Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus

VIliOLOGY

46, 275288

Separation

(1971)

of Two

Polypeptide Subunit

Chains

of influenza

from

the Hemagglutinin

Virus

W. G. LAVER Department of Microbiology, The Australian National

The John Curtin School of Medical Research, University, Canberra, A.C.T., Australia

Accepted March

24, 1971

Particles of the BEL(Ao) strain of influenza virus (grown in the allantoic sac of embryonated chicken eggs) were disrupted with cold sodium dodecyl sulphate (SDS), and the hemagglutinin subunits were isolated by electrophoresis on cellulose acetate strips. The hemagglutinin subunits were found to contain two different polypeptide chains with molecular weights of approximately 60,000 and 21,009. In the intact subunits the heavy chain was joined by disulphide bond(s) to the light chain to form a dimer, and each hemagglutinin subunit contained two of these dimers. The two chains were separated by SDS-polyacrylamide gel electrophoresis in the presence of dithiothreitol or, on a preparative scale, by centrifugation on a guanidine hydrochloridedithiothreitol density gradient. The two chains were similar in amino acid composition, with the exception that the heavy chain contained about 9 times more proline than the light chain. The heavy chain also contained much more glucosamine. Maps of the tryptic peptides from the two chains were quite different, indicating that they differed in amino acid sequence. The other protein components of the BEL(Ao) influenza virus particle (neuraminidase, internal ribonucleoprotein antigen, and a small internal protein) were each associated with a different polypeptide. Thus particles of BEL(Ao) influenza virus contain at least 5 different polypeptide chains.

little is known about the mechanism of this attachment. Second, the hemagglutinin induces antibodies t*hat are responsible for t’he neutralization of virus infectivity (Webster et al., 1968; Schild, 1970; Webster and Laver, 1967), and variation in t,he hemagglutinin antigen is the major reason for the frequent occurrence of epidemics of influenza. Variation in the neuraminidase antigen also occurs, but little is known about the nature of the antigenic determinants on either the hemagglutinin or the neuraminidase subunits or about, the molecular mechanism of antigenic variation. Third, the hemagglutinin subunit is one of the protein components (anot#her is the neuraminidase) of a biological membrane (the influenza virus envelope), and a knowledge of the structure of the proteins in the envelope and of the chemistry of their hydrophilic and hydro-

INTRODUCTION

The hemagglutinin submms of influenza virus are rood-shaped struc$ures approximately 140 A long and 40 A wide with a molecular weight of about 150,000 (Fig. 1). One end of the hemagglutinin rod appears to have hydrophobic properties, and the ot,her is hydrophilic (Laver and Valentine, 1969). It is thought that the hydrophilic end protrudes from the virus particle (forming the “spikes” visible in electron micrographs) and carries the various biological activities of the subunit, while the hydrophobic end is buried in the lipid of the viral envelope. The detailed structure of the hemagglutinin subunit of influenza virus is of particular interest for a number of reasons. First, it is by means of the hemagglutinin that the virus attaches to specific glycoprotein receptors on the surface of cells, but 275

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LAVER

FIG. 1. Electron micrograph of the isolated hemagglutinin subunits of influenza virus in the presence of SDS. The subunits were isolated from a recombinant virus (having A2/Hong Kong/l968 hemagglutinin and Ao/BEL neuraminidase) by electrophoresis on cellulose acetate strips after disruption of the virus particles with SDS at 20”. These subunits do not hemagglutinate in the presence of SDS; they are “monovalent,” attaching to red cells without bridging them. The preparation did, however, have some hemagglutinin activity. It is thought that this was due to a small number of aggregated subunits, some of which can be seen in the electron micrograph. X 250,000.

phobic regions may lead to some understanding of the mechanisms involved in the assembly of the virus envelope and of membranes in general. The experiments reported in this paper show that the hemagglutinin subunits of influenza virus consist of two different polypeptide chains. Methods for the isolation of each of these chains are described, and some of their properties are reported. MATERIALS

Viruses. (Burnet et combinant magglutinin

AND

METHODS

The BEL strain of Ao influenza al., 1942) and a number of reinfluenza viruses having the hesubunits from various influenza

A2 strains (Laver and Webster, unpublished) were grown in the allantoic sac of ll-dayold chick embryos. The virus part’icles were purified by adsorption-elution on chick erythrocytes followed by differential centrifugation and sedimentation through a sucrose gradient (lo-40 % sucrose in 0.15 M NaCl) as previously described (Laver, 1969a). Isolation of the hemagglutinin subunits. The virus particles were disrupted with 1% sodium dodecyl sulphate (SDS) at room temperature (20’), and the virus proteinwere separated by electrophoresis on cellus lose acetate strips in Tris-boric acid-EDTA buffer pH 9 containing 0.4 % SDS as previ-

POLYPEPTIDES

OF INFLUENZA

owl\- described (Laver, 1964). The fractions containing the hemagglut#inin subunits (Fig. 2) were eluted from the strips with water (25 ml) and filtered t’o remove hit’s of cellulose acetate. Cold (-20”) ethanol (50 ml) was then added, and a trace of sodium chloride, and the mixture was st,ood at -20’ overnight while the prot,ein slowly flocculated. The precipitated hemagglutinin subunit,s were then recovered by centrifuging (1500 y, 15 min), redissolved in a small volume (about 0.5 ml) of cold water, and stored frozen at, -20”. rl crylamide gel electrophoresis. Gel electrophoresis \vas done in 7.5 % polyacrylamide gels with 0.2 % N, N-bismet’hylene ncrylamide in Tris-boric acid-EDTA buffer pH 9 containing 0.1 % SDS and 0.1 mg/ml dithiothreit~ol as described previously (Laver et al., 1969). Protein samples were dissolved by heating to 100” for 2-3 min in 1% SDS containing 0.1 mg/ml dithiothreitol before being applied to the gels. Gels were stained with Coomassie brilliant blue dye (Imperial Chemical Industries) dissolved in meth:tnol-acetic acid-water (5: 1: 5) and destained with the same solvent,. For quantitative measurement, of the amounts of protein in the bands, the gels were scanned at a \\-nvrlength of 549 nm (Fazekas de St,. (;rot*ll cut al., 1963) using a Gilford automatic scanning spectrophotometer. Molecular wrights of proteins were est’imated from their mobilities in the gel by the method of Weber and &born (1969) using t’he following st’andard proteins: ribonuclease (JIW 13,700), carboxypeptidase (?IIW 34,600), cat,:dase (MW 57,500)) and hemocyanin (3lW 90,000). ;l mine acid analyses. Protein samples cwuaining 200-300 pg were hydrolyzed wit’11 6 N HCl (2.0 ml) in sealed evacuated tubes at 110” for 22 hr and the hydrolyzates jvere analyzed by the method of Moore et al. (1958) using the Model B Beckman amino acid analyzer \vit h an expanded scale (Spackman, 1960). (Jucosamine assa!ys. Samples of protein (256 pg) n-ere hydrolyzed with 4.0 N HCI in sealed evacuated t,ubes for 3 hr at 100”. Glucosamine n-us assayed by the method of liondle and Morgan (1955) as modified by Eraan :tnd l\luir (1957). Ppptirle maps. Proteins were digested with

HE:M~4GGLUTIXI~

57

trypsin, and the tryptic peptidea soluble at pH 6.5 were mapped by two-dimensional electrophoresis and chromatography on large sheets of Whatman No. 3 mm paper as previously described (Laver, 1969b). Guanitline hydrochloride density qrad ien t centrifugation. Guanidine hydrochloride was recrystallized from water and redissolved in water (at’ 20’) to give a saturated solution. Linear 5-ml density gradients were then prepared using 90%: and 45 ‘;s suturatecl guanidine hydrochloride solutions containing 0.25 mg/ml dit,hiot,hreitol. Proteins were dissolved in sat#urat,ed guanidine hydrochloride solution, dit,hiot,hreitol (0.5 rngl ml) was added, and the mixture was heated to 100” for 2-3 min. Water was then added to the mixture until its densit,y w-as less than that of 45 70 saturated guanidine hydrochloride. Samples (0.2 ml) were layered onto the gradients, centrifuged in the SW 65 swinging-bucket rotor (Spinco, Beckman Indust’ries Inc.), and harvested by drop collection through a needle piercing the bottom of t)he tube. The distribution of protein in the gradients was determined by spotting a small sample (1 ~1) of each fraction onto a dry cellulose acet’ate &rip (Toxoid Ltd., London), staining t,he dried spots with Coomassie brilliant blue dissolved in methanolacetic acid-water (5 : 1: 5) and dest,aining with the same solvent. The fractions containing protein could then be seen. This method was both sensitive and economical, so that, peaks of protein in the gradients were twsily and quickly locat,ed and very little \~aluable material was used in the process. It may also be more generally applied, for example, to sucrose or cesium chloride gradients since these substances do not int,erfere in the te*qt: and the method is capable of detecting as little as 0.2 pgIp1 of protein. RESULTS

Separation of the Proteins of REL InJEuenza T’irus Particles of the BEL strain of ho influenza virus disrupt#ed with cold (‘20’) SDS gave 5 bands of protein during electrophoresis on cellulose acetate strips in buffer containing SDS (Fig. 2). Two of these bands possessed hemagglutining activity (Lnver,

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FIG. 2. Stained cellulose acetate strip and polyacrylamide gels showing the electrophoretic separation of the proteins of the BEL strain of Ao influenza. The virus particles were first disrupted with cold (20”) SDS, and the proteins were separated by electrophoresis on cellulose acetate strips. The 5 bands of protein obtained were eluted individually, concentrated by precipitation with ethanol, redissolved in hot (100”) SDS and dithiothreitol, and reelectrophoresed on polyacrylamide gels l-5. A sample of the original virus was run on a companion gel at the same time for comparison (gel 6). HA = hemagglutinin; N = neuraminidase; RNP = internal ribonucleoprotein antigen; IP = small molecular weight internal protein (VP3 of Haslem et al., 1970b; protein 7 of Compans et al., 1970).

1964; Schild, 1970) and contained rodshaped structures measuring 140 8 X 40 8 which, it is believed, are the hemagglutinin subunits of the virus (Laver and Valentine, 1969). Identification of the other bands is described later. The reason why the hemagglutinin subunits formed 2 bands during cellulose acetate electrophoresis is not known. In all other respects, the subunits from each of the bands were found to be identical. An electron micrograph of the isolated hemagglutinin subunits is shown in Fig. 1. In the presence of SDS the hemag-

glutining subunits exist mostly as “monomers” which adsorb to, but do not agglutinate, red cells. These subunits had a sedimentation coefficient (in the presence of SDS) of 7.5 S and a molecular weight of about 150,000 (Laver and Valentine, 1969). When the hemagglutinin subunits were heated to 100” in a 1% solution of SDS containing dithiothreitol (0.1 mg/ml), the subunits dissociated into their component polypeptides, which formed two well-separated bands during polyacrylamide gel electrophoresis (Fig. 2).

POLYPEPTIDES

OF INFLUENZA

The molecular weight of the faster polypeptide was estimated to be approximately 21,000 and the slower one approximately 60,000. These values were obtained by comparing the mobilities of the 2 bands with the mobilities of standard proteins in the same gels (Weber and Osborn, 1969). The relative amount, of protein in the two bands was estimated by scanning the gels in a

HEMAGGLUTININ

279

direct recording densit’ometer. The slow band contained twice as much protein as the fast band. Isolation of the Two Polypeptides Hemcagglutinin Subunit

of the

The “light” and “heavy” polypeptide chains of the BEL virus hemagglutinin subunit could be isolated in pure form by

FIG. 3. Separation of the light and heavy chains of BEL virus hemagglutinin on a guanidine hydrochloride density gradient. A sample of the hemagglutinin subunits of BEL virus (2.0 mg) dissolved in guanidine hydrochloride-dithiothreitol solution (0.2 ml) was layered ont,o a linear 5 ml guanidine hydrochloride-dithiothreitol density gradient as described in Materials and Methods and centrifuged (Spinco SW 65 rotor) at 55,000 rpm for 10 hr at 15”. Twenty fractions (0.25 ml) were collected through a hole in the bottom of the tube, and each was tested for protein by spotting 2 ~1 onto cellulose acetate and staining with Coomassie blue. The stained spots from one gradient are shown above. Fractions containing protein (3-9 and 13-17) were pooled and the protein was concentrated by precipitation with 5 volumes of ethanol. Samples were then dissolved in hot, SDS-dithiothreitol and electrophoresed on polyacrylamide. The stained gels are shown. A sample of the original hemagglutinin subunits electrophoresed on polyacrylamide under the same conditions is shown at’ the left for comparison; 0.95 mg of the heavy chain and 0.51 mg of the light chain (assayed by the method of Lowry el al., 1951) were recovered from the gradient, an overall yield of 73y0. It can be seen that the light chain sedimented more rapidly than the heavy chain in guanidine hydrochloride. Possible reasons for this are discussed in the text.

LAVER

preparative acrylamide gel electrophoresis using a method described previously (Laver, 1970). The yields of each of the two chains were very low, however, probably because most of the material failed t,o elute from the gel, and a better method of isolating the two chains in quantities sufficient for analysis was therefore sought. It was found t,hat the subunits dissociated when dissolved in saturated guanidine hydrochloride solution containing dithiothreitol and the two chains could then be separated by centrifugation on a guanidine hydrochloride density gradient. The results (shown in Fig. 3) were surprising. Although complete separation of the two polypeptide chains was achieved, with good (75%) recovery of protein, the rate of sedimentation of the two chains in the guanidine hydrochloride gradien: was quite the opposite to that expected fram t,heir mobilities in SDS-polyacrylamide gels. Thus, the polypeptide which migrated more rapidly in the gels (the “light” chain) was found in the fraction which sedimented more rapidly in guanidine hydrochloride. Conversely, the “heavy” chain (which migrat,ed m3re slowly in t,he gels) was found near the top of t,he guanidine hydrochloride gradient (Fig. 3). The most likely explanation of these anomolous results is that the light chain aggregated in the guanidine hydrochloride and hence sedimented much more rapidly than expected, so that the excellent separat’ion achieved was entirely fortuitous. These findings are not restricted to the BEL strain of Ao influenza virus. The polypeptides of t,he hemagglutining subunits from a series of A2 influenza viruses have also been separated in this way (Laver and Webster, unpublished). Amino Acid Compositim ad Peptide Maps The amino acid composition of the two polypeptides of BEL virus hemagglutinin is given in Table 1. The two chains did not differ greatly in amino acid composition with the exception of proline, which was present mainly in the heavy chain. Glucosamine assays showed that’ the heavy chain possessed 7.6 % glucosamine (assayed as the free base). The heavy chain also contained fucose (1.1 %), mannose (4.8 %), and galac-

TABLE 1 AMIINO ACID COMPOSITION OF THE Two POLYPEPTIDE CHAINS ISOL~\TED FROM BEL VIRUS HEM~GGLUTININ* Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

Light chain Heavy chain 8.0 1.6 2.7 13.9 4.5 7.8 13.3 0.5 10.6 5.6 1.1 6.0 0.8 6.0 9.6 3.6 4.3 ND

5.8 2.0 4.3 11.7 7.2 9.2 11.3 4.6 8.5 5.5 1.5 6.0 0.5 6.6 8.9 3.4 3.3 NP

a Average values from duplicate analyses expressed as moles of amino acid per 100 moles of amino acids recovered. * Not determined.

tose (1.5%) so that the heavy chain contained at least 17 % carbohydrate. Carbohydrate analysis of the light chain has not so far been done but data obtained from amino acid analyses show that the light chain contains approximately 1.5 % glucosamine, a figure which may need to be modified when enough material can be obtained for a direct glucosamine analysis. Samples of the two polypeptide chains isolated from BEL virus hemagglutinin by guanidine hydrochloride density gradient centrifugat~ion were dige&ed with trypsin and the peptides which were soluble at pH 6.5 (the pH of the electrophoresis buffer) were mapped by tlvo-dimensional electrophoresis and chromatography. Photographs of the peptide maps show that the two chains are completely different (Fig. 4). These peptide maps were quite reproducible and duplicate maps of the two chains were exactly the same as those shown in Fig. 4, even in the most faintly staining peptides. A map of the trypt,ic peptides from the whole

282

LAVER

hemagglutinin subunit of BEL virus is also shown (Fig. 4) and peptides from the whole subunit can be assigned to one or other of the two chains. This is particularly noticeable for a group of 5 peptides which migrated toward the +ve electrode during electrophoresis: 3 of these peptides obviously came from the heavy chain and 2 from the light chain. Some peptides visible on the maps of the isolated light and heavy chains appear to be missing from the map of the whole subunits. This is probably because the more complex the mixture of peptides, the harder they are to map and more material could be applied in the case of the individual chains than when the whole subunits were mapped. Demonstration That Both the Heavy and Light Polypeptide Chains Are an Integral Part of the Hemagglutinin Subunit The experiments described above showed that the preparation of hemagglutinin subunits eluted from cellulose acetate strips after electrophoresis of SDS-disrupted BEL influenza virus contained two different kinds of polypeptide chains. Did both of these polypeptides belong to the hemagglutinin subunits, or was one of them a contaminant which migrated along with the subunits during cellulose acetate electrophoresis? In an attempt to answer this question, the isolated hemagglutinin subunits were purified further by SDS-sucrose gradient density gradient centrifugation and reexamined by polyacrylamide gel electrophoresis. The hemagglutinin subunits of BEL influenza virus eluted from cellulose acetate strips were concentrated by cold ethanol precipitation as described in Materials and Methods. The preparation of subunits was dissolved in 1% SDS at 20” and centrifuged on a sucrose gradient containing 0.2 % SDS as described in the legend to Fig. 5. Rabbit 7 S IgG antibody centrifuged on a companion gradient served as a sedimentation marker. Two peaks of protein were obtained (Fig. 5). One of these peaks had a sedimentation coefhcient of 7.7 S and coincided with the peak of hemagglutinin activity. [Hemagglutinin activity was detected in the fractions from the gradient only after removal of SDS as de-

scribed by Laver and Valentine (1969).] The other protein peak, close to the top of the gradient, possessed no hemagglutinin activity at all. The proteins in each of the two peaks were then examined by polyacrylamide gel electrophoresis. Both polypeptide chains were found in the 7.7 S hemagglutinin peak and these were present in the same ratio as in the original preparation. This indicates that both chains are associated with the 7.7 S “monovalent” hemagglutinin subunit. The protein at the top of the gradient also contained the two polypeptide chains. This material neither agglutinated red cells nor combined with antibody to the hemagglutinin, when tested in the antibody blocking test for nonhemagglutinating antigen described by Webster and Darlington (1969), and it is thought to be hemagglutinin protein denatured during the isolation .and concentration of the subunits. SDS-Polyacrylamide Electrophoresis of the Hemagglutinin Subunits in the Absence of Dithiothreitol The isolated hemagglutinin subunits of BEL virus were dissolved in hot SDS and electrophoresed on polyacrylamide gels as described in Materials and Methods with the exception that dithiothreitol was omitted from the sample and from the buffer in which the gels were soaked. A single protein band was obtained (Fig. 6) which was found, by coelectrophoresis with standard proteins, to have a molecular weight of approximately 79,000. This protein in the presence of dithiothreitol gave two bands with molecular weights of approximately 60,000 and 21,000 (Fig. 6). This experiment indicates that in the hemagglutinin subunit the light and heavy chains are held together as a dimer by disulphide bonds. Other Proteins in Particles of InJluenza Virus Besides the two bands containing the hemagglutinin subunits, three other protein bands were obtained after cellulose acetate electrophoresis of SDS-disrupted BEL virus (Fig. 2). Each of these proteins was eluted from the cellulose acetate and examined by SDS-polyacrylamide gel electrophoresis (in the presence of dithiothreitol) (gels 3,4, and

POLYPEPTIDES

OF INFLUENZA

HEMAGGLUTININ

283

FIG. 5. Demonstration that the light and heavy chains are both associat,ed with the 7.7 8 hemagglutinin subunit of BEL virus. The hemagglutinin subunits, isolated by electrophoresis on cellulose acetate strips, were concentrated by cold (-20”) ethanol precipitation and redissolved in l(j;, SDS solution. A sample of the concentrated subunits was centrifuged on a 5-ml linear SDS-sucrose gradient (7.5-30%) sucrose in 0.091 M tris buffer, pH 9 containing 0.27, SDS) in the Spinco SW 65 rotor at 50,000 rpm for 6 hr at 20”. Thirty-one fractions were collected through a hole in the bottom of the tube, and each was tested for protein by spotting 2 ~1 on cellulose acetate and st,aining with Coomassie blue (the stained spots are shown) and for hemagglutinin activity after removal of the SDS with KC1 as described (Laver and Valentine, 1969). Fractions 19-21 (peak of hemagglutinin activity) and fractions 27-28 (devoid of hemagglutinin activity even after removal of SDS) were concentrated by cold (-20”) ethanol precipitation and the proteins in these fractions were examined by SDS-polyacrylamide gel electrophoresis. Two stained gels are shown. A sample of rabbit 7 S IgG antibody to BEL virus hemagglutinin centrifuged on a companion gradient served as a sedimentation marker.

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fied antigen gave a single band of protein on SDS-polyacrylamide electrophoreais which had exactly the same mobility as the band shown above on gel 4. The protein run on gel 3 was thought to be BEL virus neuraminidase. This was not identified directly because the enzyme of BEL was inactivated during cellulose acetate electrophoresis. However, influenza type A2 neuraminidase was stable and could be isolated on cellulose acetate. This enzyme gave a band on SDS-polyacrylamide electrophoresis which had the same mobility as the protein on gel 3 above. The protein run on gel 5, which is the most abundant protein in the virus particle, had the same mobility as the small molecular weight internal protein (VP3) described by Haslem et al. (1970b) or the membrane protein (protein 7) of Compans et al. (1970). DISCUSSION

FIG. 6. Stained gels showing SIX-polyacrylamide electrophoresis of the hemagglutinin subunits of BEL influenza virus in the presence and absence of dithiothreitol. Molecular weights were est,imated by coelectrophoresis with marker proteins (ribonuclease, carboxypeptidase, catalase, and hemocyanin) according to Weber and Osborn (1969).

5, Fig. 2). Each ran as a single component, and the bands could be correlated with the bands obtained by SDS-polyacrylamide gel el&ctrophoresis of the whole BEL virus (gel 6, Fig. 2). One of the bands (gel 4) was identified as the polypeptide of the nucleocapsid (the internal or ribonucleoprotein antigen) in hhe following way. Immunologically active (in immunodiff usion tests) influenza type A soluble antigen was purified from the soluble antigen fraction of infected allantoic fluid by ammonium sulphat’e fractionation and cesium chloride density gradient centrifugation (Laver, unpublished). The puri-

The hemagglutinin subunits of the BEL (Ao) strain of influenza virus were found to contain two different polypeptide chains; one light and the other heavy. These subunits were isolated from virus particles disrupted with cold SDS, by electrophoresis on cellulose acetate strips, and it was shown previously (Laver, 1964) t,hat subunits isolated in this way contained some protein (about 30%) which would not adsorb to red cells. Therefore one or other of the chains may have been present as a contaminant in the preparation of hemagglutinin subunits eluted from cellulose acetate strips rather than as an integral experiments

part of the subunit.s. However, showed (Fig. 5) that both the

light and heavy chains were associated with the biologically active 7.7 S hemagglutinin subunits, and it is thought that the protein which failed to adsorb to red cells (Laver, 1964) was the more slowly sedimenting, biologically inactive mat’erial shown in Fig. 5 and represented subunits which had been denatured during the isolation procedure. Of course, two kinds of subunit may have been present,, one composed entirely of light chains and the other entirely

of heavy chains

but these subunits would need to the same sedimentation coefficient more likely interpretation of the that, the hemagglutinin subunits

have had and the results is of BEL

virus are a homogeneous population of molecules possessing both light and heavy polypeptide chains. During elect,rophoresis on cellulose acet’at)e the hemagglutinin subunits of REI, virus migrated as two distinct bands (Laver, 1964; Schild, 1970). The reason for this is not known : both bands had the same biological activit,y, the subunits in each sedimrnted at the same ratf’, and peptide maps of tllra two bands \vthre exact,ly the same (I,aver, 1964). The subunits in each of the t \vo bands also possessed the same proportion of light and heavy chains (Fig. 2). The light and heavy chains had molecular \veights of approximately 21,000 and 60,000. Both chains contained glucosamine, and an:dyses suggested that the heavy chain contained much more carbohydrate than the light chain. The heavy chain cont)ained 9.4 % N-:~cr~tylglucosnmine as well as neutral sugars; thus it probably cont’ained about 20’2 carbohydrate. It is not, known to what extent carboh\-drate affect,s the mobilities of proteins in SDS-polyacrylamide electrophoresis, but if it behaves in the same way as protein, then it, is likely that the polypeptide portion of the heavy chain had a molecular \veight of about 45,000. The ratio bet#ween the molecular n-rights of the heavy and light chains \i\-astherefore approximately 2: 1. The ratio bet wwn the amounts of protein in the slow and fast, bands obtained after poly:tcrylamide gel electrophoresis of the disrul)t ed hemagglutinin subunit’s \vas also about 2:1, suggesting that the two chains \vere present in the subunit in eyuimolar amounts. Xl~S-poly:lcr~lamide electrophoresis in the absent of dithiothreitol showed t,hat t#lle light and heavy chains were held together b> PmSPmmSPbonds, forming a dimer \vith a molecular weightj of about, 79,000 (Fig. 6). Since the intact1 hemagglutinin subunit has bwn estimated to have a molecular weight of approximately 150,000 (I,:rvw and Valentinr, 1969), it, is likely- that twh subunit’ is composed of two of these dimers. The light and heavy chains could be separated on :a preparative scale after disruption of the subunits in the presence of dit’hiothrcitol. I’reparative polyacrylamide gel clectrophoresis gave complete separation of

the two chains, but, was not used as t:he yields were often poor. When the subunits were disrupted with hot, SDS and dithinthreito1 and centrifuged on a sucrose densit) gradient (containing SDS and dithiothrritol) the heavy chain sedimented faster than tile light chain, as expected, but, only, a pwtial separation of the two chains n-as obt:lirwtl. The met hod finally cllosen, which gavel I)urtl preparations of esch chain in high >.icAld, consisted of dissolving the subunits in gwnidine hydrochloride (containing dit,hiot~lweitol) arid centrifuging on a guanidine li\= drochloride density gradient. Cont,nu?- to expectations, the he:lv~- chain sedimtJtltrd slower than the light chain, probable bec:lllse the light chains aggregated in guar;idine II~drochloride (I:ig. 5). The isolated light chains also shon-ed a tendency to aggregatth thwing SDS-polyacq~lamide elcctrophoresis, :r11t1 material n-as often seen streaking from tIlta origin of the gel (l’ig. 3). Webster (1970) estimated the moltw~lar weights of the polypeptide chains of the hemagglutinin subunits of BEL (:20) influenza virus by exclusion chromatography in 6 111guanidine hydrochloride containing nwrcaptoet hanol, md obtained two protrin peaks. One of these, corresponding lvith material having a molecular wright of 4i,OOO, he claimed to contain the polypeptidrs of t 11~ hemagglutinin subunit ; the other, with ;I very much greater molecular weight, he assumed to be aggregated material. It non seems likely that the two peaks obtained b> Webster represented the heavy and light chains of the hemagglutinin subunits, the light cliains aggregating during exclusion chromatograph>- in gunnidine hydrochloridt~ in the same way as they did in guanitline hydrochloride density gradient centrifug:\tion (Fig. 3). Haslcm et al. (1970qb) h:tw also studied the proteins of the BEL strain of Ao influenza. These authors claimed that the hemagglutinin subunit XLS a glycoprotein wit,li ii molecular weight in the regioli of 77,000. It is likely, however, that, the singles band with ;\lW of 77,000, obtairwci h!, Haslem et al. (1970b) during pol~:lcr~l:rrnitl~, gel electrophoresis of HEI, virus l~rm:r.gglutinin, was :t dimrr of the light chain (.\I%’ 21,000) and the heavy chain (11%’ CX~,OOO)

286

LAVER

held together by -S-Sbonds. These authors dissociated the virus particles with hot SDS and a mixture of mercaptoethanol and hydroxyethyl disulphide, but both of these latter reagents were removed by dialysis before polyacrylamide gel electrophoresis, and -SSbonds between the light and heavy chains may have reformed following the removal of the reducing agents. This interpretation of the findings of Haslem et al. is supported by the results obtained above. When the hemagglutinin subunits of BEL virus were dissolved in hot SDS without dithiothreitol, a single band having a molecular weight of approximately 79,000 was obtained following polyacrylamide gel electrophoresis (Fig. 6), whereas the same material in the presence of dithiothreitol yielded two bands of protein with molecular weights of approximately 21,000 and 60,000. Compans et al. (1970) and Schulze (1970) each found 7 polypeptides in particles of the WSN strain of Ao influenza. These corresponded well with the polypeptides of BEL virus described above with the exception that the two large molecular weight minor components of Compans et al. (1970) and Schulze (1970) were not seen. There were also minor differences in the other 5 polypeptides worth commenting on. Protein 3 of Compans et al. (1970) (VP II of Schulze, 1970) which was thought to be the polypeptide of the nucleocapsid (the internal ribonucleoprotein antigen) of WSN virus had a similar molecular weight as the polypeptide of the internal antigen of BEL virus (Fig. 2). However, the amount of protein found by Compans et al. (1970) in the nucleocapsid fraction of WSN virus was greater than that found in the internal antigen fraction of BEL virus (Fig. 2 and Haslem et al., 1970b). The reason for this might be that the preparations of WSN virus examined by Compans et al. (1970) contained very little incomplete virus, whereas the preparations of BEL virus, which were grown in embryonated eggs, almost certainly contained incomplete virus particles. Lief and Henle (1956) have shown a decreased incorporation of nucleocapsid antigen into virus particles of increasing incompleteness. It was found previously that the hemag-

glutinin fraction of BEL virus contained 37% of the virus protein (Laver, 1964). Compans et al. (1970) found that all 3 spike glyeoproteins (hemagglutinin plus neuraminidase) accounted for only about 23 % of the virus protein and they suggested that the hemagglutinin subunits isolated by electrophoresis on cellulose acetate strips from SDSdisrupted BEL virus (Laver, 1964) contained some core (“nonspike”) protein as a contaminant. This, however, seems unlikely as the experiments described above showed no evidence of any core proteins following SDSpolyacrylamide electrophoresis of the hemagglutinin subunits of BEL virus isolated on cellulose acetate strips and further purified on SDS-sucrose gradients (Fig. 5). A more likely explanation is that the proportion of spike proteins to internal proteins in preparations of influenza viruses depends on the strain of virus and on the number of incomplete virus particles present. The relative numbers of hemagglutinin and neuraminidase subunits incorporated into the virus particles have also been found to vary greatly, even with closely related strains (Webster et al., 1968). Schulze (1970) found that the hemagglut]inin subunits of WSN virus contained about 35 % of the protein of the virus, but the virus was grown in chick embryo fibroblasts, rather than in the MDBK cells of Compans et al. (1970) and hence may have contained incomplete virus particles. The preparation of WSN virus examined by Schulze (1970) did, in fact, contain less nucleocapsid protein (17 %) than the MDBK-grown WSN virus examined by Compans et al. (1970), which contained 24.1% nucleocapsid protein. The relative mobilities of the polypeptides of WSN virus found by Compans et al. (1970) and Schulze (1970) also differed slightly from the relative mobilities of the polypeptides of BEL virus. In the present work, the location of the light and heavy chains in the hemagglutinin subunit is not known. Compans et al. (1970) found that their fastest-migrating glycoprotein (protein 6), which seemed to correspond with the light chain of BEL virus hemagglutinin, was not removed from the virus particle by bromelain treatment and was

POLYPEPTIDES

OF INFLUENZA

present in the spikeless cores. This suggests that the light chains might occur at the hydrophobic end of the hemagglutinin subunit and be buried in the lipid of the viral envelope making them inaccessible to proteolytic enzymes, but further work is needed to decide this. It is not known what gives the hemagglutinin subunits their hydrophilic and hydrophobic ends. The light and heavy chains were remarkably similar in amino acid composition (with the exception of proline) but possibly the carbohydrate, which was att’ached predominantly to the heavy chain, was responsible for t,he hydrophilic region of the subunits. We have been interested in the mechanism of antigenic variation in influenza viruses and have shown previously that antigenic drift in the hemagglutinin antigen is associated wit)h changes in amino acid sequence of the proteins in the hemagglutinin subunit’s (Laver and Webster, 1968). When this work was done, however, it, was not known that the hemagglutinin subunits contained two different kinds of polypeptide chains, and one of t’he interesting questions we are now trying to answer is whether variation in amino acid sequence occurs in both the light and heavy chains or whet’her variation is restricted to one chain only. Preliminary peptide mapping experiments with t’he light and heavy chains of the hemagglutinin subunits from a number of immunologically different strains of influenza (Laver and Webster, unpublished) have been done and the results suggest that variations in amino acid sequence occur with both light and heavy chains. ACKNOWLEDGMENTS Miss Nicola Baker and Miss Kathleen Williams provided excellent technical assistance. The amino acid analyses were done by Mr. L. B. James and the carbohydrate assays by Dr. E. R. B. Graham. Electron microscopy was by Dr. N. G. Wrigley. REFERENCES BURNET, F. M., BEVERIDGE, W. I. B., BULL, D. R., and CLARK, E. (1942). Investigations of an influenza epidemic in military camps in Victoria in May, 1942. Med. J. Aust. 2,371376. COMPANS, R. W., KLENK, H. D., CALIGUIRI, L. A., and CHOPPIN, P. W. (1970). Influenza virus

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