Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion

Virus Remmh,

177

10 (1988) 177-192

EIsevier VRR 00411

Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion F. Fo~~ows~

and l-l. Meier-Ewert

Abteilung f6r Virologie, Institut fGr Medizinische Mikrobiologie der Teehntschen Univer.~ir~t Miinchen, O-8000 Mikehen, l? R.G.

(Accepted for publication 21 January 1988)

The spike glycoprotein of influenza C/Johannesburg/ l/66 was isolated in a soluble form by digestion of MDCK cell-grown vii-ions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75000 daltons determined in SDS-PAGE. Comparison to Triton X-lOOisolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-ter~nus, The soluble glycoprotein appears to possess both r~ptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed. Influenza C glycoprotein; Hemolysis

Bromelain cleavage: RDE activity; Hemagglutination;

Correspondence to: H. Meier-Ewert, Abteitung f&r Virologie, Institut Biedersteiner Str. 29, D-So00 Miinchen 40, F.R.G. 016%1702/88/$03.50

ftir Med. Mi~obiolo~e,

0 1988 Elsevier Science Publishers B.V. (Biomedicai Division)

TU,

178 Introduction

Influenza C viruses are similar to other viruses of the orthomyxovirus group, both in structure and molecular biology. The similarities include a segmented RNA genome of negative polarity, the types of major protein components and the virion morphology (For review see Air and Compans, 1983). However, influenza C virions have only one type of spike glycoprotein, in contrast to type A and B influenza viruses, which have two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The influenza C spike is arranged to form a characteristic, regular, hexagonal beehive pattern on the viral surface (Herrler et al., 1981), suggesting that the spike protein may have a trimeric structure similar to that of influenza A HA (Wiley et al., 1977; Wilson et al., 1981). The surface glycoproteins of ortho- and paramyxoviruses have three biological functions: binding to the host cell receptors, fusion with the host cell membrane, and receptor-destroying enzyme (RDE) activity. In contrast to other viruses of both myxovirus groups, where these functions are divided between two different surface glycoproteins, in influenza C virus all these functions are performed by the single spike protein, coded for by genome segment 4 (Nakada et al., 1984; Pfeifer and Compans, 1984). In addition, the RDE of influenza C is a neuraminate-oacetylesterase, which releases acetyl residues from position C-9 of 9-O-acetyl-Nacetylneuraminic acid (Neu 5,9 AC,) (Herrler et al., 1985). This is a unique property, since all other ortho- and paramyxoviruses have a neuraminidase as their RDE. Recently, inhibition studies with the serine esterase inhibitor, diisopropyl fluorophosphate (DFP) have provided evidence that the receptor-binding and receptor-destroying sites are localized on the heavy subunit of the glycoprotein but do not share the same binding site (Muchmore and Varki, 1987). In permissive host cells, post-translational cleavage of the precursor glycoprotein (gp 88) of influenza C virus produces two disulphide-linked subunits (gp 65 and gp 30). The virus particles show full biological activity only when this cleavage has occurred, a result similar to that obtained with the hemagglutinin cleavage which occurs in type A and B influenza viruses. This activating cleavage does not take place in all host cells. When influenza C virus is grown in embryonated chicken eggs all glycoprotein is present in the cleaved form. Virus grown in chicken kidney or MDCK cells possesses both cleaved and uncleaved glycoprotein, whereas virus grown in chicken embryo fibroblast (CEF) cells has uncleaved glycoprotein exclusively. The uncleaved glycoprotein produced in CEF cells can be activated by in vitro treatment with trypsin or elastase (Herrler et al., 1979; Sugawara et al., 1981) to produce virions with full biological activity. Amino acid sequence analysis of the fully active form of the influenza C virus glycoprotein has revealed that the carboxy-terminus of gp 30 is the membrane anchoring region of the molecule, and that the N-terminal sequence of this subunit contains hydrophobic residues corresponding to the HA, subunit of influenza A and B viruses. In particular, the N-terminal tripeptide sequence, Ile-Phe-Gly, of influenza C virus is found in the HA, glycoprotein of A/Victoria/ 3/75 (H3N2) virus with the addition of an N-terminal glycine residue (Herrler et al., 1981).

179 However, the F, subunit of paramyxovirus F glycoprotein (Sendai virus) shows N-terminal similarity with influenza C virus, as it also lacks the N-terminal glycine residue (Gething et al., 1978). It has been suggested that this sequence is involved in membrane fusion (Herrler et al., 1981; Pfeifer and Compans, 1984; Nakada et al., 1984). This report deals with the isolation of the influenza C envelope spike by bromelain treatment of MDCK cell grown virions. The bromelain-released glycoprotein fragment was purified and its biological and biochemical properties analysed.

Materials and Methods Virus and cells The Johannesburg/l/66 strain of influenza C virus was used throughout this study. Virus stocks were grown in the allantoic cavity of lo-day-old embryonated chicken eggs for 3 days at 33°C. The harvested allantoic fluid was centrifuged at 2000 rpm for 10 min and stored at - 70 o C. MDCK I cells (obtained from Dr. Herrler, Marburg, FRG) were grown in Dulbecco’s medium containing 10% fetal calf serum at 37 o C in a 5% CO, incubator. Confluent cell monolayers were infected with egg-grown stock virus. After an adsorption period of 30 min at room temperature, serum-free Eagle’s minimal essential medium (MEM) was added and the cells were incubated at 33°C and 5% CO, for 3 days. After removal of cell debris from the culture medium by low speed centrifugation the virus was pelleted at 100000 x g for 1 h. The pellet was resuspended in 100 mM NaCl, 10 mM CaCl,, 0.02% NaN, and 50 mM Tris-HCl, pH 8.0. These virus preparations were used in all experiments. In vitro cIeamge

of uncleared g~~Jco~rotej~ with t~psin

Uncleaved glycoprotein of MDCK cell grown virus was activated with TPCKtreated trypsin (Sigma) at a concentration of 10 pg/ml for 45 min at room temperature. The reaction was stopped by adding an equal amount of soybean trypsin inhibitor (Sigma) prior to pelleting the virus. Isolation

of the glycoprotein

The glycoprotein was released from the virus particles by digestion with bromelain (Sigma) as described for the isolation of the hemagglutinin of influenza A by Brand and Skehel (1972). Virus in 0.1 M Tris-HCl, pH 8.0, 0.05 M 2-mcrcaptoetha~ol, 0.01 M CaCI, and 0.1% NaN, was digested with bromelain with an enzyme : virus protein ratio of 1 : 2 for 18 h at 33” C. Bromelain was inhibited by 1 mM iodoacetamide and the virus cores were removed from the incubation mixture by centrifuging for 60 min at 100000 X g. The supernatant containing the soluble

180 glycoprotein (Amicon). Purification

was concentrated

by centrifugation

in a Centricon

microconcentrator

of the soluble glycoprotein

The soluble glycoprotein fragment (bgp) was purified by centrifugation through a 520% (w/v) sucrose gradient in 50 mM Tris-HCI, pH 8.0, containing 100 mM NaCl, 10 mM CaCl, and 0.02% NaN,, for 16 h at 150000 x g and 20’ C. Sedimentation coefficient was determined using catalase from bovine liver and human y-globulin, both from Serva (Heidelberg, FRG), as marker proteins according to Martin and Ames (1961). Gradients were fractionated from the top in 0.5 ml fractions, by careful pipetting from the liquid-air interface. Fractions were checked for RDE activity, for protein by protein assay and SDS-PAGE. Glycoprotein-containing fractions were pooled and concentrated. Protein assay Protein was determined as described by Bradford (1976) with the Coomassie brilliant blue G-250 reagent from Bio-Rad Laboratories (Bio-Rad protein assay dye reagent concentrate). Bovine serum albumin was used as the standard. Protein concentration in the fractions from the sucrose gradient was assayed in a microtiter plate test as described by Simpson and Sonne (1982). The absorbance was read at 600 nm in a Dynatech microtiter plate reader MR 600. Assay for determination

of the receptor-destroying

enzyme activity

The receptor-destroying enzyme (RDE) activity of the glycoprotein was assayed with mucin (BSM) from bovine submaxillary glands (Boehringer Mannheim, FRG) as substrate, as described by Herrler et al. (1985). 100 ~1 mucin (20 mg/ml) in PBS was incubated with 1 ~1 of the glycoprotein containing sample for 15 min at 33°C. Acetic acid released from the mucin was determined enzymatically using a commercially available test kit (Boehringer Mannheim, FRG). A sample of mucin served as control and was subsequently subtracted from all other aiiquots. Hemagglutination-inhibition

titration

Purified bgp at a concentration of 1 mg/ml was serially diluted 2-fold in phosphate-buffered saline in a microtiter plate. To 25 ~1 of each dilution 25 ~1 of a 0.5% suspension of chicken red blood cells were added. After incubation for 30 min at 4O C, 25 ~1 containing 4 HAU of influenza C virus were added. Hemagglutination-inhibition by bgp was read after incubation for 30 min at 4°C. Hemolysis Hemolysis was tested as described by Kitame et al. (1982). Virus and purified bgp at final concentrations of 5-40 pg/rnl were incubated with 1 ml of 2% chicken

181 erythrocytes resuspended 33” C for 60 supernatants

in PBS at 4” C for 30 min. After low speed centrifugation the pellet was in 1 ml of saline buffered with 10 mM MES, pH 5.0, and incubated at min. The mixtures were centrifuged again and the optical density of the was measured at 540 nm.

Isolation of the glycoprotein with Triton X-100 Solubilization of the viral glycoprotein by the non-ionic detergent Triton X-100 was performed using a method described by Vainstein et al. (1984). A pellet of influenza C virus (1 mg protein) was suspended in 10% (w/v) Triton X-100, 100 mM NaCl, 50 mM Tris-HCl, pH 7.4. After shaking for 1 h at room temperature the detergent-insoluble material was pelleted by centrifugation for 60 n-tin at 100 000 x g. The detergent was removed from the supematant by direct addition of 40 mg SM-2 Bio-beads (Bio-Rad). After 16 h the liquid phase was taken off with a pipette. Low plcl and trypsin treatment Purified virus and bgp-preparations were adjusted to pH 5 by addition of 0.1 M citric acid, incubated for 10 min at room temperature and readjusted to neutral pH with a 0.5 M Tris solution. Samples were incubated with TPCK-treated trypsin (Sigma) for 20 min at room temperature in a 1 : 40 (w/w) ratio of trypsin to glycoprotein, according to Ruigrok et al. (1986). The reaction was terminated by the addition of an equal amount of soybean trypsin inhibitor (Sigma) and the tryptic products were analysed on 12% polyacrylamide gels. SDS-poEyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed by the discontinuous Laemmli system (Laemmli, 1970) in a Bio-Rad mini Protean II unit for 45 min at 200 V. Protein samples were mixed with sample buffer cont~ning 125 mM Tri-HCl, pH 6.8, 20% glycerol, 10% SDS and 0.001% bromphenol blue and boiled for 5 min. To separate the proteins under reducing conditions the sample buffer contained 2% 2-mercaptoethanol. Proteins were visualized by the standard Coomassie blue staining procedure. Determination of the N-terminal sequence After electrophoresis of the supernatant of the bromelain incubation mixture under nonreducing conditions the subunits of the glycoprotein were separated in a second 10% polyacrylamide gel under reducing conditions. The separated subunits were electroblotted onto siliconized glass fiber sheets, stained with Coomassie blue, and bgp 30 was subjected to Edman degradation in a gas phase sequencer (type 470A, Applied Biosystems), according to Eckerskorn et al. (1988).

182 Results

Isolation of the glycoprotein The glycoprotein of influenza C virus was isolated in a soluble form by treating whole virus particles with the protease bromelain, using the method described by Brand and Skehel (1972) for the isolation of influenza A hemagglutinin. After incubation with bromelain and centrifugation of the incubation mixture, the supernatant contained the soluble glycoprotein and the spikeless virus cores were found in the pellet (Fig. 1). The apparent molecular weight of the soluble glycoprotein, as determined by SDS-PAGE, is 75000 daltons, indicating that the cleavage site of bromelain is near to the viral membrane and the whole ectodomain of the glycoprotein (bgp) is cleaved off (Fig. 1). Separation of the isolated glycoprotein in SDS-PAGE under reducing conditions showed that migration of gp 65, the larger subunit of the glycoprotein, is unaltered after bromelain treatment. In contrast, gp 30, the smaller subunit which is the membrane-bound portion of the glycoprotein, exhibits a difference in molecular weight of approximately 3000-4500 daltons after bromelain treatment (Fig. I), presumably due to the loss of the hydrophobic C-terminal peptide which remains in the viral membrane. The bgp 30 and gp 65 usually migrate as a double band in gel electrophoresis, possibly because of heterogeneous glycosylation.

4

b

a

I

Fig. 1. Isolation of the glycoprotein of influenza C virus by bromelain digestion of MDCK cell-grown virions. Trypsin-activated particles were incubated with bromelain and spikeless virus cores were removed by centrifugation. Proteins were analysed by SDS-PAGE under nonreducing conditions. Lane 1: control virus, lane 2: pellet of the incubation mixture, lane 3: supernatant containing the soluble glycoprotein fragment (bgp) and the enzyme bromelain. Lanes 4-7: Triton X-100 isolated glycoprotein as comparison to purified bgp. Proteins were analysed under nonreducing (4 and 5) and reducing (6 and 7) conditions. Lanes 4 and 6: Triton X-100 isolated glycoprotein. Lanes 5 and 7: purified bgp.

183

Fig. 2. Isolation of the uncleaved glycoprotein (gp 88) by bromelain treatment of influenza C virus grown in MDCK cells without trypsin. The bromelain-released glycoprotein was purified by sucrose density gradient centrifugation. Proteins were separated in SDS-PAGE under reducing conditions. Lane 1: control virus, lane 2: pellet, lane 3: purified bgp.

Cleavage products

of gp I obtained by bromelain

treatment

Unless trypsin is added to the culture medium, MDCK-cell grown influenza C virions possess a mixture of cleaved and uncleaved spike glycoprotein (ratio approximately 1: 4). Bromelain treatment, however, leads to complete cleavage of the glycoprotein molecule (Fig. 2) producing two disulphide-linked subunits, of which the larger, bgp 65, has identical gel migration properties as the gp 65 subunit of the trypsin-activated glycoprotein. The bromelain cleavage site must, therefore, be situated in close proximity to the activating cleavage site that is used by cellular proteases, or by trypsin and elastase under in vitro conditions. Amino acid sequence analysis of the N-terminus of the bromelain-cleaved gp 30 revealed that the sequence differs only by the presence of one additional arginine residue (Table 1). With influenza A virions with uncleaved precursor HA it has been shown that bromelain treatment does not produce infectious virus particles (Klenk et al., 1977). The effect of the N-terminal arginine residue on the biological activity of the influenza C virus glycoprotein needs further investigation.

TABLE

1

N-TERMINAL TEIN, WHICH

AMINO ACID SEQUENCES OF GP 30 OF INFLUENZA C VIRUS HAS BEEN CLEAVED IN VIVO a AND WITH BROMELAIN.

Proteolytic cleavage in viva b Cleavage by bromelain a Source: Meier-Ewert cells without trypsin.

’

NH2 Ite NH, Arg Ite

Phe Phe

Gly Gly

Ile Ile

et al., 1981. b Virus grown in embryonated

Asp Asp

Asp Asp

chicken

Leu Leu

Ile Ile

GLYCOPRO-

Ile... Ile...

eggs. ’ virus grown in MDCK

184 Purification of the glycoprotein The bromelain-released glycoprotein was purified by sucrose density gradient centrifugation. After fractionation of the gradient the glycoprotein was detected by measuring the protein concentration and the receptor-destroying enzyme activity of each fraction (Fig. 3a). The RDE activity was found to be proportional to the concentration of the glycoprotein. The purity of the glycoprotein was tested by SDS-PAGE of the gradient fractions (Fig. 3b). Due to incomplete dissociation by the sample buffer, trimers and dimers of the glycoprotein can be seen at the top of the gel in the bgp-containing fractions. This partial resistance to dissociation by SDS, which was also described for the hemagglutinin of influenza A by Doms and Helenius (1986) was routinely observed in gels under nonreducing conditions. In the presence of reducing agents dissociation was complete. By comparing the position of the glycoprotein trimer in the gradient with the positions of the marker proteins catalase and human y-globulin, which were centrifuged in parallel gradients, a sedimentation coefficient of 10 S was calculated, indicating a molecular weight of 206000 for the bromelain-released trimeric spike.

a

0

0 5

10

15

20

Fractton

Fig. 3. Purification of the bromelain-released glycoprotein (bgp) of MDCK cell grown influenza C virus by sucrose density gradient centrifugation. (a) Sucrose gradients were fractionated from the top and were determined. l) and receptor-destroying enzyme activity (A -A) protein concentration (oTo calculate the sedimentation coefficient, marker proteins (bovine catalase, 11.3 S; human y-globulin, 7.1 S) were centrifuged in parallel gradients. (b) SDS-PAGE of the sucrose gradient fractions.

185 7) s

II,35

a

L

5

10

15

20

Fraction structure of the bromelain-released glycoprotein Fig. 4. Stabilizing effect of Ca ‘+ ions on the quaternary of influenza C virus. Isolated glycoprotein was purified by sucrose density gradient ~nt~fugation with different Ca2+ concentrations. The gradients contained: no CaCl, (a), 2 mM CaCl, (b) and 5 mM CaCl, (c). Arrows indicate the S-values of the marker proteins.

During the sucrose density gradient cent~fugation the trimeric structure of the glycoprotein was found to be stable only in the presence of Ca2+ ions. Depending on the concentration of CaCl, in the gradient, a gradual change from the mono-

Fig. 5. Susceptibility of bromelain-released glycoprotein to tryptic digestion after incubation at low pH. Solutions containing the isolated glycoprotein were adjusted to pH 5 and readjusted to neutral pH as described in Materials and Methods. Samples were digested with trypsin and digestion products were analysed on a 12% polyacrylamide gel. Lane 1: bgp at pH 7 without trypsin, lane 2: bgp at pH 7 with trypsin, lane 3: tryptic digestion products of bgp incubated at pH 5. T: trypsin, TI: trypsin inhibitor.

186 merit to the trimeric form was observed after centrifugation (Fig. 4). The sedimentation coefficients for the monomers and dimers were 5.1 S and 7.8 S, respectively. To stabilize the trimeric structure a CaCl, concentration of 10 mM was used in the sucrose gradients. Sensitivity

to proteolytic

digestion of the glycoprotein

In order to obtain some information in regard to the structure of the spike molecule, the glycoprotein was treated with trypsin. The trimeric form was not sensitive to degradation by trypsin, whereas the monomers from sucrose gradients bands with molecular without Ca2+ were digested to produce several characteristic weights of 42 000, 32000 and 28 000 daltons, respectively. The same digestion pattern could be observed if the glycoprotein was incubated at pH 5 (Fig. 5), indicating a conformational change of the glycoprotein at low pH. A similar proteolytic susceptibility after low pH treatment was described for the HA of influenza A, where the tryptic products have been well characterized (Skehel et al., 1982). The biological activity of the glycoprotein The receptor-destroying enzyme activity, which has been shown to be a neuraminate-0-acetylesterase (Herrler et al., 1985), is characteristic for the influenza C glycoprotein and represents a useful means to detect the glycoprotein. After bromelain treatment all the RDE activity was recovered in the supernatant, and after sucrose gradient centrifugation the activity was found in the glycoprotein containing fractions. The RDE activity was directly proportional to the protein concentration (Fig. 3a). In sucrose gradients with CaCl, concentrations of less than

TABLE

2

RDE ACTIVITY OF THE ISOLATED GLYCOPROTEIN AFTER INCUBATION AT pH 5 AND TRYPSIN DIGESTION OF THE LOW pH TREATED GLYCOPROTEIN. Bgp CONTAINING SOLUTIONS WERE ADJUSTED TO pH 5. AFTER VARIOUS TIMES THE pH WAS READJUSTED TO NEUTRAL AND RDE ACTIVITY WAS DETERMINED. ADDITIONALLY, THE bgp SOLUTIONS AT NEUTRAL pH AND AFTER LOW pH TREATMENT WERE DIGESTED WITH TRYPSIN BEFORE RDE ACTIVITY DETERMINATION. Incubation time at pH 5 (min) PH 7 bgp PH 5 bgp

pH 7 bgp trypsin pH 5 bgp trypsin

10 20 30 treated treated

10 20 30

Esterase activity (a) 100 100 89 82 88 84 84 80

187 TABLE

3

HEMAGGLUTINATION-INHIBITION GLYCOPROTEIN OF INFLUENZA

bw

b

undiluted 1:lO 1:20 1:30

ACTIVITY C VIRUS.

Hemagglutination-inhibition

OF

PURIFIED

BROMELAIN-RELEASED

titer a

2

4

8

16

32

64

128

256

512

_

_

-

_

_

_

_

++

++ ++

++ ++ ++

++ ++ ++

++ ++ ++

+ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

a given as l/dilution; agglutination.

b starting

dilution;

-, no agglutination;

+, partial

agglutination;

+ + , complete

5 mM, where the glycoprotein was present in monomeric or dimeric form, RDE activity could be detected in the corresponding protein fractions. Therefore, RDE activity is apparently a property of each subunit of the glycoprotein spike (data not shown), and a trimeric structure is not required for enzyme function. After acid treatment of the isolated glycoprotein, the esterase activity was unchanged, although the glycoprotein became susceptible to trypsin digestion due to the conformational change. Even the tryptic digestion products of the glycoprotein after low pH treatment showed RDE activity, although the activity was slightly decreased (Table 2). To determine whether bgp is able to recognize the cell receptor, the hemagglutination-inhibition test was used. Table 3 summarizes the competition of bgp with virions for the receptors of the red blood cells, resulting in hemagglutinationinhibition for influenza C viruses. It is thus shown that purified bgp binds to the receptor, since RDE activity is irrelevant at 4” C. The glycoprotein after exposure to low pH as well as their tryptic digestion products showed the same hemagglutination-inhibition activity, but no visible hemagglutination was discernible with these preparations (data not shown). Hemolysis of chicken red blood cells by purified bgp could not be detected, even after incubation for 24 h at 37 o C. The same result was obtained, when the pH was dropped to the fusion pH without a centrifugation step.

Discussion Studies on the isolated glycoprotein of influenza C virus have so far been done with detergent solubilized spikes (Herrler et al., 1981). This complete form of the glycoprotein aggregates into rosettes immediately after removal of the detergent. In the present report, we describe the isolation of the glycoprotein with the protease bromelain, yielding the glycoprotein in a soluble form since the hydrophobic C-terminus remains in the viral membrane. Like the bromelain-released hemagglutinin of influenza A viruses, the bromelain fragment of the influenza C glycoprotein appears to represent the whole ectodomain of the surface glycoprotein.

188 The bromelain-released glycoprotein (bgp) sedimented in 5-20% sucrose gradients with a sedimentation coefficient of 10 S, indicating a trimeric structure with a molecular weight of 206000 for the spike molecule. A threefold symmetry of the spike molecule was first implied by the characteristic hexagonal arrangement of the spikes on the viral surface (Herrler et al., 1981). Cross-linking studies with formaldehyde were consistent with a trimeric structure (Nagele, unpublished result), which has also been convincingly demonstrated by electron microscopy (Hewat et al., 1984). The isolated trimers, however, were only stable in the presence of Ca2+ ions. With increasing concentrations of Ca2+ in the sucrose gradients a gradual shift from the monomeric to the trimeric form was observed, suggesting a requirement for Ca2+ to stabilize the quaternary structure of the glycoprotein. A contribution of calcium ions to the stabilization of the quaternary structure was also found for a type 2 neuraminidase of influenza A. The structural analysis revealed potential Ca*+ binding sites at the symmetry axis of the tetrameric molecule (Varghese et al., 1983). An additional destabilization of the bromelain fragment is possibly produced by the absence of the C-terminal membrane anchor. The importance of the transmembrane anchor in preventing dissociation of the trimer has been shown for the hemagglutinin of influenza A. Although intact HA trimers were stable after acid treatment and SDS-denaturation, BHA dissociated under the same conditions (Doms and Helenius, 1986). Three biological functions reside in the surface glycoprotein of the influenza C viruses: binding of the receptor, fusion and receptor-destroying enzyme activity. Not all of these properties could be demonstrated with the bromelain-released glycoprotein. The bgp showed no hemagglutination of chicken erythrocytes, possibly because bgp does not aggregate. In binding experiments we also found no attachment of 3H-labeled bgp to the receptors of red blood cells. When a 0.5% suspension of chicken erythrocytes was incubated with the tritiated spike, no decrease of radioactivity could be detected in the supematant after sedimentation of the cells. On the other hand, the fact that ‘bgp competes with intact influenza C virions for the receptor sites in hemagglutination-inhibition tests indicates that the receptor binding function is still present in the bromelain fragment. It seems possible, that the binding to the cell receptors is too weak to be detectable under these experimental conditions, or perhaps a critical number of spike molecules has to attach to the surface of red blood cells in very close proximity. Possibly for the same reason, a hemolytic activity of the bgp could not be demonstrated. Consistent with these results, glycoprotein rosettes of influenza C virions isolated with octylglucoside and purified by sucrose density gradient centrifugation were found to be devoid of hemagglutinating activity, but they inhibited HA activity of intact influenza C virions in competition tests. Finally, the detergent-isolated glycoprotein caused also no hemolysis of erythrocytes at low pH (Herrler et al., in press). In contrast to our findings are the biological activities of the isolated hemagglutinin of influenza A viruses. Detergent-isolated HA rosettes agglutinate erythro-

189 cytes (Laver and Valentine, 1969) and mediate hemolysis and fusion at low pH (Sato et al., 1983; Wharton et al., 1986). Though the adsorption of BHA to red blood cells was not observed (Laver et al., 1974) Wharton et al. (1986) recently reported BHA-mediated hemolysis, although at a much reduced rate compared to intact virus particles and HA rosettes. The esterase activity of the isolated influenza C glycoprotein could be demonstrated with bovine submaxillary mucin as substrate. The RDE activity was’found to be a property of each subunit of the spike trimer, and like the receptor-binding function was not destroyed by acid treatment of the glycoprotein. This suggests, that the active site of the esterase and the receptor-binding site are not affected by the irreversible conformational change which is induced at low pH. Even after tryptic digestion of the low pH treated glycoprotein, both biological activities could still be observed. Studies on the low pH structure of the influenza A HA indicated no detectable changes of the secondary structure but rather a relative movement of whole domains (Skehel et al., 1982). Major changes occur in the stem tertiary structure, i.e., stretching of the spike, while the top portion is only affected in its quaternary structure (Daniels et al., 1983; Ruigrok et al., 1986). Therefore most antigenic sites on HA,, and the sialic acid receptor remain functional after low pH treatment. Another influenza virus protein containing hemagglutinating activity is a neuraminidase (NA) molecule of the N9 subtype isolated by Laver et al. (1984). The N9 NA can be isolated in a soluble form by pronase treatment of virus particles, and these pronase released “heads” showed similar properties to the bromelain released glycoprotein of influenza C. The pronase released N9 NA did not adsorb to red blood cells, inhibited the attachment of N9 NA rosettes to cells in competition tests and showed receptor-destroying activity. The availability of a soluble form of the influenza C virus spike protein should facilitate more detailed studies to compare this glycoprotein to other influenza virus surface proteins. Crystallizing experiments are underway.

Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft (Me 422/2-2). We thank Dr. Lottspeich, Max Planck Institut, Martinsried, F.R.G., for performing the amino acid sequencing. The MDCK I cells were kindly provided by Dr. Herrler, Marburg, F.R.G.

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(Received

24 November

1987; revision

received

20 January

1988)