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Estimation of the molecular weights of the polypeptide chains from the isolated hemagglutinin and neuraminidase subunits of influenza viruses
VIROLOGY
40, 643-654 (1970)
Estimation
of the
from
the
Molecular
Isolated
Weights
Hemagglutinin
Subunits
of Influenza
of the
Polypeptide
and
Neuraminidase
Chains
Viruses
R. G. WEBSTER of Virology
Laboratories The
University
and Immunology, of Tennessee Accepted
Medical
St. Jude Units,
Children’s Memphis,
Research Tennessee
Hospital, 388101
and
November 4, 1969
Estimation of the molecular weights of the polypeptide chains of the isolated hemagglutinin and neuraminidase of influenza viruses by exclusion chromatography gave values of 47,000 for the hemagglutinin and 58,000 for the neuraminidase. The denatured hemagglutinin and neuraminidase existed in an equilibrium between lower molecular weight polypeptides and aggregates even in the presence of 6M guanidine hydrochloride and reducing agents. Studies on the neuraminidase by polyacrylamide gel electrophoresis gave two closely spaced bands that migrated slightly more rapidly than a bovine serum albumin marker protein. The evidence suggests that the neuraminidase contains at least two polypeptide chains with a molecular weight of approximately 58,OCQthat cannot be resolved by exclusion chromatography. The efficiency of the denatured hemagglutinin and neuraminidase in blocking the activity of antibodies to these subunits was very low, suggesting that the antigenic determinants were not composed of adjacent amino acids on a single polypeptide chain. INTRODUCTION
Influenza viruses have two major viruscoded antigens on the surface of the virion, the hemagglutinin and the neuraminidase. Until recently these two antigens were believed to be one and were thought of as the virus-specific or V antigen. Recent studies (Laver and Kilbourne, 1966) have shown that this is not so and that the neuraminidase and hemagglutinin reside in separate molecules that are controlled by separate genetic loci. The hemagglutinin and neuraminidase subunits can be separated from the intact virion by a number of techniques (No11 et al., 1962; Wilson and Rafelson, 1963; Laver, 1963; Seto et al., 1966; Drzeniek et al., 1966; Laver and Valentine, 1969; Webster and Darling1 This work was supported by U.S. Public Health Service Research Grants AI 05343 and AI 08831 from the National Institute of Allergy and Infectious Diseases, the Hartford Foundation, and ALSAC.
ton, 1969). The neuraminidase isolated by these different methods has a sedimention coefficient in the range of 8 to 10 S, and electron microscopic studies (Laver and Valentine, 1969; Webster and Doarlington, 1969J show structures about 80 A long by 40 A wide congected at the midpoint by a narroy rod 100 A long to a small knob about 40 A in diameter. The separated hemagglutinin subunits have a sedimentation cgefficient of 7 to 3 S and are short rods 40 A wide and 160 A long. Now that the hemagglutinin and neuraminidase of influenza viruses can be separated in a high state of purity and the morphology and biological properties of these subunits have been established, the next questions we wish to ask are (1) how many polypeptide chains are these in each subunit and (2) what are the molecular weights of the constitutive polypeptide chains? We would also like to know whether the hemagglutinin and neuraminidase are antigenic 643
644
WEBSTER
after dissociation into their constituitive poiypeptide chains and whether their antigenie sites are located on single polypeptide chains or are shared between polypeptide chains and can be detected only when the polypeptide chains are associated. The present studies on the molecular substructure of the hemagglutinin and neuraminidase show that these subunits are made up of polypeptide chains of molecular weight of approximately 47,000 for the hemagglutinin and 58,000 for the neurThe individual polypeptide aminidase. chains were found to occur in an equilibrium state between aggregates and monomers. The evidence suggests that the antigenic sites were not made up from adjacent amino acids on a single polypeptide chain. MATERIALS
AND
METHODS
Viruses. The Ao/BEL strain of influenza and recombinants X-7 (Kilbourne et al., 1967) and X-15 (Kilbourne, 1968) were used in this study. X-7 is a recombinant influenza virus possessing the neuraminidase from Az/RI/5+ virus and the hemagglutinin from A,,/NWS virus. X-15 is a recombinant influenza virus possessing the neuraminidase from Az/RI/5* virus and the hemagglutinin from A/Equine/l influenza virus. The viruses were grown in the allantoic cavity of 11-day-old chick embryos at 35” and purified by adsorption-elution on chick erythrocytes followed by differential centrifugation and then sedimentation through a sucrose gradient (Laver, 1964). Radioisotopically labeled viruses. X-7 influenza virus was grown in BHK 31/13 cells (Stoker and Macpherson, 1964). The BHK cells were grown in plastic flasks at 37” in a medium containing Eagle’s salts (Eagle, 1959), dextrose, vitamins, glutamine, and 10 % fetal calf serum. The cells were infected at a multiplicity of approximately 1 EID,, of X-7 virus per cell, and after 60 min adsorption the cells were washed three times phosphate-buffered saline (PBS) and overlaid with Hanks’ minimum essential medium diluted Z-fold with Hanks’ saline and containing 20 &Ji/ml of a mixture of 16 acids (Schwarz BioResearch, 3H-amino average specific activity approximately 1000 mCi/mmole). The virus caused cytopathic
destruction of the cell sheet, and 36 hours after infection the released virus was purified as described above. BEL influenza virus was grown in chick embryo lung cells, kindly provided by Dr. A. Portner. The cells were infected and overlaid with Hanks’ minimum essential medium containing 3H-amino acids as described above. Thirty-six hours after infection and maintenance at 35” in 5% CO2 in air the cells were scraped off the plastic dishes and sonicated to disrupt the cells, and the virus was purified as described above. Isolation of the hemagglutinin and neuranzinidase front injluxznxa viruses. The neuraminidase of AZ/RI/j+ virus was isolated from the recombinant influenza virus X-7 by disruption of the virus with sodium dodecyl sulfate (SDS) and separation of the neuraminidase from the other virus proteins by electrophoresis on cellulose acetate (Laver, 1964; Laver and Valentine, 1969). The hemagglutinin of BEL influenza virus was separated in the same way (Laver and Valentine, 1969). Purity of the isolated hemagglutinin and neurantinidase. Extensive studies have shown that the neuraminidase and hemagglutinin isolated from X-7 and BEL influenza viruses on cellulose acetate strips are pure by electrophoretic, immunological, and electron microscopic criteria. Electrophoretic separation gives a single protein band associated only with hemagglutinin or neuraminidase activity (Laver, 1964; Laver and Kilbourne, 1966; Laver and Valentine, 1969). Injection of the isolated neuraminidase into rabbits elicits the production of antibodies that react specifically with the neuraminidase in inhibition tests and immunodiff usion tests (Webster and Pereira, 1968; Kilbourne et al., 1968; Schild and Pereira, 1969). The isolated hemagglutinin reacted only with antibodies to the specific hemagglutinin and did not react with antibodies to neuraminidase or to nucleoprotein. Electron microscopic examination of the isolated hemagglutinin and neuraminidase shows a homogeneous preparation of subunits with a uniform and characteristic morphology (Laver and Valentine, 1969). The isolated hemagglutinin and neuraminidase used in these experiments
INFLUENZA
NEURAMINIDASE
fulfill the above criteria; and the isolated neuraminidase was the same preparation given to Laver and Valentine (1969) for their morphological studies. Polyacrylamide
Electrophoresis
1. Gels without reducing agents: The isolated and concentrated neuraminidase from X-7 influenza virus was treated with 1% SDS and 1% 2-mercaptoethanol in 0.008 M phosphate, pH 7.2, for 3 hours at 37” and electrophoresed on acrylamide gels by the methods described by Evans and Kingsbury (1969). The only modification employed in some experiments was a reduction in the final concentration of acrylamide from 10 to 8 %. After electrophoresis the gels were removed from the tubes and sliced into 1 mm-thick disks using the apparatus described by Groves et al. (1968), ground in a tissue homogenizer with 0.5 ml of normal saline, and dialyzed against normal saline to remove sucrose that interfered with the estimation of neuraminidase activity. 2. Gels containing reducing agents: The isolated and purified neuraminidase from X-7 virus was precipitated with ethanol at room temperature and held at room temperature for 16 hours, and the precipitate was dissolved in electrophoresis buffer containing an additional 1% SDS and 1% dithiothreitol and was finally heated to 100” in a water bath for 10 min. The buffer was composed of 75.6 g of tris(hydroxymethyl)aminomethane (Tris), 6.0 g of boric acid, 7.5 g of Disodium ethylenediaminetetraacetate (EDTA), 4.0 g of SDS in one liter of water, pH 5.9. Gels were prepared in Lucite tubes (0.6 cm X 35.5 cm) by mixing 2 volumes of solution A (15.0 g of acrylamide, 0.405 g of N, N-methylenebisacrylamide in 100 ml of water) with 2 volumes of solution B [1.25 ml of N, N, N’ , N’-tetramethylethylenediamine (TEMED), 25.0 ml of electrophoresis buffer to 100 ml with distilled water] and 1 volume of solution C (0.19 g of ammonium persulfate in 100 ml of water). After polymerization, the gels were forced out of the tubes and soaked in a 1:4 dilution of electrophoresis buffer containing 0.01% dithiothreitol for 2 days. The gels-which had expanded slightly during soaking-were sucked back into the tubes with negative
AND
HEMAGGLUTININ
645
pressure, the ends were cut off with a razor blade to give flat surfaces and mounted in the electrophoresis equipment. Denatured protein samples were applied in 10 % glycerol and electrophoresed for 24 hours at 0.6 mA per gel at 23”. After electrophoresis the gels were extruded from the tubes and stained with 0.05% Coomassie Brilliant Blue in 45.5 % methanol and 0.1% acetic acid for 24 hours. The gels were decolorized in 45.5 % methanol, 9.1% acetic acid. Exclusion chromatography. The isolated hemagglutinin from A,/BEL influenza virus and the neuraminidase from X-7 virus labeled with 3H amino acids were denatured with 6 M guanidine hydrochloride containing 0.05 M lithium chloride, 0.1 M mercaptoethanol, and 0.01 M EDTA adjusted to pH 7.2, and chromatographed in the same solution on a Biogel-A-50m 2% beaded agarose column (50 cm X 0.9 cm), according to the method described by Davison (1965). Thyroglobulin, bovine serum albumin, chymotrypsinogen, and cytochrome c (all obtained from Sigma Chemical Company) were used as reference proteins. These proteins were dissolved in the 6 M guanidine hydrochloride denaturing solvent, and 1 mg amounts of protein were applied to the column in a total volume of 0.2 ml. Lyophilized E. coli cells (washed extensively in the denaturing solvent) were used as the exclusion volume marker, and dinitrophenylalanine as the internal volume marker. The marker proteins in the eluent samples (0.5 ml) were estimated turbidimetrically at 450 rnp after addition of 2 ml of 5 % trichloroacetic acid, cytochrome c was estimated at an optical density of 415 rnp, and DNP alanine at 350 mp in a Zeiss spectrophotometer. The isotopically labeled hemagglutinin and neuraminidase samples (0.5 ml) were collected from the column and precipitated with 3 ml of 5% trichloroacetic acid; 1 ml of bovine serum albumin (500 pg) was added as carrier protein. The precipitates were taken up in 1.0 ml of NCS (AmershamSearle, Chicago), mixed with 10 ml of toluene based scintillant, and counted in a liquid scintillation spectrometer. Antisera. Hyperimmune rabbit antisera to the viruses and to the isolated neuraminidase were prepared as described previously
646
WEBSTER
(Hilbourne et al., 1968). Antiserum to the fractions of neuraminidase from polyacrylamide gels was prepared by grinding the gel slices in saline and injecting rabbits in the footpad with the geI-protein suspension in complete Freund’s adjuvant. Hyperimmunizing doses of the same antigen were injected into the rabbits by the intraperitoneal route 40 and 60 days after the primary injection. Serum samples were collected before immunization and 7 and 14 days after each subsequent injection. Serological methods. Hemagglutinin titrations and hemagglutinin inhibition assays were done in plastic trays (WHO type) using chicken erythrocytes (Fazekas de St. Groth and Webster, 1966). Neuraminidase titrations and neuraminidase inhibition tests were done with fetuin as substrate as previously described (Webster and Pereira, 1968). Neuraminidase activity was expressed as optical density readings at 549 mF. Fractions from gels were dialyzed against physiological saline to remove the sucrose which interfered with the neuraminidase assay. Antibody blocking test for denatured hemagglutinin and neuraminidase. To detect the presence of denatured antigens, the following antibody blocking test was used. Four inhibiting doses of antibody to the hemagglutinin or neuraminidase (in 0.025 ml) were mixed with serial 2-fold dilutions of the denatured hemagglutinin or neuraminidase (0.25 ml) and allowed to react together for 30 min at room temperature. Four hemagglutinating doses of whole BEL or X-15 viruses (in 0.025 ml) were then added and 0.025 ml of 5 % chicken erythrocytes, and the test was read after 35 min at 23”. BEL virus was used to detect the presence of blocking activity caused by denatured hemagglutinin from this virus. X-15 virus that has the property of showing hemagglutination inhibition with antibodies to X-7 neuraminidase was used to detect the presence of the denatured neuraminidase from X-7. Hemagglutination indicated the presence of antigen and the end point of the test was recorded as for standard hemagglutination titrations (Fazekas de St. Groth and Webster, 1966).
RESULTS
Dissociation of Neuraminidase and Estimation of the Number and Molecular Weights of the Constitutive Polypeptide Chains by Polyacrylamide Gel Electrophoresis The isolated and purified neuraminidase from X-7 influenza virus was treated with 1% SDS and 1% mercaptoethanol at 37” for 3 hours and electrophoresed on polyacrylamide gels that did not contain reducing agents. After staining, several protein bands could be detected (Fig. 1) ; there were several bands near the top of the gel, 1 (a, b, c, d) and two well separated bands (2 and 3). Several gels were electrophoresed under identical conditions, cut into l-mm slices and assayed for enzyme activity and antigenicity. The sampIes in fraction 1 contained neuraminidase enzyme activity, while no enzyme activity was detected in bands 2 and 3. The original material and fraction 1 and fraction 2 were antigenic when injected into rabbits; i.e., they induced the formation of antibodies that neutralized the enzyme activity of the isolated intact neuraminidase subunit, whereas fraction 3 failed to induce the formation of antibodies that would neutralize neuraminidase activity. The antibodies induced in rabbits by the starting material (purified neuraminidase) and by fractions 1 and 2 were specific for the neuraminidase; they did not inhibit hemagglutination by intact virus or fix complement with ribonucleoprotein antigen. Antibodies to fraction 3 could not be detected in any of the above-mentioned tests. Approximate estimation of the molecular weights by comparison of the migration of the above fractions with protein standards (Shapiro et al., 1967) suggested that fraction 3 had a molecular weight of the order of 100,000 and the other fractions are considerably larger than this. The immediate question arises: Why were there so many high molecular weight protein bands when we supposedly started with a purified protein? There are three possibilities: first, that the isolated neuraminidase was not pure and we are dealing with a mixture of proteins; second, that the various bands represent aggregates of polypeptide
INFLUENZA
NEURAMINIDASE
chains that make up the neuraminidase; or third, that there are several neuraminidases. The second possibility seems the most likely, for fractions 1 (a, b, c, d) and 2 have enzyme activity or can induce specific antineuraminidase antibodies, and the following results support this contention. Electrophoresis of Dissociated Neuranzinidase in Gels Containing Reducing Agent The higher neuraminidase
molecular weight bands of that occurred in the above
Neurammdase
AND
IIEMAGGLUTININ
647
experiments may have been due to incomplete denaturation of the protein before electrophoresis or to reaggregation due to the lack of reducing conditions during electrophoresis. To overcome these difficulties and ensure dissociation, the isolated neuraminidase was extensively denatured by precipitation with ethanol at 20” and treatment with 1% SDS, 1% dithiothreitol and heated to 100” and then electrophoresed on polyacrylamide gels that had been presoaked in buffer containing 0.1% dithiothreitol (Laver,
OCtwlty
Induchm of neurom~n!dase mhibiing anhbodies m robbits
Fro. l.-Electrophoresis of the isolated neuraminidase from influenza (X-7) on polyacrylamide gels that did not contain reducing agent. Isolated neuraminidase (0.1 ml) (containing approximately 2000 units of neuraminidase*) was treated with 1% SDS and 1% mercaptoethanol at 37” for 3 hours and electrophoresed on polyacrylamide gels. A final concentration of 8% acrylamide was used in the gels, but in all other ways the methods of Shapiro et al. (1967) were followed. The gels were sliced into l-mm disks in the apparatus described by Groves et al. (1968). Neuraminidase activity was estimated after grinding each slice in a tissue homogenizer with 0.5 ml of 0.15 M sodium chloride and dialysis overnight against 0.15 M NaCl before assay for neuraminidase. Rabbits were injected with samples mixed with complete Freund’s adjuvant into the footpad and were boosted 40 and 60 days later by the intraperitoneal route. The molecular weight determinations were done in 10% gels according to Shapiro et al. (1967) using bovine serum albumin, pepsin, and chymotrypsinogen as markers. * One unit of neuraminidase is defined as that quantity which will give a reading of 0.50 optical density units at 549 Q in the neuraminidase test with excess fetuin as substrate.
648
WEBSTER
FIG. 2. Electrophoresis of the isolated neuraminidase from influenza containing reducing agent. Isolated neuraminidase, 0.1 ml, was treated threitol as described in Materials and Methods and electrophoresed taining dithiothreitol.
1970). The results obtained are shown in Fig. 2. There were two main protein bands running closely (1, 2) together that migrate slightly more rapidly than a bovine serum albumin marker electrophoresed in a separate column under identical conditions. Very minor bands (a, b, c) could still be detected suggesting that under these conditions aggregation still occurred, or that minor contaminants of high molecular weight were present. Denaturation sf the Hemagglutinin and Neuraminidase- of In&&& Virus with 6 M Guanidine Hydrochloride The second method employed in this study to dissociate the hemagglutinin and neuraminidase subunits of influenza viruses was treatment with guanidine hydrochloride. Attempts to denature the neuraminidase subunit with 1% SDS and 1% mercaptoethanol without heating to 100’ were unsuccessful; such treatment, in fact, increased the enzyme activity of the preparation. As a denaturing agent, 6 R’I guanidine hydrochloride is more effective than SDS and breaks all noncovalent bonds (Kawahara and Tanford, 1966). Treatment of neuraminidase with this reagent abolished all biological activity when assayed in the presence of the reagent, but if the 6 M guanidine hydrochloride was first dialyzed away, about 10% of the enzyme activity of the neuraminidase activity returned. After dialysis against iodoacetamide or mercaptoethanol, no enzyme activity was detected. This experiment shows that enzyme activity was abolished by guanidine hydrochloride
(X-7) on polyacrylamide gels with 1% SDS and 170 dithioon acrylamide gels con-
and mercaptoethanol treatment and the following experiments support the contention of Kawahara and Tanford (1966) that guanidine hydrochloride is an efficient method for dissociating proteins. Exclusion Chromatography BEL Hemagglutinin Neuraminidase
of Labeled Ao/ and AzlRI/5+
The isolated and 3H-labeled hemagglutinin and neuraminidase were denatured in 6 M guanidine hydrochloride under reducing conditions and applied to beaded agarose columns (Biogel A-50m). The results are shown in Figs. 3 and 4. The denatured hemagglutininand the neuraminidase both gave three peaks of radioactivity. In each case, the first peak came through the column with the E. coli exclusion volume marker, the second peak before the thyroglobulin marker, and the main peak of radioactivity after the bovine serum albumin marker. Again, we are faced with multiple peaks of activity from isolated virus subunits (hemagglutinin and neuraminidase), and the following experiments will show that the first two peaks of activity to elute from the columns are probably aggregates. The labeled material from each peak was dialyzed to remove guanidine hydrochloride and tested for neuraminidase or hemagglutinin activity both in the direct tests and in antibody blocking tests. The major peak of activity from the neuraminidase gave very weak antibody blocking activity, but no hemagglutinin or neuraminidase activity could be detected in any of the peaks.
1NFLUENZA
NEURAMINIDASE
AND
HEMAGGLUTIN
IN
649
C&hmma
FIG. 3. Exclusion chromatography of the isolated hemagglutinin (Aa/BEL) from in6uenza virus on beaded agarose. Isolated hemagglutinin from Ao/BEL influenza virus labeled with 3H-labeled amino acids in chick lung cells was denatured with 6 M guanidine hydrochloride containing 0.05 M lithium chloride, 0.1 M mercaptoethanol, and 0.01 M ethylenediaminetetraacetic acid (EDTA) adjusted to pH 7.2 and chromatographed in the same solution on a Biogel A50m beaded 2% agarose column (50 cm X 0.9 cm). Samples (0.5 ml) were collected and precipitated with 3 ml of 57, trichloroacetic acid to which 1 ml of bovine serum albumin (500 pg) was added as carrier protein. The precipitates were taken up in 1.0 ml of NCS (Amersham-Searle, Chicago), mixed with 10 ml of a toluene-based scintillant and counted in a liquid scintillation spectrometer.
The Capacity of Denatured Neuraminidase to Reaggregate The early peaks of radioactivity from the exclusion gel chromatography experiments were a constant finding, and attempts were made to ensure denaturation of the neuraminidase subunit. Samples of neuraminidase were dialyzed againt 6 M guanidine hydrochloride denaturing reagent and then sealed in glass and heated in a boiling water bath for 30 min. The resulting material still showed three peaks of activity on beaded agarose columns in the presence of the denaturing reagent. The isolated neuraminidase from X-7 influenza virus was labeled with %I (Webster et al., 1962) and chromatographed on beaded agarose columns with 6 Ad guanidine hydro-
chloride under reducing conditions. Denaturation of the neuraminidase was less effective after labeling with 1251, and a greater proportion of the counts came through the column with the exclusion marker. However, once again three peaks of activity were obtained (Fig. 5). The eluted material in peaks 2 and 3 (fractions 1 and 2) were concentrated and reapplied to the column. One might expect that the material from the isolated peaks would be homogeneous with respect to molecular weight and should rechromatograph as single peaks. However, if the original pattern of three peaks of activity was obtained on rechromatography, one would have to conclude that there was an equilibrium between free and aggregated polypeptide chains. In each case the radio-
650
WEBSTER
.
I
I I ‘-1
1 I
I
31;
20
SAMPLE MOdw$.
E,c II
I
I
40
5c
-\.-.. s., I
6:
I
70
NUMBER
r Thyroglobul!n
?
T
S.S.A Chym?&snogen cyi bchrome
0. .P olonlne t
FIG. 4. Exclusion chromatography of the isolated neuraminidase (AZ/RI/~+) from influenza virus on beaded agarose. Isolated neuraminidase from the recombinant X-7 influenza virus labeled with SHlabeled amino acids in BHK 21/13 cells WM denatured with 6 M guanidine hydrochloride as described for Fig. 3.
was again found in three peaks (Fig. 5B and 5C) suggesting that even in the presence of 6 M guanidine hydrochloride and reducing conditions reaggregation occurs. The isolated hemagglutinin (labeled with ?I) was chromatographed under the same conditions as described above for the neuraminidase. The isolated fractions of labeled hemagglutinin from the column also reequilibrated into three peaks on rechromatography.
activity
Estimation of the Molecular Weight of Ad BEL Hemagglutinin and AJRI/5+ Neuraminidase by Exclusion Chromatography To estimate the molecular weights of the hemagglutinin and neuraminidase accurately, four separate experiments were carried out on the major peaks of activity, and the reproducibility between experiments was
very good. In each case, the peak activity of the marker proteins and the radioactive samples eluted in an identical fashion in each experiment and the results are shown in Fig. 6. The distribution coefficients for the main peaks of activity for each protein were calculated according to Davison (1968)) and the estimated molecular weight of the neuraminidase polypeptide chains was 58,000 and for the hemagglutinin the estimated molecular weight was 47,000. The implicit assumption was that the lowest molecular weight peak that contained the majority of the radioactivity also contained the monomeric forms of the hemagglutinin or the neuraminidase. This was probably justified in that 6 M guanidine hydrochloride is a very efficient denaturing agent, especially in the presence of reducing agents.
INFLUENZA
NEURAMINIDASE
Attempts to Demonstrate the Presence oj Antigenic Determinants on Dissociated Hemagglutinin and Neuraminidase Subunits In addition to determining the molecular weight of the polypeptide chains making up the hemagglutinin and neuraminidase, we would like to know where the antigenic determinants are located and whether the separated polypeptides have any activity. Antibody blocking tests were carried out after denaturation of the hemagglutinin and neuraminidase with guanidine hydrochloride under reducing conditions and on the fractions from the agarose columns. Extremely weak blocking activity was obtained with denatured neuraminidase, but denatured hemagglutinin failed to block antibodies in the hemagglutination inhibition test. Denaturation of 1000 enzyme units of neuraminidase (1 unit being that amount of neuraminidase that will give an optical density reading of 0.50 at 549 rnp after 30 min action on excess fetuin substrate) gave one blocking unit and, hence, the reaction is very inefficient. The denatured neuraminidase was also tested for antibody blocking activity in the neuraminidase inhibition test with specific antineuraminidase antibodies, but in this test no blocking activity could be detected. These results would indicate that the antigenic determinants are probably not composed of adjacent amino acids on a single polypeptide chain, for antigenic activity was lost on denaturation. The determinants may be made up from folded areas on a single polypeptide chain or could be shared among different polypeptide chains. The small amount of biological activity in the antibody blocking test with neuraminidase may have been due to random reassortment of chains into aggregates. The enormous capacity of the denatured proteins to reaggregate suggests that more biological activity should be obtained during renaturation and it might just be a case of controlling the ionic and pH conditions more carefully. DISCUSSION
Studies on the isolated and denatured hemagglutinin and neuraminidase of influ-
AND
HEMAGGLUTININ
A
FIG. 5. Reaggregation of neuraminidase subunits. Isolated neuraminidase (RI/5+) was labeled with Ias1 (Webster et al., 1962) and chromatographed on agarose columns as described in Fig. 3. Samples 28-33 (fraction 1) and 47-52 (fraction 2) were pooled, concentrated to 0.2 ml by dialysis against polyethylene glycol 20,000, and rechromatographed after dialysis against 6 M guanidine hydrochloride denaturing solution as given in Fig. 3. (A) Chromatography of rzSI-labeled neuraminidase on an agarose column. (B) Rechromatography of fraction I from A. (C) Rechromatography of fraction II from A.
enza viruses by exclusion chromatography gave three peaks of radioactivity for each type of subunit. Estimation of the molecular weight of the major peak of activity gave a
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WEBSTER
FIG. 6. Molecular weight estimates of the hemagglutinin and neuraminidase of influenza viruses. The molecular weights of the isolated and labeled hemagglutinin (BEL) and the neuraminidase (RI/5+) were determined by exclusion chromatography on beaded agarose as described by Davison (1968).
value of 47,000 for the hemagglutinin and 58,000 for the neuraminidase. The two peaks of activity that, correspond with polypeptides of higher molecular weight were probably aggregates of the lower molecular weight, polypeptides and evidence was presented to show that reaggregation of the lower molecular weight polypeptides occurs. The studies of the isolated and denatured neuraminidase by polyacrylamide gel electrophoresis in SDS under reducing conditions gave two closely spaced bands that migrated slightly more rapidly than the bovine serum albumin reference protein. These results from polyacrylamide gel electrophoresis suggest, that the neuraminidase is composed of at least, two polypeptide chains whereas exclusion chromatography gave one peak of activity that would correspond with polypeptides of this size. These apparently contradicting results could be explained on the grounds that polyacrylamide gel electrophoresis gave greater resolution than exclu-
sion chromatography. An alternative explanation would be that one of the two bands obtained on polyacrylamide gel electrophoresis is an aggregate of the other, but from the close proximity of the two bands, after prolonged electrophoresis this would seem less likely. The hemagglutinin of BEL influenza virus has also been separated into two bands on polyacrylamide gel electrophoresis (Laver, 1970), but exclusion chromatography was not, able to resolve these polypeptides. Both the hemagglutinin and the neuraminidase subunits of influenza viruses are relatively large structures and from the sedimentation data and their dimensions as determined by electron microscopy (Laver and Valentine, 1969; Webster and Darlington, 1969), they should have molecular weights in excess of 150,000. This would mean that there must, be at least, three polypeptides of approximately equal size (HA = 47,000; N = 58,000) per molecule. Electron micrographs of the isolated neuraminidase show complex structures (Laver and Valentine, 1g069). The top o,f the structures is about 80 A long and 52 A wide with a centrally attached fiber 100 A long with a knob about 40 A in diameter at the end. It is tempting to postulate that the denser band obtained on polyacrylamiie gel electrophoresis represents the 80 A structure while the Jess dense band represents the smaller 40 A knob. This can only be speculation until the substructures of the neuraminidase subunit are separated and studied independently. The ability of the denatured hemagglutinin and neuraminidase to form aggregates even under reducing conditions shows that the polypeptides have a great tendency to form some kind of quaternary structure. This type of aggregation, which was certainly not specific under reducing conditions, may be a special property of virus proteins and may be necessary during assembly in the infected cell. The low efficiency of the denatured hemagglutinin
and neuraminidase
at blocking
the
activity of antibodies to these subunits was disappointing, particularly since we are interested in studying the antigenic determinants of these subunits. The evidence would indicate that the determinants are not
INFLUENZA
NEURAMINIDASE
composed of adjacent amino acids on a single polypeptide chain and that the polypeptide chain cannot be renatured to give antigenic determinants or enzymatic activity. Estimates of the molecular weights of the virion proteins of influenza virus on polyacrylamide gel electrophoresis (White et al., 1970) gave values of 50,000 for the hemagglutinin and 20,000 for the neuraminidase monomers. Dimmock and Watson (1970) separated five structural proteins from i&uenza virions on polyacrylamide gels. One of these (V,) had a molecular weight of 81,000 and another (V,) had a molecular weight of 63,000, and these may or may not correspond to the hemagglutinin and neuraminidase subunits. The difficulties with these studies is lack of positive identification between the subunits of the viruses and the protein bands obtained on acrylamide gels. These proteins may or may not be the hemagglutinin or neuraminidase. Initial separation of the influenza virion subunits followed by characterization as was done in this report leaves no doubt as to the identification of the proteins. The different values obtained by different workers might be explained on the grounds that different strains of influenza viruses were used in the studies. However, the similarity in sedimentation coefficients and the dimensions of the hemagglutinin and neuraminidase from different strains of influenza argues against this possibility. The more likely explanations are aggregation of proteins and lack of positive identification of the proteins on acrylamide gels that would permit comparison of results. ACKNOWLEDGMENTS The author wishes to thank Dr. W. G. Laver for carrying out the electrophoresis on polyacrylamide gels containing dithiothreitol. The excellent technical assistance of Mr. Melvin C. Smith is gratefully acknowledged. REFERENCES DAVISON, P. F. (1968). Proteins in denaturing solvents: Gel exclusion studies. Science 161, 90% 907. DIMMOCK, N. J., and WATSON, D. H. (1970). Nonvirion antigens of influenza virus. “The Biology of Large RNA Viruses,” in press.
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DRZENIEK, R., SETO, J. T., and ROTT, R. (1966). Characterization of neuraminidases from myxoviruses. Biochim. Biophys. Acta 128,547~558. EAGLE, H. (1959). Amino acid metabolism in mammalian cell culture. Science 130.432437. EVANS, M. J., and KINGSBURY, D. W. (1969). Separation of Newcastle disease virus proteins by polyacrylamide gel electrophoresis. Virology 37, 597-604. FAZ~KAS DE ST. GROTH, S., and WEBSTER, R. G. (1966) Disquisitions on original antigenic sin. 1. Evidence in man. J. Exptl. Med. 124,331-345. GROVES, W. E., DAVIS, F. C., JR., and SILLS, B. H. (1968). An automatic device for sectioning analytical polyacrylamide gels: Radioactive Escherichia coli 50 S ribosomal proteins. Anal. Bioshem. 24, 462-469. KAW.AHARA, K., and TANFORU, C. (1966). The number of polypeptide chains in rabbit muscle aldolase. Biochemistry 5, 1578-1584. KILBOURNE, E. D. (1968). Recombination of influenza A viruses of human and animal origin. Science 160, 74-76. KILBOURNE, E. D., LIICF, F. S., SCHULMAN, J. L., JAHIEL, R. I., and LAVER, W. G. (1967). Antigenie hybrids of influenza viruses and their implications. Perspectives Viral. 5, 87-106. KILBOURNE, E. I~., LAVER, W. G., SCHULMAN, J. L., and WEBSTER, R. G. (1968). Antiviral activity of antiserum specific for an influenza neuraminidase. J. Viral. 2,281-288. LAVER, W. G. (1963). The structure of influenza viruses. 3. Disruption of the virus particle and separation of neuraminidase activity. Virology 20, 251-262. LAVICR, W. G. (1964). Structural studies on the protein subunits from three strains of influenza virus. J. Mol. BioZ. 9,109-124. LAVF:R, W. G. (1970). Chemistry of antigenic variation in influenza virus. Properties of the surface subunits of antigenic variants. “The Biology of Large RNA Viruses,” in press. LAV~R, W. G., aud KILBOURNE, E. D. (1966). Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30,493-501. LAVF,R, W. G., and VALt:NTINE, R. C. (1969). Morphology of the isolated hemagglutinin and neuraminidase subunits of influenza virus. Virology 38, 105-119. NOLL, H., AOYAGI, T., and OILLANDO, J. (1962). The structural relationship of sialidase to the influenza virus surface. Virology 18,154-157. SCHILD, G. C., and PEIIEIRA, H. G. (1969). Characterization of the ribonucleoprotein and neuraminidase of influenza A viruses by immunodiffusion. J. Gen. Viral. 4,355-363. SETO, J. T., DRZENIX, R., and ROTT, R. (1966).
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Isolation of a low molecular weight sialidase (neuraminidase) from influenza virus. Biochim. Biophys. Acta 113, 492-404. SHAPIRO, A. L., VI~UELA, E., and MAIZEL, J. V., JR., (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-820. STOKER, M., and MACPHERSON, I. (1964). Syrian hamster fibroblast cell line BHK 21 and its derivatives. Nature 203, 1355-1357. WEBSTER, R. G., and DARLINQTON, R. W. (1969). Disruption of myxoviruses with Tween 20 and isolation of biologically active hemagglutinin and neuraminidase subunits. J. Viral. 4, 182187.
R. G., and PEREIRA, H. G. (1968). A common surface antigen in influenza viruses from human and avian sources. J. Gen. Viral. 3, 201-208. WEBSTER, R. G., LAVER, W. G., and FAZEKAS DE ST. GROTH, S. (1962). Methods in immunochemistry of viruses. 3. Simple techniques for labelling antibodies with Ia11 and 3%. Australian J. WEBSTER,
Exptl. Biol. Med. Sci. 40,321328. WHITE, D. O., TAYLOR, J. M., HASLAM, E. A., and HAMPSON, A. W. (1970). Synthesis and transport
of influenza polypeptides. “The Biology of Large RNA Viruses,” in press. WILSON, V. W., and RAFELSON, M. E. (1963). Isolation of neuraminidase from influenza virus. Biochem Prep. 10, 113-117.
40, 643-654 (1970)
Estimation
of the
from
the
Molecular
Isolated
Weights
Hemagglutinin
Subunits
of Influenza
of the
Polypeptide
and
Neuraminidase
Chains
Viruses
R. G. WEBSTER of Virology
Laboratories The
University
and Immunology, of Tennessee Accepted
Medical
St. Jude Units,
Children’s Memphis,
Research Tennessee
Hospital, 388101
and
November 4, 1969
Estimation of the molecular weights of the polypeptide chains of the isolated hemagglutinin and neuraminidase of influenza viruses by exclusion chromatography gave values of 47,000 for the hemagglutinin and 58,000 for the neuraminidase. The denatured hemagglutinin and neuraminidase existed in an equilibrium between lower molecular weight polypeptides and aggregates even in the presence of 6M guanidine hydrochloride and reducing agents. Studies on the neuraminidase by polyacrylamide gel electrophoresis gave two closely spaced bands that migrated slightly more rapidly than a bovine serum albumin marker protein. The evidence suggests that the neuraminidase contains at least two polypeptide chains with a molecular weight of approximately 58,OCQthat cannot be resolved by exclusion chromatography. The efficiency of the denatured hemagglutinin and neuraminidase in blocking the activity of antibodies to these subunits was very low, suggesting that the antigenic determinants were not composed of adjacent amino acids on a single polypeptide chain. INTRODUCTION
Influenza viruses have two major viruscoded antigens on the surface of the virion, the hemagglutinin and the neuraminidase. Until recently these two antigens were believed to be one and were thought of as the virus-specific or V antigen. Recent studies (Laver and Kilbourne, 1966) have shown that this is not so and that the neuraminidase and hemagglutinin reside in separate molecules that are controlled by separate genetic loci. The hemagglutinin and neuraminidase subunits can be separated from the intact virion by a number of techniques (No11 et al., 1962; Wilson and Rafelson, 1963; Laver, 1963; Seto et al., 1966; Drzeniek et al., 1966; Laver and Valentine, 1969; Webster and Darling1 This work was supported by U.S. Public Health Service Research Grants AI 05343 and AI 08831 from the National Institute of Allergy and Infectious Diseases, the Hartford Foundation, and ALSAC.
ton, 1969). The neuraminidase isolated by these different methods has a sedimention coefficient in the range of 8 to 10 S, and electron microscopic studies (Laver and Valentine, 1969; Webster and Doarlington, 1969J show structures about 80 A long by 40 A wide congected at the midpoint by a narroy rod 100 A long to a small knob about 40 A in diameter. The separated hemagglutinin subunits have a sedimentation cgefficient of 7 to 3 S and are short rods 40 A wide and 160 A long. Now that the hemagglutinin and neuraminidase of influenza viruses can be separated in a high state of purity and the morphology and biological properties of these subunits have been established, the next questions we wish to ask are (1) how many polypeptide chains are these in each subunit and (2) what are the molecular weights of the constitutive polypeptide chains? We would also like to know whether the hemagglutinin and neuraminidase are antigenic 643
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WEBSTER
after dissociation into their constituitive poiypeptide chains and whether their antigenie sites are located on single polypeptide chains or are shared between polypeptide chains and can be detected only when the polypeptide chains are associated. The present studies on the molecular substructure of the hemagglutinin and neuraminidase show that these subunits are made up of polypeptide chains of molecular weight of approximately 47,000 for the hemagglutinin and 58,000 for the neurThe individual polypeptide aminidase. chains were found to occur in an equilibrium state between aggregates and monomers. The evidence suggests that the antigenic sites were not made up from adjacent amino acids on a single polypeptide chain. MATERIALS
AND
METHODS
Viruses. The Ao/BEL strain of influenza and recombinants X-7 (Kilbourne et al., 1967) and X-15 (Kilbourne, 1968) were used in this study. X-7 is a recombinant influenza virus possessing the neuraminidase from Az/RI/5+ virus and the hemagglutinin from A,,/NWS virus. X-15 is a recombinant influenza virus possessing the neuraminidase from Az/RI/5* virus and the hemagglutinin from A/Equine/l influenza virus. The viruses were grown in the allantoic cavity of 11-day-old chick embryos at 35” and purified by adsorption-elution on chick erythrocytes followed by differential centrifugation and then sedimentation through a sucrose gradient (Laver, 1964). Radioisotopically labeled viruses. X-7 influenza virus was grown in BHK 31/13 cells (Stoker and Macpherson, 1964). The BHK cells were grown in plastic flasks at 37” in a medium containing Eagle’s salts (Eagle, 1959), dextrose, vitamins, glutamine, and 10 % fetal calf serum. The cells were infected at a multiplicity of approximately 1 EID,, of X-7 virus per cell, and after 60 min adsorption the cells were washed three times phosphate-buffered saline (PBS) and overlaid with Hanks’ minimum essential medium diluted Z-fold with Hanks’ saline and containing 20 &Ji/ml of a mixture of 16 acids (Schwarz BioResearch, 3H-amino average specific activity approximately 1000 mCi/mmole). The virus caused cytopathic
destruction of the cell sheet, and 36 hours after infection the released virus was purified as described above. BEL influenza virus was grown in chick embryo lung cells, kindly provided by Dr. A. Portner. The cells were infected and overlaid with Hanks’ minimum essential medium containing 3H-amino acids as described above. Thirty-six hours after infection and maintenance at 35” in 5% CO2 in air the cells were scraped off the plastic dishes and sonicated to disrupt the cells, and the virus was purified as described above. Isolation of the hemagglutinin and neuranzinidase front injluxznxa viruses. The neuraminidase of AZ/RI/j+ virus was isolated from the recombinant influenza virus X-7 by disruption of the virus with sodium dodecyl sulfate (SDS) and separation of the neuraminidase from the other virus proteins by electrophoresis on cellulose acetate (Laver, 1964; Laver and Valentine, 1969). The hemagglutinin of BEL influenza virus was separated in the same way (Laver and Valentine, 1969). Purity of the isolated hemagglutinin and neurantinidase. Extensive studies have shown that the neuraminidase and hemagglutinin isolated from X-7 and BEL influenza viruses on cellulose acetate strips are pure by electrophoretic, immunological, and electron microscopic criteria. Electrophoretic separation gives a single protein band associated only with hemagglutinin or neuraminidase activity (Laver, 1964; Laver and Kilbourne, 1966; Laver and Valentine, 1969). Injection of the isolated neuraminidase into rabbits elicits the production of antibodies that react specifically with the neuraminidase in inhibition tests and immunodiff usion tests (Webster and Pereira, 1968; Kilbourne et al., 1968; Schild and Pereira, 1969). The isolated hemagglutinin reacted only with antibodies to the specific hemagglutinin and did not react with antibodies to neuraminidase or to nucleoprotein. Electron microscopic examination of the isolated hemagglutinin and neuraminidase shows a homogeneous preparation of subunits with a uniform and characteristic morphology (Laver and Valentine, 1969). The isolated hemagglutinin and neuraminidase used in these experiments
INFLUENZA
NEURAMINIDASE
fulfill the above criteria; and the isolated neuraminidase was the same preparation given to Laver and Valentine (1969) for their morphological studies. Polyacrylamide
Electrophoresis
1. Gels without reducing agents: The isolated and concentrated neuraminidase from X-7 influenza virus was treated with 1% SDS and 1% 2-mercaptoethanol in 0.008 M phosphate, pH 7.2, for 3 hours at 37” and electrophoresed on acrylamide gels by the methods described by Evans and Kingsbury (1969). The only modification employed in some experiments was a reduction in the final concentration of acrylamide from 10 to 8 %. After electrophoresis the gels were removed from the tubes and sliced into 1 mm-thick disks using the apparatus described by Groves et al. (1968), ground in a tissue homogenizer with 0.5 ml of normal saline, and dialyzed against normal saline to remove sucrose that interfered with the estimation of neuraminidase activity. 2. Gels containing reducing agents: The isolated and purified neuraminidase from X-7 virus was precipitated with ethanol at room temperature and held at room temperature for 16 hours, and the precipitate was dissolved in electrophoresis buffer containing an additional 1% SDS and 1% dithiothreitol and was finally heated to 100” in a water bath for 10 min. The buffer was composed of 75.6 g of tris(hydroxymethyl)aminomethane (Tris), 6.0 g of boric acid, 7.5 g of Disodium ethylenediaminetetraacetate (EDTA), 4.0 g of SDS in one liter of water, pH 5.9. Gels were prepared in Lucite tubes (0.6 cm X 35.5 cm) by mixing 2 volumes of solution A (15.0 g of acrylamide, 0.405 g of N, N-methylenebisacrylamide in 100 ml of water) with 2 volumes of solution B [1.25 ml of N, N, N’ , N’-tetramethylethylenediamine (TEMED), 25.0 ml of electrophoresis buffer to 100 ml with distilled water] and 1 volume of solution C (0.19 g of ammonium persulfate in 100 ml of water). After polymerization, the gels were forced out of the tubes and soaked in a 1:4 dilution of electrophoresis buffer containing 0.01% dithiothreitol for 2 days. The gels-which had expanded slightly during soaking-were sucked back into the tubes with negative
AND
HEMAGGLUTININ
645
pressure, the ends were cut off with a razor blade to give flat surfaces and mounted in the electrophoresis equipment. Denatured protein samples were applied in 10 % glycerol and electrophoresed for 24 hours at 0.6 mA per gel at 23”. After electrophoresis the gels were extruded from the tubes and stained with 0.05% Coomassie Brilliant Blue in 45.5 % methanol and 0.1% acetic acid for 24 hours. The gels were decolorized in 45.5 % methanol, 9.1% acetic acid. Exclusion chromatography. The isolated hemagglutinin from A,/BEL influenza virus and the neuraminidase from X-7 virus labeled with 3H amino acids were denatured with 6 M guanidine hydrochloride containing 0.05 M lithium chloride, 0.1 M mercaptoethanol, and 0.01 M EDTA adjusted to pH 7.2, and chromatographed in the same solution on a Biogel-A-50m 2% beaded agarose column (50 cm X 0.9 cm), according to the method described by Davison (1965). Thyroglobulin, bovine serum albumin, chymotrypsinogen, and cytochrome c (all obtained from Sigma Chemical Company) were used as reference proteins. These proteins were dissolved in the 6 M guanidine hydrochloride denaturing solvent, and 1 mg amounts of protein were applied to the column in a total volume of 0.2 ml. Lyophilized E. coli cells (washed extensively in the denaturing solvent) were used as the exclusion volume marker, and dinitrophenylalanine as the internal volume marker. The marker proteins in the eluent samples (0.5 ml) were estimated turbidimetrically at 450 rnp after addition of 2 ml of 5 % trichloroacetic acid, cytochrome c was estimated at an optical density of 415 rnp, and DNP alanine at 350 mp in a Zeiss spectrophotometer. The isotopically labeled hemagglutinin and neuraminidase samples (0.5 ml) were collected from the column and precipitated with 3 ml of 5% trichloroacetic acid; 1 ml of bovine serum albumin (500 pg) was added as carrier protein. The precipitates were taken up in 1.0 ml of NCS (AmershamSearle, Chicago), mixed with 10 ml of toluene based scintillant, and counted in a liquid scintillation spectrometer. Antisera. Hyperimmune rabbit antisera to the viruses and to the isolated neuraminidase were prepared as described previously
646
WEBSTER
(Hilbourne et al., 1968). Antiserum to the fractions of neuraminidase from polyacrylamide gels was prepared by grinding the gel slices in saline and injecting rabbits in the footpad with the geI-protein suspension in complete Freund’s adjuvant. Hyperimmunizing doses of the same antigen were injected into the rabbits by the intraperitoneal route 40 and 60 days after the primary injection. Serum samples were collected before immunization and 7 and 14 days after each subsequent injection. Serological methods. Hemagglutinin titrations and hemagglutinin inhibition assays were done in plastic trays (WHO type) using chicken erythrocytes (Fazekas de St. Groth and Webster, 1966). Neuraminidase titrations and neuraminidase inhibition tests were done with fetuin as substrate as previously described (Webster and Pereira, 1968). Neuraminidase activity was expressed as optical density readings at 549 mF. Fractions from gels were dialyzed against physiological saline to remove the sucrose which interfered with the neuraminidase assay. Antibody blocking test for denatured hemagglutinin and neuraminidase. To detect the presence of denatured antigens, the following antibody blocking test was used. Four inhibiting doses of antibody to the hemagglutinin or neuraminidase (in 0.025 ml) were mixed with serial 2-fold dilutions of the denatured hemagglutinin or neuraminidase (0.25 ml) and allowed to react together for 30 min at room temperature. Four hemagglutinating doses of whole BEL or X-15 viruses (in 0.025 ml) were then added and 0.025 ml of 5 % chicken erythrocytes, and the test was read after 35 min at 23”. BEL virus was used to detect the presence of blocking activity caused by denatured hemagglutinin from this virus. X-15 virus that has the property of showing hemagglutination inhibition with antibodies to X-7 neuraminidase was used to detect the presence of the denatured neuraminidase from X-7. Hemagglutination indicated the presence of antigen and the end point of the test was recorded as for standard hemagglutination titrations (Fazekas de St. Groth and Webster, 1966).
RESULTS
Dissociation of Neuraminidase and Estimation of the Number and Molecular Weights of the Constitutive Polypeptide Chains by Polyacrylamide Gel Electrophoresis The isolated and purified neuraminidase from X-7 influenza virus was treated with 1% SDS and 1% mercaptoethanol at 37” for 3 hours and electrophoresed on polyacrylamide gels that did not contain reducing agents. After staining, several protein bands could be detected (Fig. 1) ; there were several bands near the top of the gel, 1 (a, b, c, d) and two well separated bands (2 and 3). Several gels were electrophoresed under identical conditions, cut into l-mm slices and assayed for enzyme activity and antigenicity. The sampIes in fraction 1 contained neuraminidase enzyme activity, while no enzyme activity was detected in bands 2 and 3. The original material and fraction 1 and fraction 2 were antigenic when injected into rabbits; i.e., they induced the formation of antibodies that neutralized the enzyme activity of the isolated intact neuraminidase subunit, whereas fraction 3 failed to induce the formation of antibodies that would neutralize neuraminidase activity. The antibodies induced in rabbits by the starting material (purified neuraminidase) and by fractions 1 and 2 were specific for the neuraminidase; they did not inhibit hemagglutination by intact virus or fix complement with ribonucleoprotein antigen. Antibodies to fraction 3 could not be detected in any of the above-mentioned tests. Approximate estimation of the molecular weights by comparison of the migration of the above fractions with protein standards (Shapiro et al., 1967) suggested that fraction 3 had a molecular weight of the order of 100,000 and the other fractions are considerably larger than this. The immediate question arises: Why were there so many high molecular weight protein bands when we supposedly started with a purified protein? There are three possibilities: first, that the isolated neuraminidase was not pure and we are dealing with a mixture of proteins; second, that the various bands represent aggregates of polypeptide
INFLUENZA
NEURAMINIDASE
chains that make up the neuraminidase; or third, that there are several neuraminidases. The second possibility seems the most likely, for fractions 1 (a, b, c, d) and 2 have enzyme activity or can induce specific antineuraminidase antibodies, and the following results support this contention. Electrophoresis of Dissociated Neuranzinidase in Gels Containing Reducing Agent The higher neuraminidase
molecular weight bands of that occurred in the above
Neurammdase
AND
IIEMAGGLUTININ
647
experiments may have been due to incomplete denaturation of the protein before electrophoresis or to reaggregation due to the lack of reducing conditions during electrophoresis. To overcome these difficulties and ensure dissociation, the isolated neuraminidase was extensively denatured by precipitation with ethanol at 20” and treatment with 1% SDS, 1% dithiothreitol and heated to 100” and then electrophoresed on polyacrylamide gels that had been presoaked in buffer containing 0.1% dithiothreitol (Laver,
OCtwlty
Induchm of neurom~n!dase mhibiing anhbodies m robbits
Fro. l.-Electrophoresis of the isolated neuraminidase from influenza (X-7) on polyacrylamide gels that did not contain reducing agent. Isolated neuraminidase (0.1 ml) (containing approximately 2000 units of neuraminidase*) was treated with 1% SDS and 1% mercaptoethanol at 37” for 3 hours and electrophoresed on polyacrylamide gels. A final concentration of 8% acrylamide was used in the gels, but in all other ways the methods of Shapiro et al. (1967) were followed. The gels were sliced into l-mm disks in the apparatus described by Groves et al. (1968). Neuraminidase activity was estimated after grinding each slice in a tissue homogenizer with 0.5 ml of 0.15 M sodium chloride and dialysis overnight against 0.15 M NaCl before assay for neuraminidase. Rabbits were injected with samples mixed with complete Freund’s adjuvant into the footpad and were boosted 40 and 60 days later by the intraperitoneal route. The molecular weight determinations were done in 10% gels according to Shapiro et al. (1967) using bovine serum albumin, pepsin, and chymotrypsinogen as markers. * One unit of neuraminidase is defined as that quantity which will give a reading of 0.50 optical density units at 549 Q in the neuraminidase test with excess fetuin as substrate.
648
WEBSTER
FIG. 2. Electrophoresis of the isolated neuraminidase from influenza containing reducing agent. Isolated neuraminidase, 0.1 ml, was treated threitol as described in Materials and Methods and electrophoresed taining dithiothreitol.
1970). The results obtained are shown in Fig. 2. There were two main protein bands running closely (1, 2) together that migrate slightly more rapidly than a bovine serum albumin marker electrophoresed in a separate column under identical conditions. Very minor bands (a, b, c) could still be detected suggesting that under these conditions aggregation still occurred, or that minor contaminants of high molecular weight were present. Denaturation sf the Hemagglutinin and Neuraminidase- of In&&& Virus with 6 M Guanidine Hydrochloride The second method employed in this study to dissociate the hemagglutinin and neuraminidase subunits of influenza viruses was treatment with guanidine hydrochloride. Attempts to denature the neuraminidase subunit with 1% SDS and 1% mercaptoethanol without heating to 100’ were unsuccessful; such treatment, in fact, increased the enzyme activity of the preparation. As a denaturing agent, 6 R’I guanidine hydrochloride is more effective than SDS and breaks all noncovalent bonds (Kawahara and Tanford, 1966). Treatment of neuraminidase with this reagent abolished all biological activity when assayed in the presence of the reagent, but if the 6 M guanidine hydrochloride was first dialyzed away, about 10% of the enzyme activity of the neuraminidase activity returned. After dialysis against iodoacetamide or mercaptoethanol, no enzyme activity was detected. This experiment shows that enzyme activity was abolished by guanidine hydrochloride
(X-7) on polyacrylamide gels with 1% SDS and 170 dithioon acrylamide gels con-
and mercaptoethanol treatment and the following experiments support the contention of Kawahara and Tanford (1966) that guanidine hydrochloride is an efficient method for dissociating proteins. Exclusion Chromatography BEL Hemagglutinin Neuraminidase
of Labeled Ao/ and AzlRI/5+
The isolated and 3H-labeled hemagglutinin and neuraminidase were denatured in 6 M guanidine hydrochloride under reducing conditions and applied to beaded agarose columns (Biogel A-50m). The results are shown in Figs. 3 and 4. The denatured hemagglutininand the neuraminidase both gave three peaks of radioactivity. In each case, the first peak came through the column with the E. coli exclusion volume marker, the second peak before the thyroglobulin marker, and the main peak of radioactivity after the bovine serum albumin marker. Again, we are faced with multiple peaks of activity from isolated virus subunits (hemagglutinin and neuraminidase), and the following experiments will show that the first two peaks of activity to elute from the columns are probably aggregates. The labeled material from each peak was dialyzed to remove guanidine hydrochloride and tested for neuraminidase or hemagglutinin activity both in the direct tests and in antibody blocking tests. The major peak of activity from the neuraminidase gave very weak antibody blocking activity, but no hemagglutinin or neuraminidase activity could be detected in any of the peaks.
1NFLUENZA
NEURAMINIDASE
AND
HEMAGGLUTIN
IN
649
C&hmma
FIG. 3. Exclusion chromatography of the isolated hemagglutinin (Aa/BEL) from in6uenza virus on beaded agarose. Isolated hemagglutinin from Ao/BEL influenza virus labeled with 3H-labeled amino acids in chick lung cells was denatured with 6 M guanidine hydrochloride containing 0.05 M lithium chloride, 0.1 M mercaptoethanol, and 0.01 M ethylenediaminetetraacetic acid (EDTA) adjusted to pH 7.2 and chromatographed in the same solution on a Biogel A50m beaded 2% agarose column (50 cm X 0.9 cm). Samples (0.5 ml) were collected and precipitated with 3 ml of 57, trichloroacetic acid to which 1 ml of bovine serum albumin (500 pg) was added as carrier protein. The precipitates were taken up in 1.0 ml of NCS (Amersham-Searle, Chicago), mixed with 10 ml of a toluene-based scintillant and counted in a liquid scintillation spectrometer.
The Capacity of Denatured Neuraminidase to Reaggregate The early peaks of radioactivity from the exclusion gel chromatography experiments were a constant finding, and attempts were made to ensure denaturation of the neuraminidase subunit. Samples of neuraminidase were dialyzed againt 6 M guanidine hydrochloride denaturing reagent and then sealed in glass and heated in a boiling water bath for 30 min. The resulting material still showed three peaks of activity on beaded agarose columns in the presence of the denaturing reagent. The isolated neuraminidase from X-7 influenza virus was labeled with %I (Webster et al., 1962) and chromatographed on beaded agarose columns with 6 Ad guanidine hydro-
chloride under reducing conditions. Denaturation of the neuraminidase was less effective after labeling with 1251, and a greater proportion of the counts came through the column with the exclusion marker. However, once again three peaks of activity were obtained (Fig. 5). The eluted material in peaks 2 and 3 (fractions 1 and 2) were concentrated and reapplied to the column. One might expect that the material from the isolated peaks would be homogeneous with respect to molecular weight and should rechromatograph as single peaks. However, if the original pattern of three peaks of activity was obtained on rechromatography, one would have to conclude that there was an equilibrium between free and aggregated polypeptide chains. In each case the radio-
650
WEBSTER
.
I
I I ‘-1
1 I
I
31;
20
SAMPLE MOdw$.
E,c II
I
I
40
5c
-\.-.. s., I
6:
I
70
NUMBER
r Thyroglobul!n
?
T
S.S.A Chym?&snogen cyi bchrome
0. .P olonlne t
FIG. 4. Exclusion chromatography of the isolated neuraminidase (AZ/RI/~+) from influenza virus on beaded agarose. Isolated neuraminidase from the recombinant X-7 influenza virus labeled with SHlabeled amino acids in BHK 21/13 cells WM denatured with 6 M guanidine hydrochloride as described for Fig. 3.
was again found in three peaks (Fig. 5B and 5C) suggesting that even in the presence of 6 M guanidine hydrochloride and reducing conditions reaggregation occurs. The isolated hemagglutinin (labeled with ?I) was chromatographed under the same conditions as described above for the neuraminidase. The isolated fractions of labeled hemagglutinin from the column also reequilibrated into three peaks on rechromatography.
activity
Estimation of the Molecular Weight of Ad BEL Hemagglutinin and AJRI/5+ Neuraminidase by Exclusion Chromatography To estimate the molecular weights of the hemagglutinin and neuraminidase accurately, four separate experiments were carried out on the major peaks of activity, and the reproducibility between experiments was
very good. In each case, the peak activity of the marker proteins and the radioactive samples eluted in an identical fashion in each experiment and the results are shown in Fig. 6. The distribution coefficients for the main peaks of activity for each protein were calculated according to Davison (1968)) and the estimated molecular weight of the neuraminidase polypeptide chains was 58,000 and for the hemagglutinin the estimated molecular weight was 47,000. The implicit assumption was that the lowest molecular weight peak that contained the majority of the radioactivity also contained the monomeric forms of the hemagglutinin or the neuraminidase. This was probably justified in that 6 M guanidine hydrochloride is a very efficient denaturing agent, especially in the presence of reducing agents.
INFLUENZA
NEURAMINIDASE
Attempts to Demonstrate the Presence oj Antigenic Determinants on Dissociated Hemagglutinin and Neuraminidase Subunits In addition to determining the molecular weight of the polypeptide chains making up the hemagglutinin and neuraminidase, we would like to know where the antigenic determinants are located and whether the separated polypeptides have any activity. Antibody blocking tests were carried out after denaturation of the hemagglutinin and neuraminidase with guanidine hydrochloride under reducing conditions and on the fractions from the agarose columns. Extremely weak blocking activity was obtained with denatured neuraminidase, but denatured hemagglutinin failed to block antibodies in the hemagglutination inhibition test. Denaturation of 1000 enzyme units of neuraminidase (1 unit being that amount of neuraminidase that will give an optical density reading of 0.50 at 549 rnp after 30 min action on excess fetuin substrate) gave one blocking unit and, hence, the reaction is very inefficient. The denatured neuraminidase was also tested for antibody blocking activity in the neuraminidase inhibition test with specific antineuraminidase antibodies, but in this test no blocking activity could be detected. These results would indicate that the antigenic determinants are probably not composed of adjacent amino acids on a single polypeptide chain, for antigenic activity was lost on denaturation. The determinants may be made up from folded areas on a single polypeptide chain or could be shared among different polypeptide chains. The small amount of biological activity in the antibody blocking test with neuraminidase may have been due to random reassortment of chains into aggregates. The enormous capacity of the denatured proteins to reaggregate suggests that more biological activity should be obtained during renaturation and it might just be a case of controlling the ionic and pH conditions more carefully. DISCUSSION
Studies on the isolated and denatured hemagglutinin and neuraminidase of influ-
AND
HEMAGGLUTININ
A
FIG. 5. Reaggregation of neuraminidase subunits. Isolated neuraminidase (RI/5+) was labeled with Ias1 (Webster et al., 1962) and chromatographed on agarose columns as described in Fig. 3. Samples 28-33 (fraction 1) and 47-52 (fraction 2) were pooled, concentrated to 0.2 ml by dialysis against polyethylene glycol 20,000, and rechromatographed after dialysis against 6 M guanidine hydrochloride denaturing solution as given in Fig. 3. (A) Chromatography of rzSI-labeled neuraminidase on an agarose column. (B) Rechromatography of fraction I from A. (C) Rechromatography of fraction II from A.
enza viruses by exclusion chromatography gave three peaks of radioactivity for each type of subunit. Estimation of the molecular weight of the major peak of activity gave a
652
WEBSTER
FIG. 6. Molecular weight estimates of the hemagglutinin and neuraminidase of influenza viruses. The molecular weights of the isolated and labeled hemagglutinin (BEL) and the neuraminidase (RI/5+) were determined by exclusion chromatography on beaded agarose as described by Davison (1968).
value of 47,000 for the hemagglutinin and 58,000 for the neuraminidase. The two peaks of activity that, correspond with polypeptides of higher molecular weight were probably aggregates of the lower molecular weight, polypeptides and evidence was presented to show that reaggregation of the lower molecular weight polypeptides occurs. The studies of the isolated and denatured neuraminidase by polyacrylamide gel electrophoresis in SDS under reducing conditions gave two closely spaced bands that migrated slightly more rapidly than the bovine serum albumin reference protein. These results from polyacrylamide gel electrophoresis suggest, that the neuraminidase is composed of at least, two polypeptide chains whereas exclusion chromatography gave one peak of activity that would correspond with polypeptides of this size. These apparently contradicting results could be explained on the grounds that polyacrylamide gel electrophoresis gave greater resolution than exclu-
sion chromatography. An alternative explanation would be that one of the two bands obtained on polyacrylamide gel electrophoresis is an aggregate of the other, but from the close proximity of the two bands, after prolonged electrophoresis this would seem less likely. The hemagglutinin of BEL influenza virus has also been separated into two bands on polyacrylamide gel electrophoresis (Laver, 1970), but exclusion chromatography was not, able to resolve these polypeptides. Both the hemagglutinin and the neuraminidase subunits of influenza viruses are relatively large structures and from the sedimentation data and their dimensions as determined by electron microscopy (Laver and Valentine, 1969; Webster and Darlington, 1969), they should have molecular weights in excess of 150,000. This would mean that there must, be at least, three polypeptides of approximately equal size (HA = 47,000; N = 58,000) per molecule. Electron micrographs of the isolated neuraminidase show complex structures (Laver and Valentine, 1g069). The top o,f the structures is about 80 A long and 52 A wide with a centrally attached fiber 100 A long with a knob about 40 A in diameter at the end. It is tempting to postulate that the denser band obtained on polyacrylamiie gel electrophoresis represents the 80 A structure while the Jess dense band represents the smaller 40 A knob. This can only be speculation until the substructures of the neuraminidase subunit are separated and studied independently. The ability of the denatured hemagglutinin and neuraminidase to form aggregates even under reducing conditions shows that the polypeptides have a great tendency to form some kind of quaternary structure. This type of aggregation, which was certainly not specific under reducing conditions, may be a special property of virus proteins and may be necessary during assembly in the infected cell. The low efficiency of the denatured hemagglutinin
and neuraminidase
at blocking
the
activity of antibodies to these subunits was disappointing, particularly since we are interested in studying the antigenic determinants of these subunits. The evidence would indicate that the determinants are not
INFLUENZA
NEURAMINIDASE
composed of adjacent amino acids on a single polypeptide chain and that the polypeptide chain cannot be renatured to give antigenic determinants or enzymatic activity. Estimates of the molecular weights of the virion proteins of influenza virus on polyacrylamide gel electrophoresis (White et al., 1970) gave values of 50,000 for the hemagglutinin and 20,000 for the neuraminidase monomers. Dimmock and Watson (1970) separated five structural proteins from i&uenza virions on polyacrylamide gels. One of these (V,) had a molecular weight of 81,000 and another (V,) had a molecular weight of 63,000, and these may or may not correspond to the hemagglutinin and neuraminidase subunits. The difficulties with these studies is lack of positive identification between the subunits of the viruses and the protein bands obtained on acrylamide gels. These proteins may or may not be the hemagglutinin or neuraminidase. Initial separation of the influenza virion subunits followed by characterization as was done in this report leaves no doubt as to the identification of the proteins. The different values obtained by different workers might be explained on the grounds that different strains of influenza viruses were used in the studies. However, the similarity in sedimentation coefficients and the dimensions of the hemagglutinin and neuraminidase from different strains of influenza argues against this possibility. The more likely explanations are aggregation of proteins and lack of positive identification of the proteins on acrylamide gels that would permit comparison of results. ACKNOWLEDGMENTS The author wishes to thank Dr. W. G. Laver for carrying out the electrophoresis on polyacrylamide gels containing dithiothreitol. The excellent technical assistance of Mr. Melvin C. Smith is gratefully acknowledged. REFERENCES DAVISON, P. F. (1968). Proteins in denaturing solvents: Gel exclusion studies. Science 161, 90% 907. DIMMOCK, N. J., and WATSON, D. H. (1970). Nonvirion antigens of influenza virus. “The Biology of Large RNA Viruses,” in press.
AND
HEMAGGLUTININ
653
DRZENIEK, R., SETO, J. T., and ROTT, R. (1966). Characterization of neuraminidases from myxoviruses. Biochim. Biophys. Acta 128,547~558. EAGLE, H. (1959). Amino acid metabolism in mammalian cell culture. Science 130.432437. EVANS, M. J., and KINGSBURY, D. W. (1969). Separation of Newcastle disease virus proteins by polyacrylamide gel electrophoresis. Virology 37, 597-604. FAZ~KAS DE ST. GROTH, S., and WEBSTER, R. G. (1966) Disquisitions on original antigenic sin. 1. Evidence in man. J. Exptl. Med. 124,331-345. GROVES, W. E., DAVIS, F. C., JR., and SILLS, B. H. (1968). An automatic device for sectioning analytical polyacrylamide gels: Radioactive Escherichia coli 50 S ribosomal proteins. Anal. Bioshem. 24, 462-469. KAW.AHARA, K., and TANFORU, C. (1966). The number of polypeptide chains in rabbit muscle aldolase. Biochemistry 5, 1578-1584. KILBOURNE, E. D. (1968). Recombination of influenza A viruses of human and animal origin. Science 160, 74-76. KILBOURNE, E. D., LIICF, F. S., SCHULMAN, J. L., JAHIEL, R. I., and LAVER, W. G. (1967). Antigenie hybrids of influenza viruses and their implications. Perspectives Viral. 5, 87-106. KILBOURNE, E. I~., LAVER, W. G., SCHULMAN, J. L., and WEBSTER, R. G. (1968). Antiviral activity of antiserum specific for an influenza neuraminidase. J. Viral. 2,281-288. LAVER, W. G. (1963). The structure of influenza viruses. 3. Disruption of the virus particle and separation of neuraminidase activity. Virology 20, 251-262. LAVICR, W. G. (1964). Structural studies on the protein subunits from three strains of influenza virus. J. Mol. BioZ. 9,109-124. LAVF:R, W. G. (1970). Chemistry of antigenic variation in influenza virus. Properties of the surface subunits of antigenic variants. “The Biology of Large RNA Viruses,” in press. LAV~R, W. G., aud KILBOURNE, E. D. (1966). Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30,493-501. LAVF,R, W. G., and VALt:NTINE, R. C. (1969). Morphology of the isolated hemagglutinin and neuraminidase subunits of influenza virus. Virology 38, 105-119. NOLL, H., AOYAGI, T., and OILLANDO, J. (1962). The structural relationship of sialidase to the influenza virus surface. Virology 18,154-157. SCHILD, G. C., and PEIIEIRA, H. G. (1969). Characterization of the ribonucleoprotein and neuraminidase of influenza A viruses by immunodiffusion. J. Gen. Viral. 4,355-363. SETO, J. T., DRZENIX, R., and ROTT, R. (1966).
654
WEBSTER
Isolation of a low molecular weight sialidase (neuraminidase) from influenza virus. Biochim. Biophys. Acta 113, 492-404. SHAPIRO, A. L., VI~UELA, E., and MAIZEL, J. V., JR., (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-820. STOKER, M., and MACPHERSON, I. (1964). Syrian hamster fibroblast cell line BHK 21 and its derivatives. Nature 203, 1355-1357. WEBSTER, R. G., and DARLINQTON, R. W. (1969). Disruption of myxoviruses with Tween 20 and isolation of biologically active hemagglutinin and neuraminidase subunits. J. Viral. 4, 182187.
R. G., and PEREIRA, H. G. (1968). A common surface antigen in influenza viruses from human and avian sources. J. Gen. Viral. 3, 201-208. WEBSTER, R. G., LAVER, W. G., and FAZEKAS DE ST. GROTH, S. (1962). Methods in immunochemistry of viruses. 3. Simple techniques for labelling antibodies with Ia11 and 3%. Australian J. WEBSTER,
Exptl. Biol. Med. Sci. 40,321328. WHITE, D. O., TAYLOR, J. M., HASLAM, E. A., and HAMPSON, A. W. (1970). Synthesis and transport
of influenza polypeptides. “The Biology of Large RNA Viruses,” in press. WILSON, V. W., and RAFELSON, M. E. (1963). Isolation of neuraminidase from influenza virus. Biochem Prep. 10, 113-117.