBUTION OF RADIOACTIVE SUGAR PRECURSORS in CEF cells labeled with either [3H]galacINTO HA, AND HA2 OF INFLUENZA A/WSN tose or [3H]mannose. Glycopeptides were VIRUS GROWN IN CEF CELLS” prepared from each glycoprotein and then analyzed by chromatography on Bio-Gel Radioactivity (dpm) incorP-6. The glycopeptides from HA, labeled porated into with [3H]mannose showed a major peak in glycoproteins fraction 54 and a shoulder in fraction 49 (Fig. Ratio 1A). When [3H]galactose was used as label HAllHAl Sugar precursor HA, HA, (Fig. lB), a single peak was observed which corresponds to the shoulder fractions of Fig. 9178 2247 4.1 [3H]Glucosamine lA, but not the major peak. Two distinct 9353 2982 3.1 [3H]Galactose peaks were resolved with glycopeptides 1377 6.1 [3H]Mannose 3416 from HA, labeled with [3H]mannose, in a Influenza A/WSN virus was grown in CEF cells fractions 50 and 54, respectively (Fig. lC), labeled with either [3H]glucosamine, [3H]galactose, or and [3H]galactose was incorporated into the [3H]mannose. Vii-ions were purified and then treated peak which eluted in fraction 50 (Fig. 1D). with trypsin as described previously (Nakamura and These results indicate that both HA, and Compans, 19’78b). The distribution of each sugar preHA, isolated from influenza WSN virions cursor into HA, and HA, was estimated after SDSgrown in CEF cells contain type I and type polyacrylamide gel electrophoresis of trypsin-treated II glycopeptides. However, the relative virions. amounts of type I and type II glycopeptides TABLE

1

(Nakamura and Compans, 19’7813). In order to understand the basis for these differences, it is necessary to determine whether they are due to the virus strain or to the host cell types used. We therefore compared the glycopeptides associated with HA, and HA, isolated from the WSN strain grown in MDBK and CEF cells. Since our WSN virus stocks grown in MDBK cells did not yield sufficient quantities of virus from CEF cells for this purpose, the virus was serially passaged four times in allantoic sacs of embryonated hen’s eggs, and the fourth-passage allantoic harvest was used for infection of CEF cells. To examine the distribution of sugar precursors in HA, and HA, isolated from virions grown in CEF cells, virions were labeled with either [3H]mannose, [3H]glucosamine, or [3H]galactose. The distribution of the respective sugar precursors into HA, and HA2 was estimated after SDSpolyacrylamide gel electrophoresis of virions treated with trypsin. The results shown in Table 1 indicate that the ratios of label in HA,/HA, varied depending on the sugar precursor used, indicating that HA, and HA, have distinct carbohydrate compositions. To compare the glycopeptide size classes

FRACTION

NO

FRPCTION

NO

FIG. 1. Glycopeptides associated with HA, and HAI from influenza A/WSN virus grown in CEF cells. Virions labeled with either [3H]mannose or [3H]galactose were purified and treated with trypsin. HA, and HA, were isolated by gel electrophoresis, digested with Pronase, and applied to a column of Bio-Gel P-6. (A) Glycopeptides from HA, labeled with [3H]mannose; (B) glycopeptides from HA, labeled with [3H]galactose; (Cl glycopeptides from HA, labeled with PH]mannose; CD)glycopeptides from HA, labeled with [3H]galactose. The position designated V, corresponds to the elution position of mucopolysaccharides in [3H]glucosaminelabeled profiles, e.g., Fig. 2.

12

NAKAMURAANDCOMPANS

TABLE 2 contained in HA, and HA, appear to differ; HA, appears to contain more type II glycoMOLECULAR WEIGHTS OF HA, HA,, AND HA, peptides than HA1, which probably reflects OF VARIOUS INFLUENZA A VIRUSES the differences in incorporation of sugar GROWN IN MDCK CELLS” precursors into HA, and HA, (see Table 1). To exclude the possibility that passage Molecular weights of glycoproteins of stock virus in eggs affects the glycopeptide size classes associated with HA, and Serotype Strain HA HA, HA, HA, of virions grown in MDBK cells, purified WSN virions labeled with [3H]mannose A/WSN ‘75,000 55,000 28,000 H&‘, were also prepared from MDBK cells inAlPRlt3l34 73,000 54,000 27,500 fected with egg-grown stock virus. HA, and A/USSR/90/77 84,000 66,000 27,000 HA, were isolated from the virions, and H,N, A/FM/l/47 78,000 57,000 28,000 extensively digested with Pronase. The reA/FW/1/50 84,000 66,000 27,560 sultant glycopeptides were similar to those observed in our previous report (Nakamura A/.Iapl365/57 75,000 53,000 32,000 HA and Compans, 197813) in which stock virus A/RI/5+/57 75,000 53,000 30,000 grown in MDBK cells was used for infection AlVictorial3/75 78,000 62,000 28,000 H,N, of cells (data not shown). AlAichi/2/68 78,000 62,000 29,500 The finding that HA, of the WSN strain A/swine/1976/31 76,000 54,500 27,500 grown in CEF cells possesses both type I HswN, and type II glycopeptides, but that it lacks n Purified virions labeled with [3H]glucosamine were type II glycopeptides when grown in either prepared from culture fluids of MDCK cells. Molecular MDBK or MDCK cells (Nakamura and weights of either HA glycoproteins or the two cleavage Compans, 1978b), indicates that a particular products of the glycoproteins, HA, and HA*, were influenza viral glycoprotein can be differendetermined after SDS-polyacrylamide gel electrophotially glycosylated in various host cell types. resis of untreated or trypsin-treated virions, respecFurther, the fact that HA, of fowl plague tively. The polypeptides of A/WSN virions grown in virus grown in CEF cells lacks type II glycoMDBK cells labeled with ‘*C-aa were used as internal peptides (Schwarz et al., 1977), which are markers. found in large amounts on the HA, glycoprotein of WSN virus grown in these cells, suggests that qualitative differences in the HAUlml with any virus strain used in the types of glycopeptides associated with par- present study. ticular glycoproteins can be observed when Molecular weights of HA, HA,, and HA, different strains of influenza A virus are of in$uenza A viruses grown in MDCK grown in a single host cell type. This ob- cells. To compare the molecular weights of servation suggests that the amino acid se- viral glycoproteins among influenza A quence of the polypeptide backbone of viral strains, virions were purified from MDCK glycoproteins might specify the types of cells after labeling with [3H]glucosamine. oligosaccharide chains linked to these glyco- The molecular weights of HA, HA,, and proteins (Nakamura and Compans; 1978b). HA, were estimated by coelectrophoresis The following experiments were carried of trypsin-treated or untreated virions with out to further examine this possibility by 14C-amino acid-labeled WSN virions grown comparing the glycopeptides of various in- in MDBK cells (Table 2). It was apparent fluenza A viruses grown in a single host cell that the electrophoretic mobilities of the type, MDCK cells. It has been demonstrated glycoproteins of influenza A viruses varied that these cells support the growth of all depending on the antigenic subtype to which influenza A and B viruses tested (Tobita et they belong. On the other hand, the sizes al., 1975; Tobita; 1975). In fact, under one- of glycoproteins of influenza A viruses of step growth conditions, virus yields from the same serotype were fairly similar. HowMDCK cells were in the range of l-4 x 103 ever, it should be noted that among viruses

CARBOHYDRATES TABLE

OF INFLUENZA

3

DISTRIBUTION OF VARIOUS SUGAR INTO HA, AND HA, OF INFLUENZA

PRECURSORS A VIRUSES=

Ratio of HA,/HA, Serotype

Strain

Glucosamine

Galactose

Mannose

HoN,

A/WSN AlPR/8/34

2.5 4.0

2.0 3.9

3.5 4.0

H,N,

A/USSR/90/77 A/FM/l/47 A/VFW/l/50

5.0 4.2 5.7

2.7 4.0 3.1

9.3 4.0 6.6

HA

AlJapl305/57 AlRI/5+/57

4.3 4.3

4.6 3.2

5.2 4.7

H,N,

A/Victorial3/75 A/Aichti2/68

5.4 4.9

3.3 3.0

8.0 7.9

%NI

A/swine/1976/31

5.0

4.1

5.2

u Influenza A viruses were grown in MDCK cells in the presence of either [3H]glucosamine, [3H]galactose, or [3H]mannose. The HA,/HA, ratio with each sugar precursor was determined after SDS-polyacrylamide gel electrophoresis of purified virions treated with trypsin.

of the H, subtype, HA glycoproteins of FM1 virus were significantly smaller than those of either USSR or FWl virus, although they belong to the same serotype. Further, the results also suggest that the difference in HA glycoproteins between FM1 virus and USSR or FWl virus is due to a size difference in HA, rather than HA,. Distribution of sugar precursors into HA, and HA2 of influenza A viruses. To compare the sugar compositions of HA, and HA, of influenza A viruses, virions grown in MDCK cells labeled with either [3H]glucosamine, [3H]galactose, or [3H]mannose were purified. The distribution of each sugar precursor into HA, and HA2 was estimated after SDS-polyacrylamide gel electrophoresis of trypsin-treated virions. The results shown in Table 3 indicate that in some strains, the HA1/HA2 ratios were quite different, depending on the sugar precursors used. This was particularly evident with WSN, USSR, FWl, Victoria, and Aichi/Z viruses. In any of these cases, the ratio of

A VIRUSES

13

label in HA,/HA, was highest with [3H]mannose and lowest with [3H]galactose. The results also show that the HAJHA, ratio observed with certain sugar precursors can vary between strains with same serotype, as was evident with viruses of the H,N, and H,N, serotypes. Glycopeptides of injkenza A viruses with H,,N, serotype. We have previously reported that WSN virions grown in MDCK cells contain both type I and type II glycopeptides with sizes similar to those of virions grown in MDBK cells (Nakamura and Compans, 1978b). It has been also observed that both type I and type II glycopeptides are present in HA1, whereas only type I is detectable with HA,. Figures 2A, B, and C show the elution profiles of glycopeptides from purified PRS virions grown in MDCK cells labeled with [3H]glucosamine, [3H]galactose, or [3H]mannose, respectively. The glycopeptides labeled with either [3H]glucosamine or [3H]galactose eluted as a single homogeneous peak in fractions corresponding to type I glycopeptides from WSN virions. r3H]Mannose was also incorporated into this peak. However, a heterogeneous shoulder was detectable with this label in the fractions where type II glycopeptides of WSN virus eluted. These results indicate that PR8 virus contains type I and type II glycopeptides with sizes indistinguishable from those of WSN virus. However, the relative amounts of type II glycopeptides appear to be much lower in PR8 than WSN virions (see Nakamura and Compans, 197813). Figures 2D and E show the gel filtration patterns of [3H]mannose-labeled glycopeptides from HA, or HA, isolated from PR8 virions. The glycopeptides from HA, or HA2 eluted similarly, with a rather homogeneous peak in fractions consistent with type I glycopeptides, indicating that HA, as well as HA, contain type I glycopeptides as major oligosaccharide components, but little or no type II glycopeptides are present in either glycoprotein. Since previous data have demonstrated that both type I and type EI glycopeptides are clearly resolved with HA,‘ of WSN virions grown in MDCK cells, it appears that HA polypeptides of different

NAKAMURAANDCOMPANS

201)4OWWiOWSC FRACTION

NO

FIG. 2. Glycopeptides of influenza A/PR/8/34 virus. Purified virions grown in MDCK cells labeled with either [3H]glucosamine (A), [3H]galactose (B), or [3H]mannose (C) were extensively digested with Pronaae, and the resultant glycopeptides were cochromatographed with [‘C]glucosamine-labeled glycopeptides from A/WSN virions grown in MDBK cells. Glycopeptides were also prepared by Pronase digestion of HA, (D) and HA, (E) isolated from [3H]mannose-labeled virions and analyzed on Bio-Gel P-6.

influenza A strains with the same antigenic subtype can be differentially glycosylated in a single host cell type. Glycopeptides from inJuenxa A viruses with H,N, serotype. Three strains, USSR,

FWl, and FMl, were analyzed. Figure 3 shows the elution profiles of [3H]mannoselabeled glycopeptides from HA, or HA, isolated from each of these three strains. The glycopeptides from HA, isolated from either USSR or FWl virions showed similar elution profiles, with a main peak in fractions corresponding to type I glycopeptides and two additional peaks in the region where type II glycopeptides elute (Figs. 3A and C). In contrast, HA, from FM1 virions predominantly consists of type I glycopeptides (Fig. 3E). The glycoprotein also appears to contain some type II glycopeptides since a shoulder was detectable in

fractions 50 through 60, but the amount of type II glycopeptides appears to be much less in HA, of FM1 than those of the other two strains. The type I glycopeptides were also the major oligosaccharide components in HA, of either USSR or FM1 virions, and no type II glycopeptides could be clearly demonstrated in the glycoprotein of these two strains (Figs. 3B and F). On the other hand, the elution profile of the glycopeptides from HA2 of FWl virus (Fig. 3D) showed a major peak in fraction 43, and a shoulder in fractions 48-50 corresponding to one of the peaks of type II glycopeptides detected with HA, from this strain, suggesting the presence of both type I and type II glycopeptides in HA2 of FWl virions. It has been shown that endo+-N-acetylglucosaminidase-H (endoH) cleaves between the two proximal N-acetylglucos-

CARBOHYDRATES

OF INFLUENZA

A VIRUSES

15

FIG. 3. Glycopeptides from HA, and HA, isolated from influenza A viruses of the H,N, serotype. HA, and HA, were isolated from either influenza A/USSR/90/77, A/FW/1/50, or A/FM/l/47 virus grown in MDCK cells labeled with [3H]mannose. The glycopeptides obtained after Pronase digestion of isolated glycoproteins were chromatographed on Bio-Gel P-6. (A) Glycopeptides from HA, of USSR; (B) glycopeptides from HA, of USSR; (C) glycopeptides from HA, of FWl; (D) glycopeptides from HA, of FWl; (E) glycopeptides from HA, of FMl; (F) glycopeptides from HA, of FMl.

amine residues of asparagine-linked oligosaccharide chains with oligomannosyl cores containing a high amount of mannose (Tarentino and Maley, 1974). We have observed

FIG. 4. Endo-H sensitivity of glycopeptides from HA, and HA, isolated from A/USSR/90/‘7’7 and A/FW/ l/50 viruses. [3H]Mannose-labeled glycopeptides obtained from HA, and HA, isolated from either USSR or FWl virions were treated with endo-H as described in Materials and Methods and then rechromatographed on Bio-Gel P-6. (A) Endo-H treated glycopeptides from HA, of USSR; (B) endo-H treated glycopeptides from HA2 of USSR; (C) endo-H treated glycopeptides from HA, of FWl; (D) endo-H treated glycopeptides from HA, of FWl.

that type II glycopeptides of WSN virus are sensitive to this enzyme, whereas type I glycopeptides are resistant (Nakamura and Compans, 1979). It therefore appears that the sensitivity of glycopeptides from influenza virus to endo-H is a useful marker to identify the structure of the glycopeptides. The endo-H sensitivity of [3H]mannoselabeled glycopeptides from HA, and HA, isolated from either USSR or FWl virions was therefore tested to confirm the above conclusions about the types of glycopeptides present in these glycoproteins. As can be seen in Figs. 4A and C, when the glycopeptides from HA, of either USSR or FWl virions were treated endo-H, the two smaller peaks detected with control glycopeptides in the fractions corresponding to type II glycopeptides (Figs. 3A and C) completely disappeared, with the appearance of a new peak around fractions 63-64. On the other hand, the main peak which represents type I glycopeptides was not affected. The amounts of label recovered in peaks of the retarded fractions which have appeared after endo-H treatment were about 51 and 47% of the total counts with USSR and FWl viruses, respectively. When the glycopeptides from HA, of FWl virus were treated with endo-H, the

16

NAKAMURA

shoulder observed in Fig. 3D disappeared, which was accompanied by the appearance of a new peak containing about 36% of the total label in fractions 60 through ‘7’7 (Fig. 4D). Although type II glycopeptides were not clearly resolved with HA, of USSR virions (Fig. 3B), treatment of the glycopeptides with endo-H produced heterogeneous peaks in fractions 57 to ‘76, which contained approximately 22% of the total label (Fig. 4B). These results suggest that HA, of USSR as well as FWl contain both type I and type II glycopeptides in similar ratios, and that although HA, of these two strains also contain type II glycopeptides, the relative amount of type II glycopeptides was significantly higher in FWl than USSR virus. The similarity of oligosaccharide chains associated with HA, between USSR and FWl viruses suggests that the glycoproteins of influenza A viruses which are closely related serologically may acquire similar oligosaccharides during growth in a single host cell type. However, the apparent difference in the amounts of type II glycopeptides in HA, between FM1 and USSR or FWl viruses and in HA, among the three strains supports the conclusion described above that particular glycoproteins of different influenza A strains can be differentially glycosylated in a single host cell type, even if the viruses belong to the same serotype. It has been previously observed that WSN virus grown in various host cell types contains type II glycopeptides that are heterogeneous in size, and two size classes may be sometimes resolved (Nakamura and Compans, 1978b). This was more clearly demonstrated with USSR and FWl viruses (Figs. 3A and C). Although we cannot rule out the possibility that the size differences between two type II glycopeptides are due to the differences in the number of residual amino acid residues, the observation that only larger type II glycopeptides (IIa) were detectable on HA, of FWl virus (Fig. 3D), while both were found on HAI, may support the possibility that the two classes of type II glycopeptides are composed of distinct numbers of monosaccharides. The amounts of smaller type II glycopeptides (IIb) were

AND

COMPANS

significantly higher than those of larger type II glycopeptides in USSR and FWl virions (not shown). In contrast, HA, isolated from these viruses contained more larger type II glycopeptides than smaller ones (Figs. 3A and C), and only larger type II glycopeptides were detectable in HA, (Fig. 3D). These differences between virions and HA glycoproteins may reflect the glycopeptides of NA glycoproteins present in virions. When either USSR or FWl virions labeled with sugar precursors were analyzed by SDS-polyacrylamide gel electrophoresis without prior treatment with trypsin, a small peak which migrates slightly faster than HA was observed, which may represent NA glycoproteins of these viruses. Glycopeptides prepared from this peak mostly eluted in the fractions corresponding to smaller type II glycopeptides, and small amounts of type I glycopeptides were also observed (data not shown). Glycopeptides H,N, serotype.

of influenza

A viruses

with

Two strains, Japl305 and RI/5+, were studied. The elution profiles of glycopeptides from purified virions labeled with various sugar precursors indicate that both of these two strains have type I glycopeptides as major oligosaccharide components, and the sizes of the glycopeptides are similar to those of WSN virions. In addition, the elution profiles of [3H]mannose-labeled glycopeptides from these strains suggest the presence of small amounts of type II glycopeptides in the glycoproteins of these viruses (data not shown). Figure 5 shows the gel filtration patterns of glycopeptides from HA, and HA, isolated from either Jap! 305 or RI/5+ virions labeled with [“HImannose. The results indicate that oligosaccharide moieties of HA, as well as HA* of Japl305 virus predominantly consist of type I glycopeptides, although type II may be present as minor components. The glycopeptides from HA, and HA, of RI/5+ virus eluted as a quite homogeneous peak in fractions coincident with type I glycopeptides, and type II glycopeptides do not appear to be present in these glycoproteins. Glycopeptides of injluenxa A viruses with H3N2 serotype. Two strains, Victoria and Aichil2, were analyzed. The elution profiles

CARBOHYDRATES

OF INFLUENZA

A VIRUSES

17

tween the two strains. On the other hand, only type I glycopeptides were clearly resolved with HA2 isolated from either of the two strains. Glycopeptides

FIG. 5. Glycopeptides associated with HA, and HA, isolated from either influenza A/Jap/305/5’7 or A/RI/ 5+/57 virus. HA, and HA2 were isolated from either Jap/305 or RI 5+ virus grown in MDCK cells labeled with lSH]mannose. The glycoproteins were digested with Pronase and applied to a column of Bio-Gel P-6. (A) Glycopeptides from HA, of Jap/305; (B) glycopep tides from HA, of Japi305; (C) glycopeptides from HA, of RI 5+; (D) glycopeptides from HA2 of RI 5+.

of glycopeptides from Victoria virions labeled with [3H]glucosamine, [3H]galactose, or [3H]mannose (Figs. 6A, B, and C) indicate that oligosaccharides of the virions are composed of both type I and type II glycopeptides and that two distinct sizesof type II glycopeptides were resolved, as was observed with USSR and FWl viruses. Further, the relative amounts of type II glycopeptides appeared to be higher than in any other influenza A viruses tested. The results shown in Figs. 6D, E, and F indicate that Aichi/2 virions also contain both type I and type II glycopeptides as their oligosaccharide components. However, type II glycopeptides of this virus appear to consist mostly of glycopeptides of sizescorresponding to the larger of the two type II glycopeptide classes observed with Victoria virus. Figure 7 shows the elution profiles of [3H]mannose-labeled glycopeptides prepared from HA, and HA, isolated from either Victoria or Aichi/2 virus. It was apparent that both type I and type II glycopeptides are present in HA, isolated from these two strains, although the ratio of type I to type II glycopeptides was distinct be-

of swine injhtma

virus.

Figures 8A, B, and C show the elution profiles of glycopeptides from swine influenza virions (1976 strain) labeled with various sugar precursors. The results indicate that swine virus contains type I glycopeptides as its major oligosaccharide components and type II as minor components, both of which had sizes indistinguishable from those of WSN virus. The elution profiles of glycopeptides from HA, and HA, of swine virus labeled with [3H]mannose (Figs. 8D and E) indicate that both of the glycoproteins possesstype I glycopeptides as major components. It also appears that small amounts of type II glycopeptides are associated with the glycoproteins. DISCUSSION

It has been previously observed that the electrophoretic mobility of HA polypeptides varies with the host cell type (Haslam et al., 1970; Compans et al., 1970; Schulze, 1970; Schwarz et al., 1977; Nakamura and Compans, 1978b), suggesting that the host cell affects the carbohydrate content of influenza viral glycoproteins. Further, Schwarz and co-workers (1977) and our laboratory (Nakamura and Compans, 1978b) have found host-dependent differences in the sizes of influenza viral glycopeptides. The present observation that the sizesof type I and type II glycopeptides were indistinguishable among all influenza A strains grown in MDCK cells suggests that the sizes of carbohydrate side chains are primarily determined by the host cell. The present studies demonstrated that HA, of WSN virus grown in CEF cells contains both type I and type II glycopeptides, while the same glycoprotein lacks type II glycopeptides when virus is grown in MDBK cells. Similarly, Burke and Keegstra (1976) observed that glycoprotein El from Sindbis virus grown in BHK cells lacks a mannose-rich glycopeptide which is found on the glycoprotein from virus grown in CEF cells. These observa-

18

FIG. 6. Glycopeptides from influenza A virions of the H,N, serotype. Influenza A/Victoria/3/75 and A/Aichi/2/68 virus were grown in MDCK cells labeled with either [3H]glucosamine, [3H]galactose, or [3H]mannose. The glycopeptides obtained after Pronase digestion of purified virions were cochromatographed with [*4C]glucosamine-labeled gycopeptides from WSN virions grown in MDBK cells. (A) [3H]Glucosamine-labeled glycopeptides from Victoria; (B) [3H]galactose-labeled glycopeptides from Victoria; (C) [3H]mannose-labeled glycopeptides from Victoria; (D) [3H]glucosamine-labeled glycopeptides from Aichi/2; (E) [3H]galactose-labeled glycopeptides from Aichi/2; (F) 13H]mannose-labeled glycopeptides from AichiLZ.

tions indicate that whether a complex or a high-mannose type of carbohydrate side chain is linked to a particular glycoprotein can be influenced by the host cell. Important information about the basis for host-dependent differences in the types of oligosaccharide chains associated with viral glycoproteins may be obtained from further studies on the glycosylation sites in HA, molecules of WSN virus grown in CEF cells. Although both type I and type II glycopeptides were found on HA, of WSN virus grown in CEF cells, this does not necessarily indicate that each HA, molecule has both types of oligosaccharide chains. From previous results (Nakamura and Compans, 1978a, b) it can be estimated that an HA molecule of the WSN strain grown in CEF cells contains a carbohydrate moiety of about

8500-9000 daltons. The distribution of [3H]galactose into HA, and HA, suggests that HA, contains about three times more type I glycopeptide molecules than HA, (see Table 1). Further, the molecular weights of type I and type II glycopeptides of WSN virions grown in CEF cells have been estimated to be 2500 and 1650-2100 daltons, respectively (Nakamura and Compans, 1978b). Thus, if each HA, molecule contained both a type I and a type II glycopeptide, the total molecular weight of oligosaccharide chains contained in HA would exceed the expected molecular weight, 8500-9000. It therefore appears more likely that type I and type II glycopeptides are alternative structures associated with distinct HA2 molecules. It will be of interest to determine whether type I

CARBOHYDRATES

OF INFLUENZA

FIG. ‘7. Glycopeptides from HA, and HA, isolated from influenza A viruses of the H3N2 serotype. HA, and HA, were isolated from either influenza A/Victoria/3/75 or A/Aichi/2/63 virions grown in MDCK cells labeled with rH]mannose. After digestion of isolated glycoproteins with Pronase, the resultant glycopeptides were applied to a column of Bio-Gel P-6. (A) Glycopeptides from HA, of Victoria; (B) glycopeptides from HA, of Victoria; (C) glycopeptides from HA, of Aichi/2; (D) glycopeptides from HA, of Aichi/2.

and type II oligosaccharide chains in HA, of the WSN strain grown in CEF cells are linked to the same or to different positions along the polypeptide backbone. Although no significant difference was observed in the size of either type I or type II glycopeptides among the influenza A strains tested, the relative amounts of type I and type II glycopeptides in either virions or isolated HA, and HA, glycoproteins varied with the virus strain, and this variation was evident among strains of the same as well as strains of different antigenic subtypes (summarized in Table 4). It is apparent that much greater variation occurs in oligosaccharides of HA, than HA, and that the variation occurs primarily in the amounts of type II glycopeptides. In contrast, type I glycopeptides were clearly resolved in both HA, and HA, of every virus strain tested. When the nine strains of four major human A subtypes are compared, it appears that a cyclical change has occurred in the abundance of type II glycopeptides, which are less abundant in viruses of H,, and H2 subtypes, and comparatively more abundant in most viruses of the H, and H, subtypes.

A VIRUSES

19

However, further analyses will be required to determine whether these results are generally characteristic of each major subtype. It is of interest to understand how the different influenza A viruses control the glycosylation of their HA polypeptides. Muramatsu and co-workers (1976) have found with human diploid cells that large oligomannosyl cores, which are similar to the high-mannose type of oligosaccharide chains, are predominant in glycopeptides from growing cells, but that in nongrowing cells the relative amount of this type of oligosaccharide decreases, accompanied by an increase in complex oligosaccharides with small oligomannosyl cores. These observations suggest that the variation in relative amounts of type I and type II glycopeptides among influenza A strains could have been due to differences in the physiological conditions of host cells or in the time required for single cycle growth of individual virus strains. However, these possibilities appear unlikely, since no significant difference has been observed in elution profiles of WSN glycopeptides between viruses grown in sparse and dense cultures, or between viruses harvested at early and late times after infection (data not shown). The most likely explanation for the difference in the ratio of type I and type II glycopeptides among influenza A viruses may be that the amino acid sequence of the HA polypeptides specifies whether a type I or a type II oligosaccharide is attached at a given position along the polypeptide backbone (Nakamura and Compans, 19’7813).Two distinct kinds of antigenic variation occur in influenza A viruses (Webster and Laver, 1971). One has been termed a major antigenic shift, which is believed to occur by recombination between a human and an animal strain. The antigenicity of the HA glycoprotein is totally unrelated among strains with distinct HA subtypes, which arise from such major shifts. The second type of variation has been termed antigenic drift, and appears to be due to gradual mutational changes causing minor variation in amino acid sequence in HA among strains of the same subtype. The differences in oligosaccharide chains among

NAKAMURA

20

AND COMPANS

FRACTION

20

32

‘IO 32 FRACTION

60

70

80

NO

90

NO

FIG. 8. Glycopeptides of influenza A/swine/1976/31 virus. Virus was grown in MDCK cells labeled with either [3H]glucosamine (A), [3H]galactose (B), or [3H]mannose (C). Purified virions were mixed with WSN virions grown in MDBK cells labeled with [14C]glucosamine. The mixture was extensively digested with Pronase, and the resultant glycopeptides were analyzed on Bio-Gel P-6. In addition, HA, and HA, were isolated from purified virions labeled with [3H]mannose, and the glycopeptides prepared from HA, (D) or HA, (E) were analyzed on Bio-Gel P-6.

influenza A strains demonstrated in the present report indicate that not only major antigenic shifts but also antigenic drift cause detectable changes in carbohydrate side chains of influenza A viruses. Notable differences in the pattern of glycopeptides were observed among viruses with the H 1 antigenic subtype. Recently, Nakajima and co-workers (1978) have demonstrated that A/USSR/90/‘7’7 virus is genetically more closely related to AlFW/1/50 virus than A/FM/l/47 virus. Our data indicated that oligosaccharide chains from HA, as well as HA, were markedly different between USSR and FM1 viruses. In contrast, no difference in oligosaccharide chains associated with HA, was detectable between USSA and FWl viruses, and a difference was found only in the relative amount of type II glycopeptides in HA, of these vi-

ruses. The fact that USSR virus is more closely related in oligosaccharide chains as well as RNAs to FWl virus rather than FM1 virus supports the idea that viral genetic information, reflected in the primary structure of HA polypeptides, may specify the oligosaccharide types that are linked to the polypeptides. Further, the significant difference in the amount of type II glycopeptides in HA, between USSR and FWl viruses may suggest that glycosylation of HA polypeptides can also be influenced by minor genetic variation, as can be observed between these two viruses (Nakajima et al., 1978). Two possible mechanisms may be considered by which changes in amino acid sequence may cause variation in the relative amounts of type I and type II glycopeptides associated with the hemagglutinin. One is a

CARBOHYDRATES

OF INFLUENZA TABLE

SUMMARY

A VIRUSES

21

4

OF OLIGOSACCHARIDE TYPES ASSOCIATED WITH HA, AND OF INFLUENZA A VIRUSES GROWN IN MDCK CELLS

HA,

Oligosaccharide types associated with glycoproteins” HA, Serotype

Virus strain

HA,

I”

IIa”

IIbd

I

IIa

IIb

HP,

A/WSN APRi3l34

++ ++

++ -

-

++ ++

-

-

H,N,

AlUSSR/99/‘77 AlFW/1/50 A/FM/l/47

++ ++ ++

i-f ++ +

++ ++ +

++ ++ ++

+p + -

-

HA

Al.Iapl305l57 AlRI/5+157

++ ++

-

-

++ ++

-

-

HA

AlVictorial3l75 AlAichil2!63

++ ++

++ ++

++ +

++ ++

-

-

%N,

A/swine/1976/31

++

-

++

-

-

+

a The amounts of each glycopeptide type in HA, or HA, were scored on the basis of elution profiles of [3H]mannose-labeled glycopeptides from isolated glycoprotein. (+ +) Clearly resolved as a peak. (+) Not clearly resolved but demonstrable as a shoulder. (-) Not detected, but may be present in a trace amount. b Type I glycopeptides. c Larger type II glycopeptides. d Smaller type II glycopeptides. e The presence of small amounts of this type of glycopeptide was confirmed by treatment of glycopeptides with endo-H.

change in the number of glycosylation sites. A mutational change may introduce a new glycosylation site or remove a previously existing glycosylation site in the HA glycoprotein. It was found that the molecular weight of the HA glycoprotein of FM1 virus was significantly smaller than that of either USSR or FWl virus, which suggests a lower carbohydrate content in FM1 than in the other two viruses. Further, the oligosaccharides of the HA glycoprotein of FM1 virus predominantly consisted of type I glycopeptides, which are larger than type II, while the glycoprotein of USSR or FWl virus contains a high amount of type II glycopeptides in addition to type I. Therefore, HA polypeptides of FM1 virus may contain fewer glycosylation sites than those of either USSR or FWl virus. The second possibility is that a change in the primary structure of HA may cause a change in the oligo-

saccharide type which is linked to a particular glycosylation site. If this mechanism is involved, it seems likely that the type of oligosaccharide chain is determined in subsequent processing steps rather than the initial transfer of oligomannosyl cores from lipid-linked intermediates to a glycosylation site, since evidence has been obtained that type II glycopeptides are structurally related to type I glycopeptides, and the former are an immature form of the latter (Sefton, 19’7’7; Robbins et al., 1977; Nakamura and Compans, 1979). In addition, evidence has been obtained that some mannose residues are removed during maturation of the precursor to type I oligosaccharide chains on HA (Nakamura and Compans, 1979), as has been observed with vesicular stomatitis and sindbis viruses (Hunt et al., 1978; Robbins et al., 1977; Tabas et al., 1978). Such a trimming process might be affected by changes

22

NAKAMURA

in the amino acid sequence of HA polypeptides, possibly resulting in variation in the type of oligosaccharide that is linked to a particular glycosylation site. No significant difference was detected in the sizes of HA, glycoproteins between two members of the H, antigenic subtype, Victoria and Aichil2 viruses, although HA, of Victoria virus contains more type II glycopeptides than Aichil2 virus. Such variation might occur through the second mechanism described above. ACKNOWLEDGMENTS This research was supported by Grant No. AI 12680 from the National Institute of Allergy and Infectious diseases, and Grant No. PCM78-09207 from the National Science Foundation. REFERENCES ADA, G. L., and GOTTSCHALK, A. (1956). The component sugars of the influenza-virus particle. &o&em. J. 62, 686-689. BURKE, D. J., and KEEGSTRA, K. (1976). Purification and composition of the proteins from sindbis virus grown in chick and BHKcells. J. Viral. 20,676-686. CHOPPIN, P. W. (1969). Replication of influenza virus in a continuous cell line: High yield of infective virus from cells inoculated at high multiplicity. Virology 38, 130-134. COLLINS, J. K., and KNIGHT, C. A. (1978). Purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components. J. Viral.

26, 457-567.

COMPANS, R. W., KLENK, H.-D., CALIGUIRI, L. A., and CHOPPIN, P. W. (1970). Influenza virus proteins. I. Analysis of polypeptides of the virion and identification of spike glycoproteins. Vi’irology 42,880-889. COMPANS, R. W. (1973). Distinct carbohydrate components of influenza virus glycoproteins in smooth and rough cytoplasmic membranes. Virology 55, 541-545. COMPANS, R. W., and CHOPPIN, P. W. (1975). Reproduction of myxoviruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, eds.), pp. 179-252. Plenum Press, New York. FROMMHAGEN, L. H., KNIGHT, C. A., and FREEMAN, N. K. (1959). The ribonucleic acid, lipid and polysaccharide constituents of influenza virus preparations. Virology 8, 176-197. HASLAM, E. A., HAMPSON, A. W., RADISKEVICS, I., and WHITE, D. 0. (1970). The polypeptides of influenza virus III. Identification of the hemagglutinin, neuraminidase, and nucleocapsid proteins. Virology 42, 566-575.

AND COMPANS L. A., ETCHINSON, J. R., and SUMMERS, D. F. (1978). Oligosaccharide chains are trimmed during synthesis of the envelope glycoprotein of vesicular stomatitis virus. Free. Nat. Acad. Sci. USA 75, 754-758. LANDSBERGER, F. R., LENARD, J., PAXTON, J., and COMPANS, R. W. (1971). Spin label ESR study of the lipid-containing membrane of influenza virus. HUNT,

Proe.

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Acad.

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68, 2579-2583.

LAVER, W. G. (1971). Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus. Virology 45, 275-288. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 115-125. MURAMATSU, T., KOIDE, N., CECCARINI, C., and ATKINSON, P. H. (1976). Characterization of mannose-labeled glycopeptides from human diploid cells and their growth-dependent alterations. J. Biol. Chem. 251, 4673-4679. NAKAJIMA, K., DESSELBERGER, U., and PALESE, P. (1978). Recent human influenza A (H,N,) viruses are closely related genetically to strains isolated in 1950. Nature (London) 274, 334-339. NAKAMURA, K., and COMPANS, R. W. (1977). The cellular site of sulfation of influenza virus glycoproteins. Virology

79, 381-392.

NAKAMURA, K., and COMPANS, R. W. (1978a). Effects of glucosamine, 2-deoxy-D-glucose and tunicamycin on glycosylation, sulfation and assembly of influenza virus glycoproteins. Virology 84, 303-319. NAKAMURA, K., and COMPANS, R. W. (1978b). Glycopeptide components of influenza viral glycoproteins. Virology 86, 432-442. NAKAMURA, K., and COMPANS, R. W. (1979). Biosynthesis of the oligosaccharides of influenza viral glycoproteins. Virology 93, 31-47. ROBBINS, P., HUBBARD, S. C., TURCO, S. J., and WIRTH, D. F. (1977). Proposal for a common oligosaccharide intermediate in the synthesis of glycoproteins. Cell 12, 893-900. ROTT, R., and KLENK, H.-D. (1977). Structure and assembly of viral envelopes. Zn “Virus Infection and the Cell Surface”; (G. Poste 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 ofinfluenza virus. I. Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars. J. Viral. 23, 217-226. SEFTON, F. M. (1977). Immediate glycosylation of sindhis virus membrane proteins. Cell 10, 659-668.

CARBOHYDRATES

OF INFLUENZA

TABAS, I., SCHLESINGER, S., and KORNFELD, S. (19’78). Processing of high mannose oligosaccharides to form complex type oligosaccharides on the newly synthesized polypeptides of the vesicular stomatitis virus G protein and the IgG heavy chain. J. Biol. Chem. 352, 716-722. TARENTINO, A. L., and MALEY, F. (1974). Purification and properties of an endo-P-N-acetylglucosaminidase from streptomyces griseus. J. Biol. Chem. 249, 811-817. TOBITA, K. (1975). Permanent canine kidney (MDCK)

A VIRUSES

23

for isolation and plaque assay of influenza B viruses. Microbial. Immunol. 162, 23-27.

Med.

TOBITA, K., SUGIURA, A., ENOMOTO, C., and FURUYAMA, M. (19’75). Plaque assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. Med. Microbial. Immunol. 162, 9-14. WEBSTER, R. G., and LAVER, W. G. (1971). Antigenic variation in influenza virus. Progr. Med. Viral. 13, 271-333.