Sialic acid is incorporated into influenza hemagglutinin glycoproteins in the absence of viral neuraminidase

61

Virus Research, 2 (1985) 61-68 Elsevier

VRR 00166

Sialic acid is incorporated into influenza hemagglutinin glycoproteins in the absence of viral neuraminidase Sukla

Basak

‘, Milan

Tomana

2 and Richard

W. Compans

’

’ Departments of Microbiology, and ’ Medicine, University of Alabama in Birmingham, Birmingham, AL 35294, U.S.A. (Accepted 26 October 1984)

Summary We have analyzed the pronase-derived glycopeptides of the hemagglutinin glycoproteins expressed from SV40 vectors carrying cloned cDNA copies of the HA gene and of HA isolated from influenza virions (A/Jap/305/57). The glycopeptides derived from the HA glycoprotein obtained from cloned genes were heterogeneous, ranging in size from 3800 to 2800 daltons. Upon treatment with neuraminidase, sialic acid was released from the glycopeptides and their size was reduced to 2900-2400 daltons. However, under the same conditions, no sialic acid was detected in the virion HA. The presence of sialic acid was confirmed by monosaccharide analysis of the HA glycoprotein derived from products of cloned genes. These results support the idea that during replication of influenza virus, the viral neuraminidase cleaves sialic acid from the HA glycoprotein in infected cells. influenza

virus, hemagglutinin,

glycoprotein,

neuraminidase,

sialic acid

Introduction The outer surface of the influenza virion is covered by two different glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Previous studies have shown that both complex and mannose-rich oligosaccharide chains are attached to the protein core of the hemagglutinin glycoprotein. However, unlike many other complex oligosaccharide chains, the complex oligosaccharides of the HA glycoprotein are not sialylated. This has been shown by analyzing the susceptibility of the virion 0168-1702/85/$03.30

0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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glycopeptides to neuraminidase, and by determining their sugar composition and structure (Klenk et al., 1970; Nakamura and Compans, 1977; Ward and Dopheide, 1981; Basak et al., 1981; Matsumoto et al., 1983). It has been suggested that virions do not have sialic acid on their envelopes because they possess viral NA activity. Sialylation of the HA glycoprotein, however, has been carried out in vitro (Schulze, 1975) and several studies have also suggested that HA may become sialylated in vivo under conditions when NA is inactive (Palese et al., 1974; Paiese and Compans, 1976; Griffin and Compans, 1979). However, the covalent attachment of sialic acid to HA in vivo has not been directly demonstrated, and Griffin et al. (1983) were unable to demonstrate sialic acid on the carbohydrate chains of the HA glycoprotein under conditions where NA was inactive. The present study was undertaken to directly determine whether HA of influenza virus can become sialylated in vivo. The previous demonstration of in vitro sialylation of influenza HA (Schulze, 1975) as well as analysis of its carbohydrate structure (Matsumoto et al., 1983) indicate that the oligosaccharides linked to HA do not have blocked terminal sugars, such as have been reported to be present on paramyxovirus glycoproteins (Prehm et al., 1979). The possibility might exist, however, that the intracellular transport pathway of HA could bypass the cellular compartment where sialylation takes place. In order to investigate whether sialylation of the HA glycoprotein can be detected in vivo, we have analyzed the oligosaccharides of glycoproteins expressed from cloned cDNA copies of the HA gene, thereby eliminating the possibility of removal of sialic acid by viral neura~nidase. Materials and Methods Virus and cells

CV-1 cells were maintained in Eagle’s minimal medium containing 10% newborn calf serum. Stocks of influenza A/Japan/305/57 (H2N2) virus (A/Jap) were grown in the allantoic membrane of lo-day-old chicken embryos. SV40 vectors containing either full-length cDNA copies of the HA gene (SV40 HA) or a C-terminal deletion (anchor-minus) mutant (SVE HAZOA-‘) were obtained from Dr. M.J. Gething and stocks were prepared in CV-1 cells. The characte~zation of these r~ombin~t viruses has been described by Gething and Sambrook (1982). Purification of HA

For purification of HA from the A/Jap strain, egg-grown stocks were used to infect CV-1 cells at a multiplicity of infection of 10 pfu/cell. At 2 h post-infection, [ 3H]glucosamine (5.0 &i/ml) was added and the radiolabelled virions were harvested and purified 24 h post-infection according to procedures described previously (Basak and Compans, 1983). [ l4 C]Glucosamine-labelled VSV was prepared similarly and the virions were purified as previously described (McSharry et al., 1971). For isolation of HA, viral polypeptides were separated by polyacrylamide gel electrophoresis according to the procedure of Laemmli (1970). The HA band, as visualized by autoradio~aphy, was extracted from the gel by electro-eluting in the presence of 0.1 M Tris-acetate buffer, pH 8.0.

63

In order to obtain the HA glycoprotein from SV40 HA infected CV-1 cells, the infected cells were radiolabelled with [3H]glucosamine (5 /.&i/ml) from 36 to 48 h post-infection. The cells were then lysed with gel dissociation buffer as described previously (Basak and Compans, 1983). ,Cells infected with SVE HA20A- were radiolabelled at 40 h post-infection. The HA protein from the culture supernatant at 48 h post-infection was either alcohol precipitated or immune precipitated using antiserum against A/Jap virus. HA glycoproteins were then purified by polyacrylamide gel electrophoresis. G~eopeptide anaksis [3H]Glucosa~ne-labelled HA and [ “C]glucosa~n~labell~ VSV were co-digested with pronase as previously described (Nakamura and Compans, 1979). The glycopeptides were analyzed on a Bio-gel P-6 column and radioactivity of the fractions was determined. In order to determine the neuraminidase sensitivity of the glycopeptides, the peak fractions were pooled and treated with protease free V. cholerae neuraminidase (Calbiochem) according to the procedure of Nakamura et al. (1979). The treated glycopeptides were then rechromatographed on the same Bio-gel P-6 column.

ikfwmaccharide analysis The monosaccharides were determined as trifluoroacetates of methyl glycosides by gas-liquid chromatography. Methanolysis of the glycopeptides and derivatization of monosaccharides were carried out as described by Tomana et al. (1978). The analyses were performed with a Hewlett-Packard gas chromatograph (Model 5830A; Hewlett-Packard Co., Palo Alto, Calif.) equipped with a 25 m fused silica (0.22 mm inner diameter) OV-1701 WCOT column and 63Ni ECD (Tomana et al., 1984). Thin-layer chromatography (TLC) Samples of l-5 ~1 were applied to precoated TLC sheets (0.2 mm thickness) and developed with n-butanol/acetic acid/water (2 : 1: 1). Standard sugars were visualized according to the procedure of Yamada et al. (1975). To detect radiolabeled sugars, the TLC sheet was cut into 2 mm strips. The silica gels from each strip were scraped into vials and the radioactivity was detected in a liquid scintillation counter.

Results Glycopeptides of HA expressed from cloned genes and virions In order to compare the size classes of oligosaccharide moieties linked to HA expressed from cloned genes or virions, ]3H]glucosamine-labelled purified HA glycoproteins were extensively digested with pronase along with the [‘*C]glucosamine-labelled glycopeptides of VSV grown in BHK cells as markers. The resultant glycopeptides were then co-chromatographed on Bio-Gel P-6. As shown in Fig. 1, [ ‘H]~ucosa~e-labelled glycopeptides derived from HA expressed from either SV40 HA (Fig. 1A) or SVE HA20A- (Fig. 1B) were observed to be more heteroge-

64

neous than glycopeptides obtained from the virions (Fig. 1C). The molecular sizes of the glycopeptides from SV40 HA were estimated to range from 3400 to 2600 daltons and for SVE HAZOA- from 3800 to 2800 daltons, as compared with approximately 3000-2400 daltons for the vu-ion-derived glycopeptides. Because of the absence of viral NA in either SV40 HA or SVE HA20Ainfected cells, it was of interest to examine the effect of neuraminidase treatment on the elution profile of glycopeptides obtained from virions or products of cloned genes. The fractions containing glycopeptides were therefore pooled, digested with neuraminidase and rechromatographed on the same column. As shown in Fig. 1, upon neuraminidase treatment some 3H label was found to be released from the HA glycopeptides obtained from products of the cloned HA genes (fractions 90-98 in Fig. 1D and fractions 85-92 in Fig. lE), while neuraminidase had no effect on the glycopeptides of HA from influenza virions (Fig. 1F). The molecular size of the resulting HA glycopeptide after neuraminidase treatment from either SV40 HA or SVE HAZOA- infected cells was

‘H dpm x IO“

‘+C dcvn

“Id-2

FRACTION

NO

‘Ii dam

‘k

X10-'

FRACTION

NO

dom

‘H dmn

14C dom

x10-z

FRACTION

NO

Fig. 1. Glycopeptides of HA glycoproteins derived from either purified virions or HA expressed from cloned genes. CV-1 cells were infected with A/Jap virus, SV40 HA or SVE HA20A- and labelled with [3H]glucosamine. Radiolabelled HA glycoproteins were purified and glycopeptides obtained as described in the text. The resultant [3H]glycopeptides and “C-labelled VSV marker glycopeptides were analyzed by gel filtration chromatography on Bio-gel P-6. (A) [3H]Glucosamine-labelled glycopeptides from HA glycoprotein expressed from SV40 HA; (B) [3H]glucosamine-labelled glycopeptides from SVE HAMA- ; (C) [3H]glucosamine-labelled glycopeptides from purified virions. Fractions 45-75 of A, fractions 45-70 of B or fractions 40-65 of C were concentrated and digested with neuraminidase, and rechromatographed in the same columns. (D) Neuraminidase-digested glycopeptides of HA glycoprotein from SV40 HA; (E) from SVE HAZOA- and (F) from purified virions. 0, 3H radioactivity; 0, 14C radioactivity. The molecular sizes were estimated by calibrating each column with Sindbis virus marker glycopeptides, as described by Sefton and Keegstra (1979).

65

estimated to be reduced to approximately 2900-2400 daltons. Similarly the elution profile of the “C-1abelled glycopeptides of VSV was also observed to be changed after neuraminidase treatment, i.e. the glycopeptides eluted later and a new peak of 14C-label appeared near the included volume. Identification of sialic acid Previous studies with WV have shown that when virus is grown in the presence of 13H]glucosamine,some of the radioactivity is incorporated as sialic acid (Burge and Huang, 1970; Moyer and Summers, 1974). Upon treatment with neuraminidase, the desialylated ~yGopeptides shift their mobility in a gel ~ltration column and a new peak appears at a fraction near to the included volume (Keegstra et al., 1975; Robertson et al., 1976; Nakamura et al., 1979). In order to characterize the 3H-labelled camponents released from glycopeptides of either SV40 HA or SVE HAZOA- by neuraminidase treatment, fractions 90-98 in Fig. 1D and fractions 85-92 in Fig. 1E were concentrated and analysed by thin-layer chromato~aphy (Fig. 2). The R, values of the 3H label in either case were found to be similar to that of a sialic acid marker. The R, values of all other sugars were found to be very different.

Mono~~h~de analysis was also carried out on the purified HA ~ycoprote~ expressed from either cloned genes or from virus. The results (Table 1) indicate the presence of sialic acid in HA purified from either SV40 HA or SVE HA2OAinfected cells. However, no sialic acid was detected in HA purified from A/Jap virions. On the other hand, a considerable amount of sialic acid was detected in the G protein isolated from purified VSV. Thus, the mono~~h~de analysis confirms the identification of sialic acid in the glywpeptides derived from the products of the 3H cpm 1( 10v3

/

DISTANCE

Fig. 2. Thin-layer c~omato~aphy of 3H-labelled sugars released from neur~d~e treated gIycupeptides shown in Fig. 1. Fractions 90-98 as shown in Fig. 1D were concentrated, transferred to a silica gel-coated sheet and developed as described in the text. Standard sugars were nm in parallel and arrows indicate their positions: I, sialic acid; 2, galactose; 3, glucose; 4, mannose; 5, N-acetyl glucosamine; 6, fucose.

66 TABLE 1 CARBOHYDRATE

vsv HA (SVE HA20 A - ) HA (SV4OA) HA (A/J@

COMPOSITION

OF INFLUENZA

HA GLYCOPROTEINS

SiaIic acid

N-Acetyl glucosamine

Mannose

Fucose

Galactose

2.9 1.7 1.1

5.2 4.1 3.2 3.0

3.0 3.0 3.0 3.0

0.7 0.5 0.8 0.5

3.1 1.8 1.1 1.9

0.0

The compositions are expressed as molar ratios relative to mannose, which was arbitrarily assigned a value of 3.0.

cloned genes. The carbohydrate composition indicates that the product of SVE HAZOA- may contain more complex sugars, i.e. the ratio of sialic acid, glucosamine and galactose to mannose is much higher than in the product of SV40 HA. Sveda et al. (1982) have also reported differences in the glycosylation of cell associated and secreted forms of HA. This might be a consequence of the slower intracellular movement of the secretory protein product of SVE HAZOA- than the wild-type HA, as observed by Gething and Sambrook (1982). Discussion Several functions have been ascribed to the NA glycoprotein of influenza virus. It has been postulated (Davenport, 1976) that after the hemagglutinin glycoprotein of influenza virus binds to host sialic acid residues, the terminal sugar of mucins, NA cleaves the sialoglycoprotein bond freeing the bound virions. The underlying cell receptor is thus exposed and the HA can attach to it. It has also been reported that NA plays a role in membrane fusion induced by myxoviruses (Huang et al., 1980). However, several lines of evidence indicate that virus penetration is probably not mediated by NA since virions remain infectious after inhibition of the activity by specific antibody (Seto and Rott, 1966; Webster and Laver, 1967; Bucher and Palese, 1975). Schulze (1970) has also demonstrated that trypsin treatment of the influenza virions removes the NA activity while increasing the infectivity of the vii-ion. Other evidence has suggested that NA plays a role in the release of budding vii-ions, by cleaving sialic acid from the HA glycoprotein. It has been shown that influenza strains with low enzyme activity were released from cells more slowly than strains with higher enzyme activity (Palese and Schulman, 1974). Moreover, by using ts mutants of influenza virus which are defective in neuraminidase activity or by using a neuraminidase inhibitor, it was observed that the virions form aggregates at the cell surface during budding, and it was suggested that if sialic acid is not cleaved, the HA recognizes these residues on neighboring virions as receptors, and thus the release of particles is inhibited (Palese et al., 1974; Palese and Compans, 1976). Virions produced under these conditions were demonstrated to bind colloidal iron hydroxide, a stain that binds to sialic acid (Palese et al., 1974; Palese and Compans, 1976). Griffin et al. (1983) observed that in the presence of cytochalasin B, which

67

results in an inactive neuraminidase, no sialic acid was detected on the carbohydrates of the HA glycoprotein. It was suggested that the aggregation of the virus observed in the presence of the inhibitor was due to binding of HA to sialic acid residues of cellular origin, since the removal of cell surface sialic acid by neuraminidase during the latent period prevented cytochalasin B induced inhibition of virion release. Our experiments provide direct evidence that in vivo, the HA glycoprotein of the influenza virus can become sialylated in the absence of neuraminidase, since the products of the cloned HA genes were observed to be sialylated. The previous finding of the lack of sialic acid on viral HA under conditions when viral neuraminidase was inactive (Griffin et al., 1983) could be due to several reasons: (1) low levels of residual neuraminidase activity could be present which, although not detected, are sufficient to cleave siahc acid from HA glycoprotein; and (2) under the experimental conditions used, very little terminal glycosylation occurs, since the processing and elongation of the carbohydrate chains are not completed. Evidence for the latter possibility was obtained by Griffin et al, (1983) by analyzing the oligosaccharides of glycopeptides from influenza virus grown in glucose-free medium, which mimics the action of cytochalasin B.

This research was supports by grant AI 12680 from the National Institutes of Allergy and Infectious Diseases, and a grant from the Cystic Fibrosis Research Center, University of Alabama in Birmingham. S&la Basak was supported by Institutional Research Award AI 07150 from the National Institute of Allergy and Infectious Diseases. References Basalt, S. and Compans, R.W. (1983) Studies on the role of glycosylation in the functions and antigenic properties of influenza virus glycoproteins. Virology 128, 77-91. Basak, S., Pritchard, D.G., Bhown, A.S. and Compans, R.W. (1981) Glycosylation sites of influenza viral glycoproteins. Ch~ac~~tion of tryptic glycopeptides from the A/USSR (HtN,) h~a~utinin glycoprotein. J. Viol. 37, 549-558. Bucher, D. and Palese, P. (1975) The biologically active proteins of influenza virus: neuraminidase. In: Tbe Influenza Viruses and Influenza (Kilbourne, E.D., ed.), p. 83. Academic Press, New York. Burge, B.W. and Huang, A.S. (1970) Comparison of membrane protein glycopeptides of Sindbis virus and vesicular stomatitis virus. J. Virol. 6, 176-182. Davenport, F.M. (1976) Influenza virus. In: Viral Infections in Humans (Evans, A.S., ed.), pp. 273-296. Plenum Publishing Corp., New York. Gething, M.-J. and Sambrook, J. (1982) Construction of influenza hemagglutinin gene that codes for intracellular and secreted forms of the protein. Nature (London) 300, 598-603. Griffin, J.A. and Compans, R.W. (1979) Effect of cytochalasin B on the maturation of enveloped viruses. J. Exp. Med. 150,379-391. Griffin, J.A., Basalt, S. and Cornpans, R.W. (1983) Effects of hexose starvation and the role of sialic acid in influenza virus release. Virology 125, 324-334. Huang, R.T.C., Rott, R., Wahn, K., KIenk, H.-D. and Kohama, T. (1980) The function of neur~nidase in membrane fusion induced by myxoviruses. Virology 107, 313-319.

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Keegstra, K., Sefton, B. and Burke, D. (1975) Sindbis virus glycoproteins; effect of the host cell on the oligosaccharide. J. Viral. 16,613-620. Klenk, H.-D., Compans, R.W. and Choppin, F.W. (1970) An electron microscope study of the presence or absence of neuraminic acid in enveloped viruses. Virology 42, 115881162. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. Matsumoto, A., Yoshima, H. and Kobata, A. (1983) Carbohydrates of influenza virus hemagglutinin: structures of the whole neutral sugar chains. Biochemistry 22, 188-196. McSharry, J.J., Compans, R.W. and Choppin, P.W. (1971) Proteins of vesicular stomatitis virus and of phenotypically mixed vesicular stomatitis virus-simian virus 5 virions. J. Virol. 8, 722-729. Moyer, S.A., and Summers, D.F. (1974) Vesicular stomatitis virus envelope glycoprotein alteration induced by host cell transformation. Cell 2, 63-70. Nakamura, K., and Compans, R.W. (1977) The cellular site of sulfation of influenza virus glycoproteins. Virology 79, 381-392. Nakamura, K. and Cornpans, R.W. (1979) Biosynthesis of the o~go~~h~d~ of influenza viral glycoproteins. Virology 93, 31-47. Nakamura, N., Herrler, G., Petri, T., Meier-Ewert, H. and Compans, R.W. (1979) Carbohydrate components of influenza C virus. J. Virol. 29, 997-1005. Palese, P. and Compans, R.W. (1976) Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): Mechanism of action. J. Gen. Virol. 33, 159-163. Palese, P. and Schtdman, J.L. (1974) Isolation and characterization of influenza virus recombinants with high and low neuraminidase activity. Use of Z-(3”-methoxyphenyl)-N-acetylneuraminic acid to identify cloned populations. Virology 57, 227-237. Palese, P., Tobita, K., Ueda, M. and Cornpans, R.W. (1974) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61, 397-410. Prehm, P., Scheid, A. and Choppin, P.W. (1979) The carbohydrate structure of the glycoproteins of the paramyxovirus SV5 growu in bovine kidney cells. J. Biol. Chem. 254,9669-9677. Robertson, J.S., Etch&on, J.R. and Summers, D.F. (1976) Glycosylation sites of vesicular stomatitis virus glycoprotein. J. Virol. 19, 871-878. Schulze, LT. (1970) The structure of influenza virus I. The polypeptides of the virion. Virology 42, 890-904. Schulze, LT. (1975) Effects of sialylation on the biological activities of the influenza virion. In: Negative Strand Viruses (Barry, R.D. and Mahy, B.W.J., eds.), pp. 53-82. Academic Press, New York. Sefton, M.W. and Keegstra, K. (1974) Glycoproteins of Sindbis virus: preliminary characterization of the oligosaccharides. J. Virol. 14, 522-530. Seto, J.T. and Rott, R. (1966) Functional significance of sialidase during influenza virus multiplication. Virology 30, 731. Sveda, M.M., Markoff, L.J. and Lai, C.-J. (1982) Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequence. Cell 30,649-656. Tomana, M., Niedermeier, W. and Spivey, C. (1978) Microdete~nation of monosaccharides in glycoprotein by gas-liquid c~omato~aphy. Anal. B&hem. 89,110-118. Tornana, M., Prchal, J.T., Gamer, L.C., Skalka, H.W. and Barker, S.A. (1984) Gas c~omato~ap~c analysis of lens monosaccharides. J. Lab. Clin. Med. 103,137-142. Ward, C.W. and Dopheide, T.A. (1981) Amino acid sequence and oligosaccharide distribution of the haemagglutinin from an early Hong Kong variant A/Aichi/2/68 (X-31). Lochem. J. 193, 953-962. Webster, R.G. and Laver, W.G. (1967) Preparation and properties of antibody directed specifically against the neuraminidase in influenza virus. J. Immunol. 99, 49-55. Yamada, T., Hisamatsu, M. and Taki, M. (1975) A method for detection of non-reducing saccharides. J. Chromatogr. 103, 390-391. (Manuscript received 25 July 1984)