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The hemagglutinating and neuraminidase protein of a paramyxovirus: Interaction with neuraminic acid in affinity chromatography
VIROLOGY
62, 125-133 (1974)
The Hemagglutinating Paramyxovirus:
and Neuraminidase
Interaction
with
Neuraminic
Protein
of a
Acid in Affinity
Chromatography’ ANDREAS
SCHEID
The Rockefeller
AND
University,
PURNELL
W. CHOPPIN
New York, New York 10021
Accepted July 23, 1974 The two glycoproteins of paramyxoviruses can be separated and purified by affinity chromatography on fetuin which is covalently linked to Sepharose. The glycoproteins are solubilized with Triton X-100 and applied to the column in the Triton-containing solution. The large glycoprotein (HN protein) of Simian virus 5 (SV5) which possesses both hemagglutinating and neuraminidase activities is retained by fetuin-Sepharose at 0” and passes elutes when the temperature is raised to 25”. The small glycoprotein through the column at 0”. This procedure provides a simple and effective means of obtaining high yields of paramyxovirus glycoproteins in pure form. The HN protein can be displaced from the fetuin by the neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid. This indicates that a site on the HN protein binds specifically to the neuraminic acid moieties of the fetuin. This finding is compatible with the hypothesis that the same active site is involved in the neuraminidase and the hemagglutinating activities of the HN protein of paramyxoviruses. INTRODUCTION
Previous studies from this laboratory described the isolation of the individual glycoproteins from the paramyxovirus SV5 (Scheid et al., 1972) and Newcastle disease virus (NDV) (Scheid and Choppin, 1973), and showed that with each of these viruses, both neuraminidase and hemagglutinating activities of the virion are associated with the larger of the two virion glycoproteins. This finding has been confirmed with NDV (Seto et al., 1973) and extended to Sendai virus (Tozawa et al., 1973; Scheid and Choppin, 1974a,b). Because of the association of both hemagglutinating and neuraminidase activities with the larger paramyxovirus glycoprotein, the designation HN has been suggested for this protein (Scheid and Choppin, 1974a). The smaller glycoprotein of SV5 and NDV possesses neither neuraminidase nor ‘Presented in part at the Conference Strand Viruses, Cambridge, England, 1973.
on Negative July 22-27,
hemagglutinating activity, and, therefore, it was suggested earlier that this protein might be involved in cell-fusing and hemolyzing activities associated with paramyxoviruses (Scheid et al., 1972; Scheid and Choppin, 1973). Subsequently, direct evidence for the involvement of the small glycoprotein in fusion and hemolysis was obtained independently in two laboratories with Sendai virus (Homma and Ohuchi, 1973; Scheid and Choppin, 1974a, b). When grown in certain cells, Sendai virions contain a precursor glycoprotein (designated FO, Scheid and Choppin, 1974a), and such particles lack hemolyzing and cell fusing activities and are not infectious. However, when the precursor is converted to the small virion protein (F) by proteolytic cleavage in uiuo or in uitro, the virions become infectious and acquire cell-fusing and hemolyzing activities. It is of obvious interest to investigate further the physical, chemical and biological activities of these virus membrane proteins. Such studies,
126
SCHEID
AND CHOPPIN
MATERIALS AND METHODS however, would be greatly facilitated by procedures for the preparation of the proCells. Monolayer cultures of a variant of teins in pure form and in large quantity. In the MDBK line of bovine kidney cells were addition, such a procedure would have grown in plastic bottles (Falcon) in reinvalue, in producing a purified inactivated forced Eagle’s medium (REM) (Bablanian vaccine. The preparation of the individual et al., 1965) with 10% fetal calf serum as proteins by centrifugation on Triton X-100 described previously (Choppin, 1969). To containing sucrose gradients has proved propagate cells on a large scale, they were useful in analytical studies (Scheid et al., passaged twice into disposable glass roller 1972; Scheid and Choppin, 1973), but is bottles (Bellco) with a growth area of 630 limited by the fact that only small quancm2 and the confluent monolayers of the tities of glycoprotein can be separated on second passage were used for the growth of each gradient. virus. Growth and purification of virus. Cells This communication describes a procedure, using SV5 as a model, by which large were infected with the W3 strain of the parainfluenza virus SV5 at a multiplicity quantities of the two virion glycoproteins of approximately 1 PFU per cell. After an can be separated by affinity chromatography. The same procedure has been success- adsorption period of 2 hr, 50 ml of REM fully applied to the isolation of the HN with 2% calf serum containing [3H]leucine, protein of Sendai virus (Scheid and Chop- 5 pCi/ml, was added. After 3 days the pin, 1974a). The rationale behind using medium was harvested, and the virus was purified as described previously (Scheid fetuin as a ligand in affinity chromatography is that, owing to its neuraminic acid and Choppin, 1973). Purified virus was content, fetuin can serve as a substrate for stored in 30-40s potassium tartrate at neuraminidase and as a receptor for the -70”. Isolation of the virus glycoproteins. Purihemagglutinin of myxo- and paramyxoviruses. It was, therefore, anticipated that fied virus at a concentration of 2 mg/ml of protein (Lowry et al., 1951) in 0.5 M it would specifically bind the paramyxovirus protein possessing these activities. Fe- potassium chloride and 0.01 M phosphate tuin-Sepharose has also been used to ob- buffer, pH 7.2, was disrupted with Triton tain aggregates of influenza virus hemag- X-100 (Scheid et al., 1972) at a final glutinin and neuraminidase (Becht and concentration of 2%. The fraction containRott, 1972), however individual glyco- ing the virion glycoproteins was prepared as described previously for NDV (Scheid proteins were not isolated in those studies. To avoid the formation of aggregates which and Choppin, 1973). Fetuin-Sepharose. The fetuin used in are mixtures of the different virion glycothe experiments reported here was preproteins, the nonionic detergent Triton pared by the method of Spiro (1960) from X-100 was used throughout the procedure fetal calf serum. From 350 ml of serum, 3.0 described here. In addition to describing the preparative procedure, this report pre- g protein with a neuraminic acid content of sents evidence which indicates that the 0.197 mmole per g protein was obtained. In some instances, fetuin obtained from retention of the HN protein on fetuin-Sepharose is due to the interaction between Grand Island Biological Company (Grand neuraminic acid-specific binding sites on Island, NY) was used. Sepharose 4B (Pharthe HN protein, and the neuraminic acid macia) was activated with cyanogen broresidues of fetuin. The results are compatimide, 25 mg per ml of beads (Axen et al., ble with the hypothesis that the site re- 1967), and washed with 0.1 M sodium sponsible for binding to neuraminic acid bicarbonate, pH 8.7. The activated Sephareceptors in the hemagglutination reaction rose was suspended in 1.5 vol of 0.15 M and the active site of the enzyme neuramisodium chloride and 0.1 M sodium bicarnidase may be one and the same. bonate containing 18 mg fetuin per ml
AFFINITY
CHROMATOGRAPHY
added Sepharose. The reaction was allowed to proceed for 3 hr at room temperature under gentle agitation. Solid glycine was added and allowed to react overnight at 4”. The beads were washed with 0.5 M NaCl and 0.1 M sodium acetate, pH 4.5; with 0.5 M NaCl and 0.1 M Tris, pH 9, and subsequently with water. The aqueous suspensions were stored at 4’ with 0.02% sodium azide added. Under these conditions fetuin-Sepharose proved to be stable for 1 yr as judged by its neuraminic acid content and its undiminished effectiveness as an adsorbant for neuraminidase. Column chromatography. Fetuin-Sepharose was suspended in running buffer (0.15 M sodium chloride and 0.05 M sodium acetate, pH 4.5, containing 0.1% Triton X-100), packed into columns and washed with the same buffer. Solutions of virus glycoproteins in 2% Triton X-100 and water were adjusted in their salt content to that of running buffer by the addition of l/9 vol of 1.5 ,‘M sodium chloride with 0.5 M sodium acetate, pH 4.5. Flow rates were controlled through the use of a peristaltic pump and the column temperatures by immersing the column into ice-water or a temperature-regulated water bath. Fractions were collected at selected time intervals and aliquots were analyzed for their neuraminidase and radioactivity content. Pooled fractions were dialyzed against 0.01 M phosphate buffer, pH 7.2, with 0.1% Triton X-100 and concentrated by dialysis against buffer containing 20% polyethyleneglycol. Hemagglutination titrations. Tritoncontaining samples were freed of the detergent with n-butanol as described previously (Scheid and Choppin, 1973), and titrations were done as described (Choppin, 1964) except that plastic plates with round bottom cups, 0.3-ml capacity (Lindbro Chemical Co., New Haven, CT) were used and serial dilutions were made in 0.1 ml and 0.1 ml of chicken erythrocytes was added. Neuraminic acid was determined by the procedure of Aminoff (1961). Fetuin and fetuin-Sepharose were incubated with SV5 glycoproteins in 1% Triton X-100 at pH 4.5,
OF SV5 GLYCOPROTEINS
127
and at various times the neuraminic acid released by the viral enzyme was measured. The total neuraminic acid content of fetuin and fetuin-Sepharose was determined after hydrolysis with 0.1 N sulfuric acid at 80” for 1 hr. Neuraminidase assays, polyacrylamide gel electrophoresis, and radioactivity measurements were done as described previously (Scheid et al., 1972). Chemicals and isotopes. 14C-Reconstituted protein hydrolyzate and [3H]leutine, 51 Ci per mmole, were purchased from Schwarz BioResearch, Orangeburg, NY. N-Acetylneuraminic acid was obtained from Sigma Chemical Co., St. Louis, MO. 2-Deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) was kindly supplied by Dr. Peter Palese. RESULTS
Separation of SV5 glycoproteins by affinity chromatography. Fetuin-Sepharose was characterized with respect to its content of neuraminic acid. The total neuraminic acid was determined after acid hydrolysis and 1.93 pmoles were found per ml of derivatized beads. This represented 49% of the neuraminic acid in the fetuin used in the coupling reaction. Steric changes are known to occur to ligands upon their covalent linkage to solid matrixes and this may render their properties different from the properties in solution. Thus, it is important to know whether the neuraminic acid residues remain accessible to the viral HN protein when fetuin is coupled to Sepharose. To evaluate this, fetuin and fetuin-Sepharose were exposed to SV5 glycoproteins until the amount of neuraminic acid released by the viral neuraminidase had reached a constant level. From fetuin, 46% of the total neuraminic acid could be released by neuraminidase, and from fetuin-Sepharose, 45%. This indicates that the efficacy of fetuin as a specific receptor is preserved after the covalent attachment to Sepharose. A separation of the SV5 glycoproteins was considered feasible because in Triton X-loo-containing solutions, each of the proteins exists as physically distinct enti-
128
SCHEID
AND CHOPPIN
ties (Scheid et al., 1972). The design of the chromatographic procedure took into account the fact that the hemagglutination reaction with paramyxoviruses is usually stable in the cold, and not at room temperature and, therefore, binding of the HN protein to the receptor was expected to be stronger in the cold. Other conditions of the chromatographic procedure were arrived at empirically. In the chromatographic procedure that was finally adopted, radioactively labeled virion glycoproteins in a Triton-containing solution at pH 4.5 were applied to fetuinSepharose at 0”. Figure 1 shows the elution profile and the experimental details. Much of the labeled material did not interact with the fetuin and was not retarded at the initial temperature of 0”. The column was washed at the same temperature with approximately three bed volumes of buffer. The column temperature was then raised to 25”, and the protein which was bound to the fetuin in the cold then eluted. As shown in Fig. 1, the peak of protein eluted when the temperature was raised coincides with the peak of neuraminidase activity. The two glycoprotein components which were separated in the experiment shown in Fig. 1 were analyzed by polyacrylamide gel electrophoresis (Fig. 2). The column effluent at O”, left panel, contained the smaller glycoprotein, protein F, as the only protein component. The column eluate at 25” contained the larger glycoprotein, the HN protein. The small peak at fraction 15 represents a dimer of the HN protein (Scheid et al., 1972). The proteins found in the two fractions obtained from the column were analyzed for their hemagglutinating and neuraminidase activities (Table 1). Hemagglutinin was detected only in the 25” eluate fraction, which also contained the neuraminidase activity and which, as shown in Fig. 2, is constituted solely by the HN protein. Taking the neuraminidase activity recovered with the HN protein in the eluate at 25” as an indicator for the recovery of this protein, a yield of 65% was obtained in this experiment, and similar yields were obtained in other experiments.
Yo Fract!on
20 No
30
FIG. 1. Fractionation of SV5 glycoproteins by affinity chromatography on fetuinSepharose. The glycoprotein fraction (4.5 ml) obtained from 8.0 mg of SV5 was mixed with 0.5 ml of 10 x running buffer and applied to a column 1.5 cm diameter, containing 14 ml of fetuin-Sepharose. The temperature during the sample application and up to fraction 16 was 0” and the flow rate 28 ml/cm2 hr. After fraction 16, the temperature was raised from 0” to 25”, and the flow rate was reduced to 14 ml/cm2 hr. Fractions of 4.8 ml (6 min per fraction up to fraction 16, 12 min per fraction afterwards) were collected. The arrow indicates the temperature shift from 0” to 25”. Twentyfive-microliter aliquots were analyzed for their neuraminidase content and 25.~1 aliquots were used for radioactivity determinations. Some of the OD,,, is caused by neuraminic acid that was eluted from the column through the action of the viral enzyme. However, it was established that this was negligible compared to the NANA released in the assay, and, therefore, no corrections were made for this. The fractions comprising the effluent at 0” (fractions 3-5) and the eluate at 25” (fractions 17-21) were pooled separately, dialyzed, and concentrated as described in Materials and Methods. After the concentration steps, the volumes were adjusted to 4.5 ml. Analyses of these two fractions are shown in Table 1 and Fig. 2.
Since a temperature shift was used in preparative chromatography, the following experiment was performed to demonstrate that this temperature shift was indeed responsible for the elution of the HN protein. The HN protein, which was isolated in the experiment shown in Fig. 1 was rechromatographed under the same conditions on a smaller scale (Fig. 3), and again retention at 0” was observed, and elution occurred when the temperature was raised to 25” after fraction 10 (right panel). In contrast, when the column temperature was kept at 25’ throughout the experiment, the HN protein eluted shortly after
AFFINITY
CHROMATOGRAPHY
129
OF SV5 GLYCOPROTEINS
8 I
iI
HN F 1 1
I30 Froctlon
60
90
i
No
FIG. 2. Polyacrylamide gel electrophoresis of the glycoproteins of SV5 obtained by fractionation on fetuin-Sepharose. One-hundred microliters of the fractions obtained in the experiment shown in Fig. 1 were prepared for electrophoresis by precipitation with n-butanol in the presence of 1% 2-mercaptoethanol. The left panel shows the polypeptide composition of the eluent at 0”, the right panel shows the polypeptide that was retained by fetuin-Sepharose and which eluted at ‘25”. [“C]Amino acid-labeled virions were added as a coelectrophoresis marker. The arrows indicate the position of the two marker glycoproteins. The origins are on the left and the anode on the right. TABLE
1
that temperature. Thus, initial experiments at pH 7.2 showed only incomplete retention in the cold, and therefore pH 4.5, the pH optimum of the SV5-neuraminidase, was selected. Also, a reduction of the Fraction Activity initial flow rate to one half or less leads to NeuramiHemagthe elution of the HN protein at 0”. glutinin nidase No attempts have been made to reuse (units) (HAU x 102) the beads because of the consideration that Sample applied to 252 575 some of the protein which is lost in the column procedure may have remained on the colEffluent at 0” <0.3 <2 umn and contaminate subsequent preparaEffluent at 25” 171 575 tions. Also, during each run a small but measurable part of the neuraminic acid is o The isolated proteins from the experiment in Fig. hydrolyzed through the action of the viral 1 were analyzed. Ten- and 25-p] aliquots of the pooled and concentrated fractions were analyzed for neuraenzyme. minidase. Hemagglutination was assayed after preThe nature of the interaction between cipitation of the proteins from 25-~1 aliquots with fetuin and the HN glycoprotein. The nan-butanol and resuspension of the proteins with ture of the interaction between fetuin-sebuffer. pharose and the HN protein was studied further by the use of the neuraminidase the void volume and before fraction 10 inhibitor 2-deoxy-2,3-dehydro-N-tri(Fig. 3, left panel). fluoroacetylneuraminic acid (FANA) which is a synthetic derivative of the natuThe affinity of the HN protein for fetuin rally occurring N-acetylneuraminic acid is low even at O”, and deviations from the conprocedure described may cause elution at (Meindl et al., 1974). The inhibitory BIOLOGICAL ACTIVITIES OF Sv5 GLYCOPROTEINS SEPARATED BY AFFINITY CHROMATOGRAPHYON FETUIN-SEPHAROSE"
130
SCHEID
AND CHOPPIN
Fractton
No
FIG. 3. Temperature dependence of the affinity of the HN glycoprotein of SV5 for fetuin-Sepharose. Identical columns of 0.38.cm diameter containing 0.6 ml fetuin-Sepharose were used. Right panel: The column temperature during the sample application and up to fraction 10 was O”, after fraction 10 it was raised to 25’. Left panel: The column temperature was 25” throughout. The samples, 0.1.ml aliquots of the HN protein isolated in experiment Fig. 1 (cf. Fig. 2 and Table I), were neutralized with 10 ~160 mM acetic acid, and 12 ~1 of 10 x running buffer were added. The initial flow rates and the collecting times for each sample were the same as in experiment of Fig. 1. After fraction 10 the flow was reduced to one half and the collecting times of the fractions doubled. Radioactivity was measured in 50-~1 samples.
stant of FANA for SV5 neuraminidase and hemagglutination were determined, and found to be 5 x 10m6 and 2 x 10m6 M, respectively. Purified HN protein was applied to fetuin-Sepharose, and the column temperature was maintained at 0” throughout the experiment (Fig. 4, left panel). After fraction 10, FANA was included in the running buffer at a concentration of 10m4 M, and this caused the prompt elution of the HN protein. In contrast, when N-acetylneuraminic acid (NANA), which does not inhibit neuraminidase or hemagglutination, was tested for its ability to interfere with the binding of the HN protein at 0” at a lo-fold higher concentration (Fig. 4, right panel), no elution occurred. Thus, the neuraminidase inhibitor FANA can displace the HN protein from fetuin-Sepharose, but NANA cannot. DISCUSSION
The procedure described here for the separation of the two glycoproteins of the paramyxovirus SV5 by affinity chromatography using a ligand which contains the receptor for one of the proteins has a number of advantages. It is a simple procedure which provides in one step prepara-
tive quantities of the proteins. The yield is high; the HN protein is obtained in a high degree of purity because of the specificity of the viral protein-receptor interaction; and the biological activities, i.e., hemagglutinating and neuraminidase activities, are preserved. A similar procedure has also been successful with Sendai virus glycoproteins (Scheid and Choppin, 1974a), and it thus should be applicable to other paramyxoviruses, and under certain conditions perhaps to influenza viruses also. In addition to providing a means for obtaining virion glycoproteins in quantities for further studies of their physical, chemical, and biological properties, the procedure should also prove valuable for the production of vaccines consisting of purified proteins, since the glycoproteins are the antigens of importance in inducing immunity to infection by enveloped RNA viruses. In principle, affinity chromatography using a virus receptor for isolation of receptor binding proteins of other viruses should be possible; however, the limitation at present with many viruses is lack of knowledge of a specific receptor to use as a ligand. Once it was found that in the case of paramyxoviruses, one glycoprotein, the HN protein, possessed both hemag-
AFFINITY 6-
CHROMATOGRAPHY
* NANA
+ FANA
10
20
10 Fraction
131
OF SV5 GLYCOPROTEINS . FANA
20
30
No
FIG. 4. Desorption of the HN glycoprotein of SV5 from fetuin-Sepharose with 2-deoxy-2.3-dehydro-N-trifluoroacetylneuraminic acid (FANA). Left panel:-After fraction 10. running buffer containing 0.1 mM FANA was used for further elution. Right panel: After fraction 10. the running buffer contained N-acetylneuraminic acid, 1 mM, and after fraction 20 the running buffer contained 0.1 mM FANA. The experimental conditions were identical to those of the experiment in Fig. 3 except that the column temperature was kept at 0” throughout. 3H was measured in 100.~1 aliquots.
glutinating and neuraminidase activities. (Scheid et al., 1972; Scheid and Choppin, 1973) the question arose as to whether the same site on the molecule was involved in both reactions. This is, is the binding to cellular receptors simply an enzyme-substrate reaction between the viral neuraminidase and neuraminic acid-containing substrate, or, alternatively, are different portions of the molecule involved in hemagglutinating and neuraminidase activities. In the case of influenza virus, two different active sites clearly exist, since the hemagglutinin and neuraminidase activities reside on different proteins. However, the evidence obtained here and previously (Scheid et al., 1972; Scheid and Choppin, 1973; 1974a.b) is compatible not only with both activities being present as a single protein, but also with the involvement of the same site in both reactions. The nature of the interaction of the HN protein with fetuin has been explored in the present studies by the use of 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA). This analog of N-acetyl-neuraminic acid has been shown previously to inhibit the neuraminidase activity of myxoviruses and paramyxoviruses, as well as hemagglutination by SV5 and
NDV (Meindl et al., 1974). The present finding that FANA interferes with the retention of the HN protein on fetuin-Sepharose excludes the possibility that fetuin-Sepharose is acting simply as an ion exchanger, and indicates the following mode of interaction. The active moieties on the immobilized fetuin are the neuraminic acid residues, and the HN protein reacts with these receptors through a neuraminic acid-specific binding site. This finding and the inhibition of hemagglutination by SV5 with FANA (Meindl et al., 19741, strongly suggest that in the case of parainfluenza viruses. a neuraminic acid-specific site on the HN protein is responsible for the hemagglutination reaction. Although this evidence does not prove that the active site of the neuraminidase is identical to the neuraminic acid-specific binding involved in the hemagglutination reaction, there are other results that are compatible with this view. First, there is the observation that with paramyxoviruses. hemagglutination titrations usually must be done in the cold to obtain full titers. in contrast to results with influenza virus. Further, in early studies it was found that, in contrast to influenza virus, NDV could not be converted to “indicator virus”
132
SCHEID
AND CHOPPIh
by heating (Francis. 1947; Anderson. 1948). “Indicator virus” was the term used for virus whose neuraminidase activity, but not hemagglutinating activity. was inactivated so that it became a sensitive indicator of competitive inhibition by glycoprotein inhibitors of hemagglutination. Both of these observations would be expected if the paramyxovirus hemagglutination reaction is a neuraminidase-substrate interaction. In addition, several ts mutants of parainfluenza viruses with defects in both neuraminidase and hemagglutinin activties have been isolated, but no mutant has been described in which only one of the two functions is affected (Pierce and Haywood. 1973; Preble and Youngner, 1973; Portner et al., 1974). It was shown previously that the HN protein exhibits full neuraminidase activities in the presence of the nonionic detergent Triton X-100 (Scheid et al., 1972; Scheid and Choppin, 1973). The present experiments have demonstrated that the binding of the HN protein to receptors is not prevented by the presence of Triton, since binding to fetuin-Sepharose occurred in the presence of the detergent. This question could not be investigated previously by the hemagglutination reaction, because it was essential to remove the detergent so that aggregates of the protein which were multivalent could form. It has thus now been established that both binding to receptors and neuraminidase activity can be expressed in the presence of the nonionic detergent. ACKNOWLEDGMENTS Supported by Research Grant AI-05600 from the National Institute of Allergy and Infectious Diseases. U.S.P.H.S. We thank Mrs. Ariane A. Cody for her excellent technical assistance. REFERENCES AMINOFF, D. (19611. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biothem. J. 81, 384-392. ANDERSOK, S. G. (1948). Mucins and mucoids in relation to influenza virus action. I. Inactivation by RDE and by viruses of the influenza group. of the
serum inhibitor of haemagglutination. /lust. J. Exp. Bioi. Med. Sri. 26, 34;~33. AXEK. R., PORATH. J.. and ERSBACK. s. (19671. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature iLondon) 214, 1302~1:104. BABLANIAX. R.. EGGER~;. H. J.. and TAMM. I. ( 1965,. Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirusinduced metabolic and morphological alterations in cultured cells. Virology 26, 100-l 13. BECHT. H.. and Ron, R. (1972). Purification of influenza virus hemagglutinin by affinity chromatography. Med. M~robiol. Immunoi. 158, 67-X. CHOPPIN, P. W. (1964). Multiplication of a myxovirus (SV5) with minimal cytopathic effects and without interference. Virolog), 23, 224-233. CHOPPI~, P. W. (1969). Replication of influenza virus in a continuous cell line: High yield of infective virus from cells inoculated at high multiplicity. Virolog> 39, 130-134. FRANCIS. T.. JR. (1947). Dissociation of hemagglutinating and antibody-measuring capacities of influenza virus. J. Enp. Med. 85, l-7. HOMMA. M.. and OHLCHI. M. (1973). Trypsin action on the growth of Sendai virus in tissue culture cells III. Structural difference of Sendai viruses grown in eggs and tissue culture cells. J. Viroi. 12, 1457- 1465. LOWRY, 0. H.. ROSEBROLGH, N. J.. FARH. A. L.. and RANDALL. R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MEINDL. P.. BODO. G.. PALESE. P.. SCHVLMA~, J.. and TC~PPY, H. (1974). Inhibition of neuraminidase activity by derivatives of Zdeoxy-2.3.dehydro-Nacetglneuraminic acid. Vuvlogy 58, 455-463. PIERCE, J. S.. and HAYHOOD. A. M. (197.3). Thermal inactivation of Newcastle disease virus. I. Coupled inactivation rates of hemagglutinating and neuraminidase activities. J. Viral. 11, 168~176. PORTKEK. A., MAHX. P. A.. and KI~CSBLH~, D. u’. (1974,. Isolation and characterization of Sendai virus temperature sensitive mutants. J. Krol. 13, 298-304. PREBLE, 0. T.. and YO~NNEK, ,J. S. 11973). Selection of tt mperature-sensitive mutants during persistent infection: Role in maintainance of persistent Newcastle disease virus infections of L cells. J. V~rol. 12, 481-491. SCHEID. A., CALICURI. L. A.. COMPANS, R. W., and CHOPPlh, P. u’. (1972). Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger Sv5 glycoprotein. Vlrolog3 50, 640-652. SCHEID, A.. and CHOPPIN P. u’. (1953). Isolation and purification of the envelope proteins of Newcastle disease virus. J. Viol. 11, 263-271.
AFFINITY
CHROMATOGRAPHY
SCHEID, A., and CHOPPIN, P. W. (1974a). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion. hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57, 475-490. SCHEID. A., and CHOPPIN. P. W. (1974b). Isolation of paramyxovirus glycoproteins and identification of their biological properties. In “Negative Strand Viruses” (R. I). Barry and B. W. J. Mahy. eds.), Academic Press, New York, in press. SETO, J. T., BECHT, H.. and ROTT, R. (1973). Isolation
OF SV5 GLYCOPROTEINS
133
and purification of surface antigens from disrupted paramyxoviruses. 2. Med. Mikrobiol. Immunol. 159, 1-12. SPIRO, R. G. (1960). Studies on fetuin, a glycoprotein of fetal serum. I. Isolation, chemical composition and physicochemical properties. J. Biol. Chem. 235, 2860-2869. TOZAWA, H., WATANABE, M., and ISHIDA, N. (1973). Structural components of Sendai virus. Serological and physiochemical characterization of hemagglutinin subunit associated with neuraminidase activity. Virology 55, 242-253.
62, 125-133 (1974)
The Hemagglutinating Paramyxovirus:
and Neuraminidase
Interaction
with
Neuraminic
Protein
of a
Acid in Affinity
Chromatography’ ANDREAS
SCHEID
The Rockefeller
AND
University,
PURNELL
W. CHOPPIN
New York, New York 10021
Accepted July 23, 1974 The two glycoproteins of paramyxoviruses can be separated and purified by affinity chromatography on fetuin which is covalently linked to Sepharose. The glycoproteins are solubilized with Triton X-100 and applied to the column in the Triton-containing solution. The large glycoprotein (HN protein) of Simian virus 5 (SV5) which possesses both hemagglutinating and neuraminidase activities is retained by fetuin-Sepharose at 0” and passes elutes when the temperature is raised to 25”. The small glycoprotein through the column at 0”. This procedure provides a simple and effective means of obtaining high yields of paramyxovirus glycoproteins in pure form. The HN protein can be displaced from the fetuin by the neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid. This indicates that a site on the HN protein binds specifically to the neuraminic acid moieties of the fetuin. This finding is compatible with the hypothesis that the same active site is involved in the neuraminidase and the hemagglutinating activities of the HN protein of paramyxoviruses. INTRODUCTION
Previous studies from this laboratory described the isolation of the individual glycoproteins from the paramyxovirus SV5 (Scheid et al., 1972) and Newcastle disease virus (NDV) (Scheid and Choppin, 1973), and showed that with each of these viruses, both neuraminidase and hemagglutinating activities of the virion are associated with the larger of the two virion glycoproteins. This finding has been confirmed with NDV (Seto et al., 1973) and extended to Sendai virus (Tozawa et al., 1973; Scheid and Choppin, 1974a,b). Because of the association of both hemagglutinating and neuraminidase activities with the larger paramyxovirus glycoprotein, the designation HN has been suggested for this protein (Scheid and Choppin, 1974a). The smaller glycoprotein of SV5 and NDV possesses neither neuraminidase nor ‘Presented in part at the Conference Strand Viruses, Cambridge, England, 1973.
on Negative July 22-27,
hemagglutinating activity, and, therefore, it was suggested earlier that this protein might be involved in cell-fusing and hemolyzing activities associated with paramyxoviruses (Scheid et al., 1972; Scheid and Choppin, 1973). Subsequently, direct evidence for the involvement of the small glycoprotein in fusion and hemolysis was obtained independently in two laboratories with Sendai virus (Homma and Ohuchi, 1973; Scheid and Choppin, 1974a, b). When grown in certain cells, Sendai virions contain a precursor glycoprotein (designated FO, Scheid and Choppin, 1974a), and such particles lack hemolyzing and cell fusing activities and are not infectious. However, when the precursor is converted to the small virion protein (F) by proteolytic cleavage in uiuo or in uitro, the virions become infectious and acquire cell-fusing and hemolyzing activities. It is of obvious interest to investigate further the physical, chemical and biological activities of these virus membrane proteins. Such studies,
126
SCHEID
AND CHOPPIN
MATERIALS AND METHODS however, would be greatly facilitated by procedures for the preparation of the proCells. Monolayer cultures of a variant of teins in pure form and in large quantity. In the MDBK line of bovine kidney cells were addition, such a procedure would have grown in plastic bottles (Falcon) in reinvalue, in producing a purified inactivated forced Eagle’s medium (REM) (Bablanian vaccine. The preparation of the individual et al., 1965) with 10% fetal calf serum as proteins by centrifugation on Triton X-100 described previously (Choppin, 1969). To containing sucrose gradients has proved propagate cells on a large scale, they were useful in analytical studies (Scheid et al., passaged twice into disposable glass roller 1972; Scheid and Choppin, 1973), but is bottles (Bellco) with a growth area of 630 limited by the fact that only small quancm2 and the confluent monolayers of the tities of glycoprotein can be separated on second passage were used for the growth of each gradient. virus. Growth and purification of virus. Cells This communication describes a procedure, using SV5 as a model, by which large were infected with the W3 strain of the parainfluenza virus SV5 at a multiplicity quantities of the two virion glycoproteins of approximately 1 PFU per cell. After an can be separated by affinity chromatography. The same procedure has been success- adsorption period of 2 hr, 50 ml of REM fully applied to the isolation of the HN with 2% calf serum containing [3H]leucine, protein of Sendai virus (Scheid and Chop- 5 pCi/ml, was added. After 3 days the pin, 1974a). The rationale behind using medium was harvested, and the virus was purified as described previously (Scheid fetuin as a ligand in affinity chromatography is that, owing to its neuraminic acid and Choppin, 1973). Purified virus was content, fetuin can serve as a substrate for stored in 30-40s potassium tartrate at neuraminidase and as a receptor for the -70”. Isolation of the virus glycoproteins. Purihemagglutinin of myxo- and paramyxoviruses. It was, therefore, anticipated that fied virus at a concentration of 2 mg/ml of protein (Lowry et al., 1951) in 0.5 M it would specifically bind the paramyxovirus protein possessing these activities. Fe- potassium chloride and 0.01 M phosphate tuin-Sepharose has also been used to ob- buffer, pH 7.2, was disrupted with Triton tain aggregates of influenza virus hemag- X-100 (Scheid et al., 1972) at a final glutinin and neuraminidase (Becht and concentration of 2%. The fraction containRott, 1972), however individual glyco- ing the virion glycoproteins was prepared as described previously for NDV (Scheid proteins were not isolated in those studies. To avoid the formation of aggregates which and Choppin, 1973). Fetuin-Sepharose. The fetuin used in are mixtures of the different virion glycothe experiments reported here was preproteins, the nonionic detergent Triton pared by the method of Spiro (1960) from X-100 was used throughout the procedure fetal calf serum. From 350 ml of serum, 3.0 described here. In addition to describing the preparative procedure, this report pre- g protein with a neuraminic acid content of sents evidence which indicates that the 0.197 mmole per g protein was obtained. In some instances, fetuin obtained from retention of the HN protein on fetuin-Sepharose is due to the interaction between Grand Island Biological Company (Grand neuraminic acid-specific binding sites on Island, NY) was used. Sepharose 4B (Pharthe HN protein, and the neuraminic acid macia) was activated with cyanogen broresidues of fetuin. The results are compatimide, 25 mg per ml of beads (Axen et al., ble with the hypothesis that the site re- 1967), and washed with 0.1 M sodium sponsible for binding to neuraminic acid bicarbonate, pH 8.7. The activated Sephareceptors in the hemagglutination reaction rose was suspended in 1.5 vol of 0.15 M and the active site of the enzyme neuramisodium chloride and 0.1 M sodium bicarnidase may be one and the same. bonate containing 18 mg fetuin per ml
AFFINITY
CHROMATOGRAPHY
added Sepharose. The reaction was allowed to proceed for 3 hr at room temperature under gentle agitation. Solid glycine was added and allowed to react overnight at 4”. The beads were washed with 0.5 M NaCl and 0.1 M sodium acetate, pH 4.5; with 0.5 M NaCl and 0.1 M Tris, pH 9, and subsequently with water. The aqueous suspensions were stored at 4’ with 0.02% sodium azide added. Under these conditions fetuin-Sepharose proved to be stable for 1 yr as judged by its neuraminic acid content and its undiminished effectiveness as an adsorbant for neuraminidase. Column chromatography. Fetuin-Sepharose was suspended in running buffer (0.15 M sodium chloride and 0.05 M sodium acetate, pH 4.5, containing 0.1% Triton X-100), packed into columns and washed with the same buffer. Solutions of virus glycoproteins in 2% Triton X-100 and water were adjusted in their salt content to that of running buffer by the addition of l/9 vol of 1.5 ,‘M sodium chloride with 0.5 M sodium acetate, pH 4.5. Flow rates were controlled through the use of a peristaltic pump and the column temperatures by immersing the column into ice-water or a temperature-regulated water bath. Fractions were collected at selected time intervals and aliquots were analyzed for their neuraminidase and radioactivity content. Pooled fractions were dialyzed against 0.01 M phosphate buffer, pH 7.2, with 0.1% Triton X-100 and concentrated by dialysis against buffer containing 20% polyethyleneglycol. Hemagglutination titrations. Tritoncontaining samples were freed of the detergent with n-butanol as described previously (Scheid and Choppin, 1973), and titrations were done as described (Choppin, 1964) except that plastic plates with round bottom cups, 0.3-ml capacity (Lindbro Chemical Co., New Haven, CT) were used and serial dilutions were made in 0.1 ml and 0.1 ml of chicken erythrocytes was added. Neuraminic acid was determined by the procedure of Aminoff (1961). Fetuin and fetuin-Sepharose were incubated with SV5 glycoproteins in 1% Triton X-100 at pH 4.5,
OF SV5 GLYCOPROTEINS
127
and at various times the neuraminic acid released by the viral enzyme was measured. The total neuraminic acid content of fetuin and fetuin-Sepharose was determined after hydrolysis with 0.1 N sulfuric acid at 80” for 1 hr. Neuraminidase assays, polyacrylamide gel electrophoresis, and radioactivity measurements were done as described previously (Scheid et al., 1972). Chemicals and isotopes. 14C-Reconstituted protein hydrolyzate and [3H]leutine, 51 Ci per mmole, were purchased from Schwarz BioResearch, Orangeburg, NY. N-Acetylneuraminic acid was obtained from Sigma Chemical Co., St. Louis, MO. 2-Deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) was kindly supplied by Dr. Peter Palese. RESULTS
Separation of SV5 glycoproteins by affinity chromatography. Fetuin-Sepharose was characterized with respect to its content of neuraminic acid. The total neuraminic acid was determined after acid hydrolysis and 1.93 pmoles were found per ml of derivatized beads. This represented 49% of the neuraminic acid in the fetuin used in the coupling reaction. Steric changes are known to occur to ligands upon their covalent linkage to solid matrixes and this may render their properties different from the properties in solution. Thus, it is important to know whether the neuraminic acid residues remain accessible to the viral HN protein when fetuin is coupled to Sepharose. To evaluate this, fetuin and fetuin-Sepharose were exposed to SV5 glycoproteins until the amount of neuraminic acid released by the viral neuraminidase had reached a constant level. From fetuin, 46% of the total neuraminic acid could be released by neuraminidase, and from fetuin-Sepharose, 45%. This indicates that the efficacy of fetuin as a specific receptor is preserved after the covalent attachment to Sepharose. A separation of the SV5 glycoproteins was considered feasible because in Triton X-loo-containing solutions, each of the proteins exists as physically distinct enti-
128
SCHEID
AND CHOPPIN
ties (Scheid et al., 1972). The design of the chromatographic procedure took into account the fact that the hemagglutination reaction with paramyxoviruses is usually stable in the cold, and not at room temperature and, therefore, binding of the HN protein to the receptor was expected to be stronger in the cold. Other conditions of the chromatographic procedure were arrived at empirically. In the chromatographic procedure that was finally adopted, radioactively labeled virion glycoproteins in a Triton-containing solution at pH 4.5 were applied to fetuinSepharose at 0”. Figure 1 shows the elution profile and the experimental details. Much of the labeled material did not interact with the fetuin and was not retarded at the initial temperature of 0”. The column was washed at the same temperature with approximately three bed volumes of buffer. The column temperature was then raised to 25”, and the protein which was bound to the fetuin in the cold then eluted. As shown in Fig. 1, the peak of protein eluted when the temperature was raised coincides with the peak of neuraminidase activity. The two glycoprotein components which were separated in the experiment shown in Fig. 1 were analyzed by polyacrylamide gel electrophoresis (Fig. 2). The column effluent at O”, left panel, contained the smaller glycoprotein, protein F, as the only protein component. The column eluate at 25” contained the larger glycoprotein, the HN protein. The small peak at fraction 15 represents a dimer of the HN protein (Scheid et al., 1972). The proteins found in the two fractions obtained from the column were analyzed for their hemagglutinating and neuraminidase activities (Table 1). Hemagglutinin was detected only in the 25” eluate fraction, which also contained the neuraminidase activity and which, as shown in Fig. 2, is constituted solely by the HN protein. Taking the neuraminidase activity recovered with the HN protein in the eluate at 25” as an indicator for the recovery of this protein, a yield of 65% was obtained in this experiment, and similar yields were obtained in other experiments.
Yo Fract!on
20 No
30
FIG. 1. Fractionation of SV5 glycoproteins by affinity chromatography on fetuinSepharose. The glycoprotein fraction (4.5 ml) obtained from 8.0 mg of SV5 was mixed with 0.5 ml of 10 x running buffer and applied to a column 1.5 cm diameter, containing 14 ml of fetuin-Sepharose. The temperature during the sample application and up to fraction 16 was 0” and the flow rate 28 ml/cm2 hr. After fraction 16, the temperature was raised from 0” to 25”, and the flow rate was reduced to 14 ml/cm2 hr. Fractions of 4.8 ml (6 min per fraction up to fraction 16, 12 min per fraction afterwards) were collected. The arrow indicates the temperature shift from 0” to 25”. Twentyfive-microliter aliquots were analyzed for their neuraminidase content and 25.~1 aliquots were used for radioactivity determinations. Some of the OD,,, is caused by neuraminic acid that was eluted from the column through the action of the viral enzyme. However, it was established that this was negligible compared to the NANA released in the assay, and, therefore, no corrections were made for this. The fractions comprising the effluent at 0” (fractions 3-5) and the eluate at 25” (fractions 17-21) were pooled separately, dialyzed, and concentrated as described in Materials and Methods. After the concentration steps, the volumes were adjusted to 4.5 ml. Analyses of these two fractions are shown in Table 1 and Fig. 2.
Since a temperature shift was used in preparative chromatography, the following experiment was performed to demonstrate that this temperature shift was indeed responsible for the elution of the HN protein. The HN protein, which was isolated in the experiment shown in Fig. 1 was rechromatographed under the same conditions on a smaller scale (Fig. 3), and again retention at 0” was observed, and elution occurred when the temperature was raised to 25” after fraction 10 (right panel). In contrast, when the column temperature was kept at 25’ throughout the experiment, the HN protein eluted shortly after
AFFINITY
CHROMATOGRAPHY
129
OF SV5 GLYCOPROTEINS
8 I
iI
HN F 1 1
I30 Froctlon
60
90
i
No
FIG. 2. Polyacrylamide gel electrophoresis of the glycoproteins of SV5 obtained by fractionation on fetuin-Sepharose. One-hundred microliters of the fractions obtained in the experiment shown in Fig. 1 were prepared for electrophoresis by precipitation with n-butanol in the presence of 1% 2-mercaptoethanol. The left panel shows the polypeptide composition of the eluent at 0”, the right panel shows the polypeptide that was retained by fetuin-Sepharose and which eluted at ‘25”. [“C]Amino acid-labeled virions were added as a coelectrophoresis marker. The arrows indicate the position of the two marker glycoproteins. The origins are on the left and the anode on the right. TABLE
1
that temperature. Thus, initial experiments at pH 7.2 showed only incomplete retention in the cold, and therefore pH 4.5, the pH optimum of the SV5-neuraminidase, was selected. Also, a reduction of the Fraction Activity initial flow rate to one half or less leads to NeuramiHemagthe elution of the HN protein at 0”. glutinin nidase No attempts have been made to reuse (units) (HAU x 102) the beads because of the consideration that Sample applied to 252 575 some of the protein which is lost in the column procedure may have remained on the colEffluent at 0” <0.3 <2 umn and contaminate subsequent preparaEffluent at 25” 171 575 tions. Also, during each run a small but measurable part of the neuraminic acid is o The isolated proteins from the experiment in Fig. hydrolyzed through the action of the viral 1 were analyzed. Ten- and 25-p] aliquots of the pooled and concentrated fractions were analyzed for neuraenzyme. minidase. Hemagglutination was assayed after preThe nature of the interaction between cipitation of the proteins from 25-~1 aliquots with fetuin and the HN glycoprotein. The nan-butanol and resuspension of the proteins with ture of the interaction between fetuin-sebuffer. pharose and the HN protein was studied further by the use of the neuraminidase the void volume and before fraction 10 inhibitor 2-deoxy-2,3-dehydro-N-tri(Fig. 3, left panel). fluoroacetylneuraminic acid (FANA) which is a synthetic derivative of the natuThe affinity of the HN protein for fetuin rally occurring N-acetylneuraminic acid is low even at O”, and deviations from the conprocedure described may cause elution at (Meindl et al., 1974). The inhibitory BIOLOGICAL ACTIVITIES OF Sv5 GLYCOPROTEINS SEPARATED BY AFFINITY CHROMATOGRAPHYON FETUIN-SEPHAROSE"
130
SCHEID
AND CHOPPIN
Fractton
No
FIG. 3. Temperature dependence of the affinity of the HN glycoprotein of SV5 for fetuin-Sepharose. Identical columns of 0.38.cm diameter containing 0.6 ml fetuin-Sepharose were used. Right panel: The column temperature during the sample application and up to fraction 10 was O”, after fraction 10 it was raised to 25’. Left panel: The column temperature was 25” throughout. The samples, 0.1.ml aliquots of the HN protein isolated in experiment Fig. 1 (cf. Fig. 2 and Table I), were neutralized with 10 ~160 mM acetic acid, and 12 ~1 of 10 x running buffer were added. The initial flow rates and the collecting times for each sample were the same as in experiment of Fig. 1. After fraction 10 the flow was reduced to one half and the collecting times of the fractions doubled. Radioactivity was measured in 50-~1 samples.
stant of FANA for SV5 neuraminidase and hemagglutination were determined, and found to be 5 x 10m6 and 2 x 10m6 M, respectively. Purified HN protein was applied to fetuin-Sepharose, and the column temperature was maintained at 0” throughout the experiment (Fig. 4, left panel). After fraction 10, FANA was included in the running buffer at a concentration of 10m4 M, and this caused the prompt elution of the HN protein. In contrast, when N-acetylneuraminic acid (NANA), which does not inhibit neuraminidase or hemagglutination, was tested for its ability to interfere with the binding of the HN protein at 0” at a lo-fold higher concentration (Fig. 4, right panel), no elution occurred. Thus, the neuraminidase inhibitor FANA can displace the HN protein from fetuin-Sepharose, but NANA cannot. DISCUSSION
The procedure described here for the separation of the two glycoproteins of the paramyxovirus SV5 by affinity chromatography using a ligand which contains the receptor for one of the proteins has a number of advantages. It is a simple procedure which provides in one step prepara-
tive quantities of the proteins. The yield is high; the HN protein is obtained in a high degree of purity because of the specificity of the viral protein-receptor interaction; and the biological activities, i.e., hemagglutinating and neuraminidase activities, are preserved. A similar procedure has also been successful with Sendai virus glycoproteins (Scheid and Choppin, 1974a), and it thus should be applicable to other paramyxoviruses, and under certain conditions perhaps to influenza viruses also. In addition to providing a means for obtaining virion glycoproteins in quantities for further studies of their physical, chemical, and biological properties, the procedure should also prove valuable for the production of vaccines consisting of purified proteins, since the glycoproteins are the antigens of importance in inducing immunity to infection by enveloped RNA viruses. In principle, affinity chromatography using a virus receptor for isolation of receptor binding proteins of other viruses should be possible; however, the limitation at present with many viruses is lack of knowledge of a specific receptor to use as a ligand. Once it was found that in the case of paramyxoviruses, one glycoprotein, the HN protein, possessed both hemag-
AFFINITY 6-
CHROMATOGRAPHY
* NANA
+ FANA
10
20
10 Fraction
131
OF SV5 GLYCOPROTEINS . FANA
20
30
No
FIG. 4. Desorption of the HN glycoprotein of SV5 from fetuin-Sepharose with 2-deoxy-2.3-dehydro-N-trifluoroacetylneuraminic acid (FANA). Left panel:-After fraction 10. running buffer containing 0.1 mM FANA was used for further elution. Right panel: After fraction 10. the running buffer contained N-acetylneuraminic acid, 1 mM, and after fraction 20 the running buffer contained 0.1 mM FANA. The experimental conditions were identical to those of the experiment in Fig. 3 except that the column temperature was kept at 0” throughout. 3H was measured in 100.~1 aliquots.
glutinating and neuraminidase activities. (Scheid et al., 1972; Scheid and Choppin, 1973) the question arose as to whether the same site on the molecule was involved in both reactions. This is, is the binding to cellular receptors simply an enzyme-substrate reaction between the viral neuraminidase and neuraminic acid-containing substrate, or, alternatively, are different portions of the molecule involved in hemagglutinating and neuraminidase activities. In the case of influenza virus, two different active sites clearly exist, since the hemagglutinin and neuraminidase activities reside on different proteins. However, the evidence obtained here and previously (Scheid et al., 1972; Scheid and Choppin, 1973; 1974a.b) is compatible not only with both activities being present as a single protein, but also with the involvement of the same site in both reactions. The nature of the interaction of the HN protein with fetuin has been explored in the present studies by the use of 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA). This analog of N-acetyl-neuraminic acid has been shown previously to inhibit the neuraminidase activity of myxoviruses and paramyxoviruses, as well as hemagglutination by SV5 and
NDV (Meindl et al., 1974). The present finding that FANA interferes with the retention of the HN protein on fetuin-Sepharose excludes the possibility that fetuin-Sepharose is acting simply as an ion exchanger, and indicates the following mode of interaction. The active moieties on the immobilized fetuin are the neuraminic acid residues, and the HN protein reacts with these receptors through a neuraminic acid-specific binding site. This finding and the inhibition of hemagglutination by SV5 with FANA (Meindl et al., 19741, strongly suggest that in the case of parainfluenza viruses. a neuraminic acid-specific site on the HN protein is responsible for the hemagglutination reaction. Although this evidence does not prove that the active site of the neuraminidase is identical to the neuraminic acid-specific binding involved in the hemagglutination reaction, there are other results that are compatible with this view. First, there is the observation that with paramyxoviruses. hemagglutination titrations usually must be done in the cold to obtain full titers. in contrast to results with influenza virus. Further, in early studies it was found that, in contrast to influenza virus, NDV could not be converted to “indicator virus”
132
SCHEID
AND CHOPPIh
by heating (Francis. 1947; Anderson. 1948). “Indicator virus” was the term used for virus whose neuraminidase activity, but not hemagglutinating activity. was inactivated so that it became a sensitive indicator of competitive inhibition by glycoprotein inhibitors of hemagglutination. Both of these observations would be expected if the paramyxovirus hemagglutination reaction is a neuraminidase-substrate interaction. In addition, several ts mutants of parainfluenza viruses with defects in both neuraminidase and hemagglutinin activties have been isolated, but no mutant has been described in which only one of the two functions is affected (Pierce and Haywood. 1973; Preble and Youngner, 1973; Portner et al., 1974). It was shown previously that the HN protein exhibits full neuraminidase activities in the presence of the nonionic detergent Triton X-100 (Scheid et al., 1972; Scheid and Choppin, 1973). The present experiments have demonstrated that the binding of the HN protein to receptors is not prevented by the presence of Triton, since binding to fetuin-Sepharose occurred in the presence of the detergent. This question could not be investigated previously by the hemagglutination reaction, because it was essential to remove the detergent so that aggregates of the protein which were multivalent could form. It has thus now been established that both binding to receptors and neuraminidase activity can be expressed in the presence of the nonionic detergent. ACKNOWLEDGMENTS Supported by Research Grant AI-05600 from the National Institute of Allergy and Infectious Diseases. U.S.P.H.S. We thank Mrs. Ariane A. Cody for her excellent technical assistance. REFERENCES AMINOFF, D. (19611. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biothem. J. 81, 384-392. ANDERSOK, S. G. (1948). Mucins and mucoids in relation to influenza virus action. I. Inactivation by RDE and by viruses of the influenza group. of the
serum inhibitor of haemagglutination. /lust. J. Exp. Bioi. Med. Sri. 26, 34;~33. AXEK. R., PORATH. J.. and ERSBACK. s. (19671. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature iLondon) 214, 1302~1:104. BABLANIAX. R.. EGGER~;. H. J.. and TAMM. I. ( 1965,. Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirusinduced metabolic and morphological alterations in cultured cells. Virology 26, 100-l 13. BECHT. H.. and Ron, R. (1972). Purification of influenza virus hemagglutinin by affinity chromatography. Med. M~robiol. Immunoi. 158, 67-X. CHOPPIN, P. W. (1964). Multiplication of a myxovirus (SV5) with minimal cytopathic effects and without interference. Virolog), 23, 224-233. CHOPPI~, P. W. (1969). Replication of influenza virus in a continuous cell line: High yield of infective virus from cells inoculated at high multiplicity. Virolog> 39, 130-134. FRANCIS. T.. JR. (1947). Dissociation of hemagglutinating and antibody-measuring capacities of influenza virus. J. Enp. Med. 85, l-7. HOMMA. M.. and OHLCHI. M. (1973). Trypsin action on the growth of Sendai virus in tissue culture cells III. Structural difference of Sendai viruses grown in eggs and tissue culture cells. J. Viroi. 12, 1457- 1465. LOWRY, 0. H.. ROSEBROLGH, N. J.. FARH. A. L.. and RANDALL. R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MEINDL. P.. BODO. G.. PALESE. P.. SCHVLMA~, J.. and TC~PPY, H. (1974). Inhibition of neuraminidase activity by derivatives of Zdeoxy-2.3.dehydro-Nacetglneuraminic acid. Vuvlogy 58, 455-463. PIERCE, J. S.. and HAYHOOD. A. M. (197.3). Thermal inactivation of Newcastle disease virus. I. Coupled inactivation rates of hemagglutinating and neuraminidase activities. J. Viral. 11, 168~176. PORTKEK. A., MAHX. P. A.. and KI~CSBLH~, D. u’. (1974,. Isolation and characterization of Sendai virus temperature sensitive mutants. J. Krol. 13, 298-304. PREBLE, 0. T.. and YO~NNEK, ,J. S. 11973). Selection of tt mperature-sensitive mutants during persistent infection: Role in maintainance of persistent Newcastle disease virus infections of L cells. J. V~rol. 12, 481-491. SCHEID. A., CALICURI. L. A.. COMPANS, R. W., and CHOPPlh, P. u’. (1972). Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger Sv5 glycoprotein. Vlrolog3 50, 640-652. SCHEID, A.. and CHOPPIN P. u’. (1953). Isolation and purification of the envelope proteins of Newcastle disease virus. J. Viol. 11, 263-271.
AFFINITY
CHROMATOGRAPHY
SCHEID, A., and CHOPPIN, P. W. (1974a). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion. hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57, 475-490. SCHEID. A., and CHOPPIN. P. W. (1974b). Isolation of paramyxovirus glycoproteins and identification of their biological properties. In “Negative Strand Viruses” (R. I). Barry and B. W. J. Mahy. eds.), Academic Press, New York, in press. SETO, J. T., BECHT, H.. and ROTT, R. (1973). Isolation
OF SV5 GLYCOPROTEINS
133
and purification of surface antigens from disrupted paramyxoviruses. 2. Med. Mikrobiol. Immunol. 159, 1-12. SPIRO, R. G. (1960). Studies on fetuin, a glycoprotein of fetal serum. I. Isolation, chemical composition and physicochemical properties. J. Biol. Chem. 235, 2860-2869. TOZAWA, H., WATANABE, M., and ISHIDA, N. (1973). Structural components of Sendai virus. Serological and physiochemical characterization of hemagglutinin subunit associated with neuraminidase activity. Virology 55, 242-253.