Vol. 27, ]No. 1
JOURNAL OF VIROLOGY, July 1978, p. 164-171 0022-538X/78/0027-0164$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.r.S.A.
Removal of Carbohydrate from Influenza A Virus and Its Hemagglutinin and the Effect on Biological Activities JAMES K. COLLINSt* AND C. A. KNIGHT Department of Molecular Biology, University of California, Berkeley, California 94720 Received for publication 25 November 1977
Treatment of influenza virus and its purified hemagglutinin with glycosidases from Diplococcus pneumoniae, which included ,B-galactosidase, f8-N-acetylglucosaminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were heterogeneous in size, whereas the released fucose-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.
Influenza virus is an enveloped virus which contains glycoprotein spikes projecting from the viral surface. Hemagglutinin (HA) is the predominant spike (13, 26) and is composed of glycosylated protein subunits which may be present in uncleaved form (HA) with a molecular weight of about 75,000 or in cleaved form, HA1 and HA2, with molecular weights of about 50,000 and 23,000, respectively. HA1 and HA2 are held together by disulfide bonds (12). The HA spike contains the binding sites for attachment of the virus to host cell neuraminic acidcontaining receptors in the process of infection and to erythrocytes in hemagglutination (25, 26). The HA also constitutes the major surface antigen (14, 24), and it can stimulate antibody which will neutralize infectivity. The biological importance of carbohydrate of the influenza virus and the HA has been studied by using inhibitors of glycosylation during synthesis of the virus. Inhibition with glucosamine or 2-deoxyglucose leads to the production of HA in an unglycosylated form (8, 10, 27, 30). Cells in which the unglycosylated or partially glycosylated HA is synthesized do not hemadsorb (10), and the HA does not become incorporated into virus particles (10, 27, 30). Tunicamycin, an inhibitor which prevents transfer of the core region of oligosaccharide chains to protein, also prevents physical particle formation of influenza virus (28). These inhibitors severely limit virus production and may also interfere with viral
synthesis in nonspecific ways (1, 20, 23). The use of specific glycosidases to remove carbohydrates (16, 19, 20) provides another approach to analyzing the function as well as the structure of these viral components. It has been reported from this laboratory (2) that treatment of influenza PR8 virus with a glycosidase preparation from Aspergillus niger resulted in partial degradation of the HA component with a concomitant loss of hemagglutinating capacity. This was attributed to removal of carbohydrate from the HA. However, difficulties encountered by using the acid conditions required for the enzymes as well as the finding of proteolytic activity in the enzyme preparations which degraded HA, (J. K. Collins, Ph.D. thesis, University of California, Berkeley, 1977) prompted the use of different glycosidase enzymes which were protease free. In the present study it was found that free amino sugar, neutral sugar, and large oligosaccharides could be removed from virus or from isolated HA by treatment with glycosidases from Diplococcus pneumoniae. Release of 50% of the HA carbohydrates did not significantly affect hemagglutinating activity. MATERIALS AND METHODS Cells and virus. Influenza A/PR8 was grown and labeled, using Madin-Darby canine kidney cells, and purified from infected culture fluids as described previously (4). Hemagglutination assay. HA was titrated by combining 0.1 ml of serial twofold dilutions of virus in 0.85% saline (in microtiter dishes) with 0.1 ml of 0.5%
t Present address: Rocky Mountain Laboratory, Hamilton, MT 59840.
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chicken erythrocytes. After 1 h at 4°C, the end point was estimated visually, and the reciprocal of the end point dilution was defined as the titer. Polyacrylamide gel electrophoresis. Slab polyacrylamide gel electrophoresis was performed with a 1-cm 3% stacking gel and a 10-cm 10% resolving gel as described previously (4). Gel filtration of carbohydrates released from virus or HA. Samples of virus or HA treated with glycosidases were clarified by centrifugation and chromatographed on a Bio-Gel P-6 (100 to 200 mesh) column (1.2 by 110 cm) equilibrated with 0.1 M ammonium acetate. Fractions (1.1 ml) were collected at a flow rate of 5.5 ml/h. Fractions were analyzed for radioactivity by transferring each to a scintillation vial, evaporating it until dry at 60°C, and then adding 7.5 ml of 3a70B scintillation fluid (Research Products International) and counting it in an Intertechnique scintillation counter. Oligosaccharides used as markers were from fetuin and ovalbumin and were prepared as described previously (4). Preparation of glycosidases. D. pneumoniae type I was obtained through the courtesy of C. E. Schwerdt from the Department of Medical Microbiology, Stanford University, Stanford, Calif. (culture collection no. H-19). The organism was maintained with biweekly subculturing in brain heart infusion broth (Difco). For preparation of glycosidases, 2 liters of broth was inoculated with 1 ml of a previous culture and incubated at 37°C for 30 h. Assay for the enzymes 4l-galactosidase and ,B-N-acetylglucosaminidase was as described by Koide and Muramatsu (11). A glycosidase preparation containing these enzymes and endoglycosidase D was prepared by ammonium sulfate precipitation from the culture fluid and Sephadex G-100 gel filtration (11, 17). The glycosidase preparation contained 1.3 mg of protein per ml and 3.4 U of,-galactosidase and 94 U off,-N-acetylglucosaminidase per ml when assayed in 0.02 M Tris-maleate buffer (pH 6.8). No proteolytic activity could be detected by a casein substrate assay. Purification of HA. The HA spike glycoprotein was purified after disruption of the virus with 1% sodium deoxycholate and extraction with tri-n-butyl phosphate according to Collins and Knight (4). Treatment with glycosidases. Virus or HA was treated with glycosidase enzymes by dilution to 0.05 to 0.2 mg/ml in a final volume of 2 ml in 0.2 M Trismaleate buffer (pH 6.8) and addition of 10 ,pl of glycosidase enzymes. Incubation at 37°C was carried out for 24 h, with a second 10-ul enzyme addition at 10 to 12 h. To terminate the reaction, samples containing the virus were pelleted by centrifugation at 80,000 x g for 20 min and then suspended in gel electrophoresis buffer for gel analysis. Samples were removed at various times for hemagglutination tests or infectivity tests by plaque assay for whole virus. Digestion of HA samples was terminated by addition of 5 volumes of cold ethanol, and the precipitated protein was analyzed by gel electrophoresis. lodination. HA was iodinated by the procedure of Hunter (6). The glycoprotein was concentrated to 1 mg/ml and dialyzed against 0.2 M phosphate buffer (pH 7.5) before 50 ,L of the glycoprotein solution was reacted with 0.2 mCi of Na'25I in the presence of 50
165
,ug of chloramine-T. Immediately after adding the reactants, the reaction was terminated by adding 240 Ig of potassium metabisulfite in 840 ,ul and 100 ,ug of potassium iodide in 100 ul. The entire mixture was put through a Sephadex G-10 column (10 by 0.6 cm) and eluted with 0.1 M phosphate buffer (pH 7.5). Fractions (1 ml) were collected. The labeled HA eluted in fractions 3 to 4, and these were pooled. HA prepared in this manner was still active and contained 1 x 105 to 1.5 x 105 cpm/mg of protein. Quantitation of neutral and amino sugar released from virus and HA. After treatment with glycosidases, virus was pelleted by centrifugation at 80,000 x g for 20 min. The supernatant fluid, or the entire digestion mixture when purified HA was treated with glycosidases, was deproteinated according to Spiro (29) by passage over a colunu (0.6 by 2.0 cm) of charcoal-Celite (1:1). The effluent and a 2-ml wash with 10% ethanol were then passed directly onto coupled ion exchange columns (0.6 by 1 cm) of AG50X2(H+) and AG1-X8(HCOO-). The columns were washed with 50% methanol, from which the neutral sugars were recovered, and assayed with the ParkJohnson ferricyanide reducing test according to Spiro (29). The AG50 column quantitatively bound amino sugar, and this was recovered by elution with 2 N HCl and was assayed by the Park-Johnson method. Parallel samples containing enzymes alone and virus or HA alone were carried through the procedure, and the amount of sugars found after incubation was negligible.
RESULTS Effect of glycosidases on virus and purified HA. Influenza virus was treated with the glycosidase mixture from D. pneumoniae, pelleted by centrifugation to separate the particles from the enzymes, and then analyzed by gel electrophoresis. The HA, of the treated virus was found to be more heterogeneous in size (Fig. 1, track 1) than the HA1 of the untreated virus (track 2). The HA1 band spread further in the gel, indicating a decrease in size, whereas the HA2 appeared to remain unaffected. Glycosidase treatment of the pure HA also caused a decrease in size of the glycoprotein. When the HA was treated with glycosidases and analyzed by sodium dodecyl sulfate gel electrophoresis under nonreducing conditions, the glycoprotein spread further down the gel (Fig. 1, track 3). Analysis of the treated HA under reducing conditions, which separate HA1 and HA2, was difficult due to glycosidase bands which migrated to the same region as HA1, as shown in Coomassie brilliant blue-stained gels (Fig. 2). Therefore, the purified HA was radiolabeled with 1251, treated with glycosidases, and analyzed together with the untreated ['"I]HA on the same slab polyacrylamide gel. Differences were found in both HA, and HA2 after glycosidase digestion (Fig. 2). These data corresponded with the finding of released carbohydrates (see below) and sug-
166
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COLLINS AND KNIGHT
gested that both of the HA glycoproteins were degraded by the glycosidases. Release of carbohydrates from virus and purified HA. Treatment of purified virus with glycosidases from D. pneumoniae was found to release neutral and amino sugars. The enzymes released 1 to 1.4 ,ug of neutral sugar and 0.4 to 0.5 ,g of amino sugar per 100 ,ug of virus after 24 h of incubation. The amount of carbohydrate released corresponded to approximately 20 to 25% of the total carbohydrates of the virus, using 4
_p
pi. M_IM
NP NAs
HA,-
HA2----
FIG. 1. SDS-polyacrylamide gel electrophoresis of influenza A/PR8 and its purified HA after treatment with glycosidases. Virus or purified HA was treated for 24 h with glycosidases from D. pneumoniae. Tracks: 1, virus treated with glycosidases; 2, untreated virus; 3, HA treated with glycosidases; 4, untreated HA. HA samples were run under nonreducing conditions so that the HA would migrate as one glycoprotein. Gels were stained with Coomassie brilliant blue.
a value of 7.2 ± 0.9 ,ug of total carbohydrate per 100 ,tg of virus (4). To deterrnine which carbohydrates were released, virus which had been labeled with [3H]galactose, [3H]fucose, or [3H]glucosamine was incubated with the enzymes. These labels remain in the same chemical form upon incorporation into virus, except for galactose, of which only 40 to 50% can be recovered as carbohydrate in the virus (4). After digestion with the glycosidase mixture, the virus was subjected to BioGel P-6 gel filtration to determine the size and amount of the released labeled carbohydrate (Fig. 3). Virus labeled with [3H]galactose or [3H]glucosamine and treated with glycosidases showed release of labeled monosaccharides. No labeled monosaccharides could be detected from fucose-labeled virus. Label released from [3H]galactose-labeled virus was identified by thin-layer chromatography as consisting solely of galactose. The amount of each sugar released is shown in Table 1. Monosaccharides and oligosaccharides were also released from the purified HA. Monosaccharides were assayed after separation into neutral and amino sugars and included 1.5 to 2 ,g of neutral sugar and 0.5 to 1.1 jig of amino sugar per 100 ,ug of HA. Release of both [3H]glucosamine and a large, heterogeneous [3H]glucosamine-labeled oligosaccharide was found after glycosidase treatment of [3H]glucosamine-labeled HA (Fig. 4a). Approximately 57% of all the glucosamine label was released, as judged by material which eluted in the included volume of the column. Gel filtration of [3H]fucose-labeled HA which had been treated with glycosidases (Fig. 4b) indicated that the large oligosaccharides that were released by the glycosidase treatment also contained fucose. Approximately 42% of all the fucose label could be released from the HA (Table 1). However, no free fucose was released, which was similar to the result obtained after treatment of whole virions with glycosidases. The elution profile of the fucosecontaining oligosaccharides was markedly different from the profile of the glucosamine-containing oligosaccharides. This suggested heterogeneity in the released glucosamine-containing oligosaccharide chains. From the P-6 gel filtration of glycosidasetreated HA, it was apparent that only approximately half of the total glucosamine or fucose label was released. The material appearing at the void volume of the column represented carbohydrate not released by the enzymes. The glucosamine label left could represent the innermost sugar linked to asparagine, which would be expected to remain if endoglycosidase D cleaved
REMOVAL OF INFLUENZA VIRUS CARBOHYDRATE
VOL. 27, 1978
167
I-0 m
20(
'I
0
x
E
E 15
I-I
v
L
CN '-l
10. 5
LA IN
5.
FRACTI ON NUMBER SDS-gel electrophoresis of [I251]HA treated with D. pneumoniae glycosidases run under conditions
FIG. 2. to separate HAI and HA2. Iodinated HA (3 x 106 to 6 x 106 cpm) was treated with glycosidases for 24 h (0), and an untreated sample (0) was run adjacent to the treated sample. After fixation in acid, the gel was sliced horizontally, and each slice was cut vertically to separate the two tracks. Each slice was then eluted and counted in a scintillation counter. The upperpanel shows a Coomassie brilliant blue-stained gel ofglycosidasetreated (top) and untreated (bottom) HA.
the oligosaccharide chains. However, it was conditions except without enzymes (Fig. 6). In found that there was glucosamine label which contrast, Pronase-digested virus showed a rapid remained as part of a large chain, since Pronase loss of infectivity. digestion of the glucosamine-labeled material in DISCUSSION the void-volume fractions left large oligosacchaof Removal up to 50% of the carbohydrate rides detectable on P-6 gel filtration (data not shown). Therefore, it appeared that oligosaccha- from the purified HA of influenza virus could be rides which were resistant to endoglycosidase D accomplished with a mixture of glycosidases from D. pneumoniae which included ,8-galactoor to combined endoglycosidase D, ,8-galactosidsidase, f)-N-acetylglucosaminidase, and endoase, and f?-N-acetylglucosaminidase may have glycosidase D. When HA was treated, heterobeen present on the HA. Effect on biological activities. The effect geneous oligosaccharides .containing glucosaof removal of carbohydrate on the biological mine and fucose as well as monosaccharides activities of the virus and the HA was measured were released from both HA1 and HA2. The after treatment with the D. pneumoniae en- monosaccharides released included glucosamine and neutral sugar. zymes. Both HA and virus retained full hemagThe release of large, glucosamine-containing glutinating activity during incubation at 37°C with or without glycosidase treatment but rap- oligosaccharides indicated that endoglycosidase idly lost HA activity if Pronase was added in- D may have cleaved carbohydrate chains. The stead of glycosidase enzymes (Fig. 5). The HA released oligosaccharides containing glucosatests were carried out on the same samples mine were heterogeneous in size; however, the which were used for gel electrophoresis or ana- fucose-containing oligosaccharides released had lyzed for release of carbohydrate to insure that a much more limited size distribution. Fucose the tests were made on material from which may have been present only on oligosaccharides of a discrete size class such that when these were carbohydrates had been cleaved. Plaque assays on samples of virus treated with released only a single peak was detected by gel the enzymes showed infectivities similar to that filtration. Glucosamine, on the other hand, could of the control, which was treated under identical have been in chains of varying length, including
168
COLLINS AND KNIGHT 25
monosaccharides from the ends of the large released chains. Incomplete removal of multiple galactose or glucosamine termini could produce heterogeneous chains. The limited size distribution of fucose-containing chains suggested that these may not have been as susceptible to this type of degradation. The presence of terminal fucose linked to galactose (4) may have prevented degradation by the exoglycosidases.
Vt
VOV
Vf
(a) V0
J. VIROL.
420 1
15-
64-
TABLE 1. Amount of labeled monosaccharide released from influenza virus and its HA by D. pneumoniae glycosidases
2 CM N
x
0
10
(b)
% of total label released by enzymes Virus HA
Label
E 0. 8
[3H]glucosamine
26
57
[3H]galactose 22 0 [3H]fucose a ND, Not determined.
6in
4
NDa 42
2 0
8-
-
c
642-
0. 10
20
30
40
50
60
70
Fraction number FIG. 3. Bio-Gel P-6 filtration of influenza A/PR8 after treatment with glycosidases. Purified labeled virus was treated for 24 h with glycosidases from D. pneumoniae and then run on a Bio-Gel P-6 column. The treated virus preparations were labeled with [:3H]glucosamine (a), [3H]galactose (b), or [3H]fucose (c). Markers included the fetuin (Vf) and ovalbumin (V,,,) oligosaccharides. The included volume and void volume are designated V, and V., respectively.
small, high-mannose-containing oligosaccharides. If endoglycosidase D was responsible for removing the large oligosaccharides found after treatment with mixed glycosidases, then this would suggest that some of the chains contain the di-N-acetylchitobiose linkage at the core region of the oligosaccharide, since this enzyme cleaves this linkage specifically (7). Heterogeneity in the released oligosaccharides may also have been generated during incubation with the glycosidase mixture. The specific exoglycosidases, ,B-galactosidase and ,f-N-acetylglucosaminidase, may have cleaved their respective
10
20
30
40
50
60
70
Fraction number FIG. 4. Bio-Gel P-6 filtration of the influenza A/PR8 HA after treatment with glycosidases. Purified labeled viral HA was treated for 24 h with glycosidases from D. pneumoniae and then run on a Bio-Gel P-6 column. The treated HA preparations were labeled with [3HJglucosamine (a) and [1H]fucose (b). Markers included the fetuin (Vf) and ovalbumin (V.,,) oligosaccharides and stachyose (V1). The included volume and void volume are designated V, and V.,, respectively.
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REMOVAL OF INFLUENZA VIRUS CARBOHYDRATE
169
1 E
x
a-
D 0.1 I 10
-- (b) w
I
w
w
:
0.1-
10 )
30 Incubation, hours
10
20
40
FIG. 5. Effect of glycosidase treatment on the hemagglutinating activity of influenza virus and its HA. Virus (a) or HA (b) was incubated at 37°C with (0) or without (0) glycosidases from D. pneumoniae or with Pronase (A), and samples were removed at various times for hemagglutination assay.
Approximately 20 to 25% of the carbohydrates intact virus could be released. These were released as galactose and glucosamine. The galactose and glucosamine which were not released could be located at internal positions or on oligosaccharides inaccessible to the enzymes. Since large oligosaccharides were not released from the virus, whereas they were readily found after treatment of the HA, the virion structure may have prevented the enzymes from acting on the carbohydrate chains. This is supported by the fact that the HA2 on the virus was not altered by glycosidases, in contrast to the HA2 when pure HA was digested. This may reflect a lack of accessibility of the HA2 carbohydrates on the intact virus, compared with those on purified soluble HA. Oligosaccharides which were not released from the virus or HA may also be resistant to endoglycolytic cleavage. Similar resistant oligosaccharides have been found on vesicular stomatitis virus (19) and Sindbis virus (16). No change in hemagglutinating activity of the HA or virus was seen after removal of carbohydrate. This suggested that the carbohydrates on the
20
30
40
Incubation, hours FIG. 6. Effect ofglycosidase treatment on infectivity of influenza virus. Virus-infected culture fluid was diluted in 0.02 M Tris-maleate buffer, pH 6.8, and incubated in the presence of glycosidases from D. pneumoniae (A) with no glycosidases (0) or with Pronase (U). For Pronase treatment, the pH was adjusted to 8.0, and 10 jig of Pronase was added at time 0. Samples were removed at various times and titrated by plaque assay. may not have been directly involved in hemagglutination. However, since virus particles are multivalent and contain many HA spikes, the incomplete removal of carbohydrate may reflect a loss of carbohydrate from only some of the spikes. Since it is likely that purified HA also consists of aggregated spikes, a similar explanation could apply. Complete biological activity of the remaining carbohydrate-containing spikes could still allow each aggregate or particle to be fully active. This possibility could not be ruled out, since increased incubation with glycosidases did not release more carbohydrate. Since monosaccharides could be released from HA1 on the intact virus, the effect of their removal on infectivity was assayed. No significant decrease in infectivity occurred over the control untreated virus. The monosaccharides, galactose and glucosamine, which may be located at the nonreducing terminal of oligosaccharide chains may, thus, not be directly required for infectivity. However, if HA spikes were left which had intact carbohydrate, these could possibly allow full infectivity, and if only 50% of the particles
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COLLINS AND KNIGHT
had the critical carbohydrate removed, a corresponding loss in infectivity would be difficult to detect. The inessential nature for some biological functions of at least part of the carbohydrate of influenza virus is at variance with earlier conclusions (2). The Aspergillus glycosidases used previously contained other degradative proteolytic enzymes which acted on the HA (Collins, Ph.D. thesis). In addition, the low pH requirement of these enzymes destroyed some of the HA activity. These two facts make interpretation of previous results very difficult. The possible involvement of carbohydrate in functional properties of other viral glycoproteins has been investigated with several viruses. Removal of sialic acid from vesicular stomatitis virus was reported to reduce infectivity 100-fold and to abolish hemagglutinating activity (21, 22). However, although these results were confirmed in one laboratory (32), they could not be repeated by another (3). If sialic acid is not essential for vesicular stomatitis virus, this result would agree with results of experiments with other enveloped viruses, such as Sindbis virus (31), Semliki Forest virus (9), and Friend leukemia virus (20), since removal of sialic acid from these viruses has no effect on infectivity or attachment to cells. A second approach to defining the role of carbohydrates on viral glycoproteins has been to grow viruses in the presence of inhibitors of glycosylation, such as 2-deoxyglucose and glucosamine (5, 8, 10, 23, 27). Although inhibition of virus glycosylation occurs, these inhibitors are not entirely specific, and they interfere with virus particle formation through depletion of the UTP pool (1), leading to less viral RNA synthesis (23) as well as depletion of the ATP pool. Tunicamycin, an inhibitor which may specifically prevent the transfer of the linkage sugar, glucosamine, and core region of the oligosaccharide chains, has also been used to interfere with the synthesis of several viruses. Production of Sindbis and vesicular stomatitis viruses (15) as well as influenza virus (27) is drastically reduced with this compound, and incompletely glycosylated proteins appear mainly in infected cells. Recently, influenza virus particles showing a markedly reduced specific hemagglutinating activity have been obtained from inhibitor-treated cells (18). Although the relative amount of HA glycoproteins per particle was also reduced on these particles, this suggests that when nearly all of the carbohydrate is absent the protein may not have its biological functions. The use of specific glycosidases in removing carbohydrates may prove to be a fruitful approach in critically dissecting the structures and
J. VIROI,.
functions of carbohydrate residues. Extensive removal of carbohydrates has been carried out with Sindbis virus (16), Friend leukemia virus (20), and vesicular stomatitis virus (19), using the glycosidases from D. pneumoniae. Here we have shown that these enzymes acted on influenza virus carbohydrates under conditions which preserved biological activity. Further work on influenza viral glycoproteins and carbohydrates with these purified glycosidase enzymes should elucidate additional structural features as well as biological roles. ACKNOWLEDGMENTS This work was supported in part by Public Health Service (PHS) research grants AI 00634 from the National Institute of Allergy and Infectious Diseases and CA 14097 from the National Cancer Institute; by PHS training grant GM 01389 from the National Institute of General Medical Sciences; by biomedical sciences support grant FR-7006 from the General Research Support Branch, Division of Research Facilities and Resources, Bureau of Health Professions Education and Manpower Training, National Institutes of Health; and by a grant to J.K.C. from the Chancellor's Patent Fund, University of California. LITERATURE CITED 1. Bekesi, J. G., E. Bekesi, and R. J. Winzler. 1969. Inhibitory effect of D-glucosamine and other sugars on the biosynthesis of protein, ribonucleic acid and deoxyribonucleic acid in normal and neoplastic tissues. J. Biol. Chem. 244:3766-3772. 2. Bikel, I., and C. A. Knight. 1973. Differential action of Aspergillus glycosidases on the hemagglutinating and neuraminidase activities of influenza and Newcastle disease viruses. Virology 49:326-332. 3. Brown, F., and B. Cartwright. 1977. Role of sialic acid in infection with vesicular stomatitis virus. J. Gen. Virol. 35:197-199. 4. Collins, J. K., and C. A. Knight. 1978. Purification of' the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components. 26:457-467. 5. Duda, E., and M. J. Schlesinger. 1975. Alterations in Sindbis viral envelope proteins by treating BHK cells with glucosamine. J. Virol. 15:416-419. 6. Hunter, W. M. 1973. Chapter 17, p. 1-36. In D. W. Weir (ed.), Handbook of experimental immunology, vol. 4. Blackwell Scientific, New York. 7. Ito, S., T. Muramatsu, and A. Kobata. 1975. Release of
galactosyl oligosaccharides by endo-f8-N-acetylglucosaminidase D. Biochem. Biophys. Res. Commun. 63:938-944. 8. Kaluza, G., C. Scholtissek, and R. Rott. 1972. Inhibition of the multiplication of enveloped RNA viruses by glucosamine and 2-deoxy-D-glucose. J. Gen. Virol. 14:251-259. 9. Kennedy, S. L. T. 1974. The effect of enzymes on structural and biological properties of Semliki forest virus. J. Gen. Virol. 23:129-143. 10. Klenk, H.-D., C. Scholtissek, and R. Rott. 1972. Inhibition of glycoprotein biosynthesis of influenza virus by D-glucosamine and 2-deoxy-D-glucose. Virology 49:723-734. 11. Koide, N., and T. Muramatsu. 1974. Endo-,f-N-acetylglucosaminidase acting on carbohydrate moieties of glycoproteins. J. Biol. Chem. 249:4897-4904. 12. Laver, W. G. 1971. Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus. Virology 45:275-288. 13. Laver, W. G. 1973. The polypeptides of influenza virus.
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Adv. Virus Res. 18:57-98. 14. Laver, W. G., and E. D. Kilbourne. 1966. Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30:493-501. 15. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J. Virol. 21:375-385. 16. McCarthy, M., and S. C. Harrison. 1977. Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus. J. Virol. 23:61-73. 17. Muramatsu, T. 1971. Demonstration of an endoglycosidase acting on a glycoprotein. J. Biol. Chem. 246:5535-5537. 18. Nakamura, K., and R. W. Compans. 1978. Effects of glucosamine, 2-deoxyglucose and tunicamycin on glycosylation sulfation, and assembly of influenza viral proteins. Virology 84:303-319. 19. Robertson, J. S., J. R. Etchison, and D. F. Summers. 1976. Glycosylation sites of vesicular stomatitis virus glycoprotein. J. Virol. 19:871-878. 20. Schiifer, W., P. J. Fischinger, J. J. Collins, and D. P. Bolognesi. 1977. Role of carbohydrate in biological functions of Friend murine leukemia virus gp7l. J. Virol. 21:35-40. 21. Schloemer, R. H., and R. R. Wagner. 1974. Sialoglycoprotein of vesicular stomatitis virus: role of the neuraminic acid in infection. J. Virol. 14:270-281. 22. Schloemer, R. H., and R. R. Wagner. 1975. Cellular adsorption function of the sialoglycoprotein of vesicular stomatitis virus and its neuraminic acid. J. Virol.
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15:882-893. 23. Scholtissek, C. 1975. Inhibition of the multiplication of enveloped viruses by glucose derivatives. Curr. Top. Microbiol. Immunol. 70:101-119. 24. Schulman, J. L., and E. D. Kilbourne. 1969. Independent variation in nature of hemagglutinin and neuraminidase antigens of influenza virus. Proc. Natl. Acad. Sci. U.S.A. 63:326-333. 25. Schulze, I. T. 1970. The structure of influenza virus. I. The polypeptides of the virion. Virology 42:890-904. 26. Schulze, I. T. 1972. The structure of the influenza virus. II. A model based on the morphology and composition of subviral particles. Virology 47:181-196. 27. Schwarz, R. T., and H.-D. Klenk. 1974. Inhibition of glycosylation of the influenza virus hemagglutinin. J. Virol. 14:1023-1034. 28. Schwarz, R. T., J. M. Rohrschneider, and M. F. G. Schmidt. 1976. Suppression of glycoprotein formation of Semliki forest, influenza, and avian sarcoma virus by tunicamycin. J. Virol. 19:782-791. 29. Spiro, R. G. 1966. Analysis of sugars found in glycoproteins. Methods Enzymol. 8:3-26. 30. Stanley, P., S. S. Ghandi, and D. 0. White. 1973. The polypeptides of influenza virus. VII. Synthesis of the hemagglutinin. Virology 53:92-106. 31. Stollar, V., B. D. Stollar, R. Koo, K. A. Harrap, and R. W. Schlesinger. 1976. Sialic acid contents of Sindbis virus from vertebrate and mosquito cells. Virology 69:104-115. 32. Wagner, R. R. 1976. Third International Colloquium on Rhabdoviruses. ASM News 42:616-619.
JOURNAL OF VIROLOGY, July 1978, p. 164-171 0022-538X/78/0027-0164$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.r.S.A.
Removal of Carbohydrate from Influenza A Virus and Its Hemagglutinin and the Effect on Biological Activities JAMES K. COLLINSt* AND C. A. KNIGHT Department of Molecular Biology, University of California, Berkeley, California 94720 Received for publication 25 November 1977
Treatment of influenza virus and its purified hemagglutinin with glycosidases from Diplococcus pneumoniae, which included ,B-galactosidase, f8-N-acetylglucosaminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were heterogeneous in size, whereas the released fucose-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.
Influenza virus is an enveloped virus which contains glycoprotein spikes projecting from the viral surface. Hemagglutinin (HA) is the predominant spike (13, 26) and is composed of glycosylated protein subunits which may be present in uncleaved form (HA) with a molecular weight of about 75,000 or in cleaved form, HA1 and HA2, with molecular weights of about 50,000 and 23,000, respectively. HA1 and HA2 are held together by disulfide bonds (12). The HA spike contains the binding sites for attachment of the virus to host cell neuraminic acidcontaining receptors in the process of infection and to erythrocytes in hemagglutination (25, 26). The HA also constitutes the major surface antigen (14, 24), and it can stimulate antibody which will neutralize infectivity. The biological importance of carbohydrate of the influenza virus and the HA has been studied by using inhibitors of glycosylation during synthesis of the virus. Inhibition with glucosamine or 2-deoxyglucose leads to the production of HA in an unglycosylated form (8, 10, 27, 30). Cells in which the unglycosylated or partially glycosylated HA is synthesized do not hemadsorb (10), and the HA does not become incorporated into virus particles (10, 27, 30). Tunicamycin, an inhibitor which prevents transfer of the core region of oligosaccharide chains to protein, also prevents physical particle formation of influenza virus (28). These inhibitors severely limit virus production and may also interfere with viral
synthesis in nonspecific ways (1, 20, 23). The use of specific glycosidases to remove carbohydrates (16, 19, 20) provides another approach to analyzing the function as well as the structure of these viral components. It has been reported from this laboratory (2) that treatment of influenza PR8 virus with a glycosidase preparation from Aspergillus niger resulted in partial degradation of the HA component with a concomitant loss of hemagglutinating capacity. This was attributed to removal of carbohydrate from the HA. However, difficulties encountered by using the acid conditions required for the enzymes as well as the finding of proteolytic activity in the enzyme preparations which degraded HA, (J. K. Collins, Ph.D. thesis, University of California, Berkeley, 1977) prompted the use of different glycosidase enzymes which were protease free. In the present study it was found that free amino sugar, neutral sugar, and large oligosaccharides could be removed from virus or from isolated HA by treatment with glycosidases from Diplococcus pneumoniae. Release of 50% of the HA carbohydrates did not significantly affect hemagglutinating activity. MATERIALS AND METHODS Cells and virus. Influenza A/PR8 was grown and labeled, using Madin-Darby canine kidney cells, and purified from infected culture fluids as described previously (4). Hemagglutination assay. HA was titrated by combining 0.1 ml of serial twofold dilutions of virus in 0.85% saline (in microtiter dishes) with 0.1 ml of 0.5%
t Present address: Rocky Mountain Laboratory, Hamilton, MT 59840.
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chicken erythrocytes. After 1 h at 4°C, the end point was estimated visually, and the reciprocal of the end point dilution was defined as the titer. Polyacrylamide gel electrophoresis. Slab polyacrylamide gel electrophoresis was performed with a 1-cm 3% stacking gel and a 10-cm 10% resolving gel as described previously (4). Gel filtration of carbohydrates released from virus or HA. Samples of virus or HA treated with glycosidases were clarified by centrifugation and chromatographed on a Bio-Gel P-6 (100 to 200 mesh) column (1.2 by 110 cm) equilibrated with 0.1 M ammonium acetate. Fractions (1.1 ml) were collected at a flow rate of 5.5 ml/h. Fractions were analyzed for radioactivity by transferring each to a scintillation vial, evaporating it until dry at 60°C, and then adding 7.5 ml of 3a70B scintillation fluid (Research Products International) and counting it in an Intertechnique scintillation counter. Oligosaccharides used as markers were from fetuin and ovalbumin and were prepared as described previously (4). Preparation of glycosidases. D. pneumoniae type I was obtained through the courtesy of C. E. Schwerdt from the Department of Medical Microbiology, Stanford University, Stanford, Calif. (culture collection no. H-19). The organism was maintained with biweekly subculturing in brain heart infusion broth (Difco). For preparation of glycosidases, 2 liters of broth was inoculated with 1 ml of a previous culture and incubated at 37°C for 30 h. Assay for the enzymes 4l-galactosidase and ,B-N-acetylglucosaminidase was as described by Koide and Muramatsu (11). A glycosidase preparation containing these enzymes and endoglycosidase D was prepared by ammonium sulfate precipitation from the culture fluid and Sephadex G-100 gel filtration (11, 17). The glycosidase preparation contained 1.3 mg of protein per ml and 3.4 U of,-galactosidase and 94 U off,-N-acetylglucosaminidase per ml when assayed in 0.02 M Tris-maleate buffer (pH 6.8). No proteolytic activity could be detected by a casein substrate assay. Purification of HA. The HA spike glycoprotein was purified after disruption of the virus with 1% sodium deoxycholate and extraction with tri-n-butyl phosphate according to Collins and Knight (4). Treatment with glycosidases. Virus or HA was treated with glycosidase enzymes by dilution to 0.05 to 0.2 mg/ml in a final volume of 2 ml in 0.2 M Trismaleate buffer (pH 6.8) and addition of 10 ,pl of glycosidase enzymes. Incubation at 37°C was carried out for 24 h, with a second 10-ul enzyme addition at 10 to 12 h. To terminate the reaction, samples containing the virus were pelleted by centrifugation at 80,000 x g for 20 min and then suspended in gel electrophoresis buffer for gel analysis. Samples were removed at various times for hemagglutination tests or infectivity tests by plaque assay for whole virus. Digestion of HA samples was terminated by addition of 5 volumes of cold ethanol, and the precipitated protein was analyzed by gel electrophoresis. lodination. HA was iodinated by the procedure of Hunter (6). The glycoprotein was concentrated to 1 mg/ml and dialyzed against 0.2 M phosphate buffer (pH 7.5) before 50 ,L of the glycoprotein solution was reacted with 0.2 mCi of Na'25I in the presence of 50
165
,ug of chloramine-T. Immediately after adding the reactants, the reaction was terminated by adding 240 Ig of potassium metabisulfite in 840 ,ul and 100 ,ug of potassium iodide in 100 ul. The entire mixture was put through a Sephadex G-10 column (10 by 0.6 cm) and eluted with 0.1 M phosphate buffer (pH 7.5). Fractions (1 ml) were collected. The labeled HA eluted in fractions 3 to 4, and these were pooled. HA prepared in this manner was still active and contained 1 x 105 to 1.5 x 105 cpm/mg of protein. Quantitation of neutral and amino sugar released from virus and HA. After treatment with glycosidases, virus was pelleted by centrifugation at 80,000 x g for 20 min. The supernatant fluid, or the entire digestion mixture when purified HA was treated with glycosidases, was deproteinated according to Spiro (29) by passage over a colunu (0.6 by 2.0 cm) of charcoal-Celite (1:1). The effluent and a 2-ml wash with 10% ethanol were then passed directly onto coupled ion exchange columns (0.6 by 1 cm) of AG50X2(H+) and AG1-X8(HCOO-). The columns were washed with 50% methanol, from which the neutral sugars were recovered, and assayed with the ParkJohnson ferricyanide reducing test according to Spiro (29). The AG50 column quantitatively bound amino sugar, and this was recovered by elution with 2 N HCl and was assayed by the Park-Johnson method. Parallel samples containing enzymes alone and virus or HA alone were carried through the procedure, and the amount of sugars found after incubation was negligible.
RESULTS Effect of glycosidases on virus and purified HA. Influenza virus was treated with the glycosidase mixture from D. pneumoniae, pelleted by centrifugation to separate the particles from the enzymes, and then analyzed by gel electrophoresis. The HA, of the treated virus was found to be more heterogeneous in size (Fig. 1, track 1) than the HA1 of the untreated virus (track 2). The HA1 band spread further in the gel, indicating a decrease in size, whereas the HA2 appeared to remain unaffected. Glycosidase treatment of the pure HA also caused a decrease in size of the glycoprotein. When the HA was treated with glycosidases and analyzed by sodium dodecyl sulfate gel electrophoresis under nonreducing conditions, the glycoprotein spread further down the gel (Fig. 1, track 3). Analysis of the treated HA under reducing conditions, which separate HA1 and HA2, was difficult due to glycosidase bands which migrated to the same region as HA1, as shown in Coomassie brilliant blue-stained gels (Fig. 2). Therefore, the purified HA was radiolabeled with 1251, treated with glycosidases, and analyzed together with the untreated ['"I]HA on the same slab polyacrylamide gel. Differences were found in both HA, and HA2 after glycosidase digestion (Fig. 2). These data corresponded with the finding of released carbohydrates (see below) and sug-
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COLLINS AND KNIGHT
gested that both of the HA glycoproteins were degraded by the glycosidases. Release of carbohydrates from virus and purified HA. Treatment of purified virus with glycosidases from D. pneumoniae was found to release neutral and amino sugars. The enzymes released 1 to 1.4 ,ug of neutral sugar and 0.4 to 0.5 ,g of amino sugar per 100 ,ug of virus after 24 h of incubation. The amount of carbohydrate released corresponded to approximately 20 to 25% of the total carbohydrates of the virus, using 4
_p
pi. M_IM
NP NAs
HA,-
HA2----
FIG. 1. SDS-polyacrylamide gel electrophoresis of influenza A/PR8 and its purified HA after treatment with glycosidases. Virus or purified HA was treated for 24 h with glycosidases from D. pneumoniae. Tracks: 1, virus treated with glycosidases; 2, untreated virus; 3, HA treated with glycosidases; 4, untreated HA. HA samples were run under nonreducing conditions so that the HA would migrate as one glycoprotein. Gels were stained with Coomassie brilliant blue.
a value of 7.2 ± 0.9 ,ug of total carbohydrate per 100 ,tg of virus (4). To deterrnine which carbohydrates were released, virus which had been labeled with [3H]galactose, [3H]fucose, or [3H]glucosamine was incubated with the enzymes. These labels remain in the same chemical form upon incorporation into virus, except for galactose, of which only 40 to 50% can be recovered as carbohydrate in the virus (4). After digestion with the glycosidase mixture, the virus was subjected to BioGel P-6 gel filtration to determine the size and amount of the released labeled carbohydrate (Fig. 3). Virus labeled with [3H]galactose or [3H]glucosamine and treated with glycosidases showed release of labeled monosaccharides. No labeled monosaccharides could be detected from fucose-labeled virus. Label released from [3H]galactose-labeled virus was identified by thin-layer chromatography as consisting solely of galactose. The amount of each sugar released is shown in Table 1. Monosaccharides and oligosaccharides were also released from the purified HA. Monosaccharides were assayed after separation into neutral and amino sugars and included 1.5 to 2 ,g of neutral sugar and 0.5 to 1.1 jig of amino sugar per 100 ,ug of HA. Release of both [3H]glucosamine and a large, heterogeneous [3H]glucosamine-labeled oligosaccharide was found after glycosidase treatment of [3H]glucosamine-labeled HA (Fig. 4a). Approximately 57% of all the glucosamine label was released, as judged by material which eluted in the included volume of the column. Gel filtration of [3H]fucose-labeled HA which had been treated with glycosidases (Fig. 4b) indicated that the large oligosaccharides that were released by the glycosidase treatment also contained fucose. Approximately 42% of all the fucose label could be released from the HA (Table 1). However, no free fucose was released, which was similar to the result obtained after treatment of whole virions with glycosidases. The elution profile of the fucosecontaining oligosaccharides was markedly different from the profile of the glucosamine-containing oligosaccharides. This suggested heterogeneity in the released glucosamine-containing oligosaccharide chains. From the P-6 gel filtration of glycosidasetreated HA, it was apparent that only approximately half of the total glucosamine or fucose label was released. The material appearing at the void volume of the column represented carbohydrate not released by the enzymes. The glucosamine label left could represent the innermost sugar linked to asparagine, which would be expected to remain if endoglycosidase D cleaved
REMOVAL OF INFLUENZA VIRUS CARBOHYDRATE
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I-0 m
20(
'I
0
x
E
E 15
I-I
v
L
CN '-l
10. 5
LA IN
5.
FRACTI ON NUMBER SDS-gel electrophoresis of [I251]HA treated with D. pneumoniae glycosidases run under conditions
FIG. 2. to separate HAI and HA2. Iodinated HA (3 x 106 to 6 x 106 cpm) was treated with glycosidases for 24 h (0), and an untreated sample (0) was run adjacent to the treated sample. After fixation in acid, the gel was sliced horizontally, and each slice was cut vertically to separate the two tracks. Each slice was then eluted and counted in a scintillation counter. The upperpanel shows a Coomassie brilliant blue-stained gel ofglycosidasetreated (top) and untreated (bottom) HA.
the oligosaccharide chains. However, it was conditions except without enzymes (Fig. 6). In found that there was glucosamine label which contrast, Pronase-digested virus showed a rapid remained as part of a large chain, since Pronase loss of infectivity. digestion of the glucosamine-labeled material in DISCUSSION the void-volume fractions left large oligosacchaof Removal up to 50% of the carbohydrate rides detectable on P-6 gel filtration (data not shown). Therefore, it appeared that oligosaccha- from the purified HA of influenza virus could be rides which were resistant to endoglycosidase D accomplished with a mixture of glycosidases from D. pneumoniae which included ,8-galactoor to combined endoglycosidase D, ,8-galactosidsidase, f)-N-acetylglucosaminidase, and endoase, and f?-N-acetylglucosaminidase may have glycosidase D. When HA was treated, heterobeen present on the HA. Effect on biological activities. The effect geneous oligosaccharides .containing glucosaof removal of carbohydrate on the biological mine and fucose as well as monosaccharides activities of the virus and the HA was measured were released from both HA1 and HA2. The after treatment with the D. pneumoniae en- monosaccharides released included glucosamine and neutral sugar. zymes. Both HA and virus retained full hemagThe release of large, glucosamine-containing glutinating activity during incubation at 37°C with or without glycosidase treatment but rap- oligosaccharides indicated that endoglycosidase idly lost HA activity if Pronase was added in- D may have cleaved carbohydrate chains. The stead of glycosidase enzymes (Fig. 5). The HA released oligosaccharides containing glucosatests were carried out on the same samples mine were heterogeneous in size; however, the which were used for gel electrophoresis or ana- fucose-containing oligosaccharides released had lyzed for release of carbohydrate to insure that a much more limited size distribution. Fucose the tests were made on material from which may have been present only on oligosaccharides of a discrete size class such that when these were carbohydrates had been cleaved. Plaque assays on samples of virus treated with released only a single peak was detected by gel the enzymes showed infectivities similar to that filtration. Glucosamine, on the other hand, could of the control, which was treated under identical have been in chains of varying length, including
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COLLINS AND KNIGHT 25
monosaccharides from the ends of the large released chains. Incomplete removal of multiple galactose or glucosamine termini could produce heterogeneous chains. The limited size distribution of fucose-containing chains suggested that these may not have been as susceptible to this type of degradation. The presence of terminal fucose linked to galactose (4) may have prevented degradation by the exoglycosidases.
Vt
VOV
Vf
(a) V0
J. VIROL.
420 1
15-
64-
TABLE 1. Amount of labeled monosaccharide released from influenza virus and its HA by D. pneumoniae glycosidases
2 CM N
x
0
10
(b)
% of total label released by enzymes Virus HA
Label
E 0. 8
[3H]glucosamine
26
57
[3H]galactose 22 0 [3H]fucose a ND, Not determined.
6in
4
NDa 42
2 0
8-
-
c
642-
0. 10
20
30
40
50
60
70
Fraction number FIG. 3. Bio-Gel P-6 filtration of influenza A/PR8 after treatment with glycosidases. Purified labeled virus was treated for 24 h with glycosidases from D. pneumoniae and then run on a Bio-Gel P-6 column. The treated virus preparations were labeled with [:3H]glucosamine (a), [3H]galactose (b), or [3H]fucose (c). Markers included the fetuin (Vf) and ovalbumin (V,,,) oligosaccharides. The included volume and void volume are designated V, and V., respectively.
small, high-mannose-containing oligosaccharides. If endoglycosidase D was responsible for removing the large oligosaccharides found after treatment with mixed glycosidases, then this would suggest that some of the chains contain the di-N-acetylchitobiose linkage at the core region of the oligosaccharide, since this enzyme cleaves this linkage specifically (7). Heterogeneity in the released oligosaccharides may also have been generated during incubation with the glycosidase mixture. The specific exoglycosidases, ,B-galactosidase and ,f-N-acetylglucosaminidase, may have cleaved their respective
10
20
30
40
50
60
70
Fraction number FIG. 4. Bio-Gel P-6 filtration of the influenza A/PR8 HA after treatment with glycosidases. Purified labeled viral HA was treated for 24 h with glycosidases from D. pneumoniae and then run on a Bio-Gel P-6 column. The treated HA preparations were labeled with [3HJglucosamine (a) and [1H]fucose (b). Markers included the fetuin (Vf) and ovalbumin (V.,,) oligosaccharides and stachyose (V1). The included volume and void volume are designated V, and V.,, respectively.
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REMOVAL OF INFLUENZA VIRUS CARBOHYDRATE
169
1 E
x
a-
D 0.1 I 10
-- (b) w
I
w
w
:
0.1-
10 )
30 Incubation, hours
10
20
40
FIG. 5. Effect of glycosidase treatment on the hemagglutinating activity of influenza virus and its HA. Virus (a) or HA (b) was incubated at 37°C with (0) or without (0) glycosidases from D. pneumoniae or with Pronase (A), and samples were removed at various times for hemagglutination assay.
Approximately 20 to 25% of the carbohydrates intact virus could be released. These were released as galactose and glucosamine. The galactose and glucosamine which were not released could be located at internal positions or on oligosaccharides inaccessible to the enzymes. Since large oligosaccharides were not released from the virus, whereas they were readily found after treatment of the HA, the virion structure may have prevented the enzymes from acting on the carbohydrate chains. This is supported by the fact that the HA2 on the virus was not altered by glycosidases, in contrast to the HA2 when pure HA was digested. This may reflect a lack of accessibility of the HA2 carbohydrates on the intact virus, compared with those on purified soluble HA. Oligosaccharides which were not released from the virus or HA may also be resistant to endoglycolytic cleavage. Similar resistant oligosaccharides have been found on vesicular stomatitis virus (19) and Sindbis virus (16). No change in hemagglutinating activity of the HA or virus was seen after removal of carbohydrate. This suggested that the carbohydrates on the
20
30
40
Incubation, hours FIG. 6. Effect ofglycosidase treatment on infectivity of influenza virus. Virus-infected culture fluid was diluted in 0.02 M Tris-maleate buffer, pH 6.8, and incubated in the presence of glycosidases from D. pneumoniae (A) with no glycosidases (0) or with Pronase (U). For Pronase treatment, the pH was adjusted to 8.0, and 10 jig of Pronase was added at time 0. Samples were removed at various times and titrated by plaque assay. may not have been directly involved in hemagglutination. However, since virus particles are multivalent and contain many HA spikes, the incomplete removal of carbohydrate may reflect a loss of carbohydrate from only some of the spikes. Since it is likely that purified HA also consists of aggregated spikes, a similar explanation could apply. Complete biological activity of the remaining carbohydrate-containing spikes could still allow each aggregate or particle to be fully active. This possibility could not be ruled out, since increased incubation with glycosidases did not release more carbohydrate. Since monosaccharides could be released from HA1 on the intact virus, the effect of their removal on infectivity was assayed. No significant decrease in infectivity occurred over the control untreated virus. The monosaccharides, galactose and glucosamine, which may be located at the nonreducing terminal of oligosaccharide chains may, thus, not be directly required for infectivity. However, if HA spikes were left which had intact carbohydrate, these could possibly allow full infectivity, and if only 50% of the particles
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COLLINS AND KNIGHT
had the critical carbohydrate removed, a corresponding loss in infectivity would be difficult to detect. The inessential nature for some biological functions of at least part of the carbohydrate of influenza virus is at variance with earlier conclusions (2). The Aspergillus glycosidases used previously contained other degradative proteolytic enzymes which acted on the HA (Collins, Ph.D. thesis). In addition, the low pH requirement of these enzymes destroyed some of the HA activity. These two facts make interpretation of previous results very difficult. The possible involvement of carbohydrate in functional properties of other viral glycoproteins has been investigated with several viruses. Removal of sialic acid from vesicular stomatitis virus was reported to reduce infectivity 100-fold and to abolish hemagglutinating activity (21, 22). However, although these results were confirmed in one laboratory (32), they could not be repeated by another (3). If sialic acid is not essential for vesicular stomatitis virus, this result would agree with results of experiments with other enveloped viruses, such as Sindbis virus (31), Semliki Forest virus (9), and Friend leukemia virus (20), since removal of sialic acid from these viruses has no effect on infectivity or attachment to cells. A second approach to defining the role of carbohydrates on viral glycoproteins has been to grow viruses in the presence of inhibitors of glycosylation, such as 2-deoxyglucose and glucosamine (5, 8, 10, 23, 27). Although inhibition of virus glycosylation occurs, these inhibitors are not entirely specific, and they interfere with virus particle formation through depletion of the UTP pool (1), leading to less viral RNA synthesis (23) as well as depletion of the ATP pool. Tunicamycin, an inhibitor which may specifically prevent the transfer of the linkage sugar, glucosamine, and core region of the oligosaccharide chains, has also been used to interfere with the synthesis of several viruses. Production of Sindbis and vesicular stomatitis viruses (15) as well as influenza virus (27) is drastically reduced with this compound, and incompletely glycosylated proteins appear mainly in infected cells. Recently, influenza virus particles showing a markedly reduced specific hemagglutinating activity have been obtained from inhibitor-treated cells (18). Although the relative amount of HA glycoproteins per particle was also reduced on these particles, this suggests that when nearly all of the carbohydrate is absent the protein may not have its biological functions. The use of specific glycosidases in removing carbohydrates may prove to be a fruitful approach in critically dissecting the structures and
J. VIROI,.
functions of carbohydrate residues. Extensive removal of carbohydrates has been carried out with Sindbis virus (16), Friend leukemia virus (20), and vesicular stomatitis virus (19), using the glycosidases from D. pneumoniae. Here we have shown that these enzymes acted on influenza virus carbohydrates under conditions which preserved biological activity. Further work on influenza viral glycoproteins and carbohydrates with these purified glycosidase enzymes should elucidate additional structural features as well as biological roles. ACKNOWLEDGMENTS This work was supported in part by Public Health Service (PHS) research grants AI 00634 from the National Institute of Allergy and Infectious Diseases and CA 14097 from the National Cancer Institute; by PHS training grant GM 01389 from the National Institute of General Medical Sciences; by biomedical sciences support grant FR-7006 from the General Research Support Branch, Division of Research Facilities and Resources, Bureau of Health Professions Education and Manpower Training, National Institutes of Health; and by a grant to J.K.C. from the Chancellor's Patent Fund, University of California. LITERATURE CITED 1. Bekesi, J. G., E. Bekesi, and R. J. Winzler. 1969. Inhibitory effect of D-glucosamine and other sugars on the biosynthesis of protein, ribonucleic acid and deoxyribonucleic acid in normal and neoplastic tissues. J. Biol. Chem. 244:3766-3772. 2. Bikel, I., and C. A. Knight. 1973. Differential action of Aspergillus glycosidases on the hemagglutinating and neuraminidase activities of influenza and Newcastle disease viruses. Virology 49:326-332. 3. Brown, F., and B. Cartwright. 1977. Role of sialic acid in infection with vesicular stomatitis virus. J. Gen. Virol. 35:197-199. 4. Collins, J. K., and C. A. Knight. 1978. Purification of' the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components. 26:457-467. 5. Duda, E., and M. J. Schlesinger. 1975. Alterations in Sindbis viral envelope proteins by treating BHK cells with glucosamine. J. Virol. 15:416-419. 6. Hunter, W. M. 1973. Chapter 17, p. 1-36. In D. W. Weir (ed.), Handbook of experimental immunology, vol. 4. Blackwell Scientific, New York. 7. Ito, S., T. Muramatsu, and A. Kobata. 1975. Release of
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Adv. Virus Res. 18:57-98. 14. Laver, W. G., and E. D. Kilbourne. 1966. Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30:493-501. 15. Leavitt, R., S. Schlesinger, and S. Kornfeld. 1977. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J. Virol. 21:375-385. 16. McCarthy, M., and S. C. Harrison. 1977. Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus. J. Virol. 23:61-73. 17. Muramatsu, T. 1971. Demonstration of an endoglycosidase acting on a glycoprotein. J. Biol. Chem. 246:5535-5537. 18. Nakamura, K., and R. W. Compans. 1978. Effects of glucosamine, 2-deoxyglucose and tunicamycin on glycosylation sulfation, and assembly of influenza viral proteins. Virology 84:303-319. 19. Robertson, J. S., J. R. Etchison, and D. F. Summers. 1976. Glycosylation sites of vesicular stomatitis virus glycoprotein. J. Virol. 19:871-878. 20. Schiifer, W., P. J. Fischinger, J. J. Collins, and D. P. Bolognesi. 1977. Role of carbohydrate in biological functions of Friend murine leukemia virus gp7l. J. Virol. 21:35-40. 21. Schloemer, R. H., and R. R. Wagner. 1974. Sialoglycoprotein of vesicular stomatitis virus: role of the neuraminic acid in infection. J. Virol. 14:270-281. 22. Schloemer, R. H., and R. R. Wagner. 1975. Cellular adsorption function of the sialoglycoprotein of vesicular stomatitis virus and its neuraminic acid. J. Virol.
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15:882-893. 23. Scholtissek, C. 1975. Inhibition of the multiplication of enveloped viruses by glucose derivatives. Curr. Top. Microbiol. Immunol. 70:101-119. 24. Schulman, J. L., and E. D. Kilbourne. 1969. Independent variation in nature of hemagglutinin and neuraminidase antigens of influenza virus. Proc. Natl. Acad. Sci. U.S.A. 63:326-333. 25. Schulze, I. T. 1970. The structure of influenza virus. I. The polypeptides of the virion. Virology 42:890-904. 26. Schulze, I. T. 1972. The structure of the influenza virus. II. A model based on the morphology and composition of subviral particles. Virology 47:181-196. 27. Schwarz, R. T., and H.-D. Klenk. 1974. Inhibition of glycosylation of the influenza virus hemagglutinin. J. Virol. 14:1023-1034. 28. Schwarz, R. T., J. M. Rohrschneider, and M. F. G. Schmidt. 1976. Suppression of glycoprotein formation of Semliki forest, influenza, and avian sarcoma virus by tunicamycin. J. Virol. 19:782-791. 29. Spiro, R. G. 1966. Analysis of sugars found in glycoproteins. Methods Enzymol. 8:3-26. 30. Stanley, P., S. S. Ghandi, and D. 0. White. 1973. The polypeptides of influenza virus. VII. Synthesis of the hemagglutinin. Virology 53:92-106. 31. Stollar, V., B. D. Stollar, R. Koo, K. A. Harrap, and R. W. Schlesinger. 1976. Sialic acid contents of Sindbis virus from vertebrate and mosquito cells. Virology 69:104-115. 32. Wagner, R. R. 1976. Third International Colloquium on Rhabdoviruses. ASM News 42:616-619.
