72 hemagglutinin heavy chain

VIROLOGY

93,458-465 (1979)

Antigenic

Determinants

of Influenza

Virus Hemagglutinin

II. Antigenic Reactivity of the Isolated N-Terminal Cyanogen Bromide Peptide of A/Memphis/72 Hemagglutinin Heavy Chain D. C. JACKSON,**’ T. A. DOPHEIDE,t ROBYN J. RUSSELL,* D. 0. WHITE,* AND C. W. WARDt * School of Microbiology, University of Melbourne, -&S Royal Parade, Parkville,

and tDivision of Protein Chemistry, Victoria, 9052, Australia

Accepted November

C.S.I.R.O.,

8, 1978

Gel filtration of a cyanogen bromide digest of pure intact hemagglutinin from A/Memphis/ 102/72influenza virus allowed the isolation of a variety of fragments. One of these fragments consists of three cyanogen bromide peptides (CNl and CN3 from HA, and CNl from HA,) which remain linked together by disulphide bonds. This fragment was found to be antigenically active, as it was able to form antigen-antibody complexes (detected by affinity chromatography of radioiodinated peptide-IgG mixtures on protein A-Sepharose) with IgG directed against the protein moiety of viral hemagglutinin. The three cyanogen bromide peptides present in this disulphide-linked fragment were separated by gel filtration, carried out under reducing conditions, and tested for antigenic activity after controlled reoxidation of the individual peptides. Only one cyanogen bromide peptide, CNl from HA,, showed significant binding to antibody. The results indicate that antigenic activity of A/Mem/lOW72 hemagglutinin resides within the N-terminal 170 amino acid residues of the hemagglutinin heavy chain.

strains of influenza virus, and a study of the antigenic properties of various sequence The hemagglutinin (HA) molecules of segments within the molecule, should open different strains of influenza virus differ the way to an understanding of antigenic in structure. This is reflected at two levels: “shift” and “drift” mechanisms. (a) Antisera raised against the hemagglutinin In a previous pacer (Jackson et al., 1978) of one strain of virus will react to varying we reported that the isolated hemagglutinin extents with other strains of the same sub- heavy chain (HA,) from A/Memphis/102/72 type, and not at all with strains of a dif- was readily bound by antibodies directed ferent subtype (for review, see Schild and against the polypeptide portion of whole Dowdle, 1975); and (b) tryptic peptide maps HA. Furthermore, a mixture of peptides indicate that differences exist in the amino derived from HA, by cyanogen bromide acid sequence of different HA types (for cleavage also exhibited specific binding to review, see Webster and Laver, 1975). antibody. Carboxymethylation of these The mechanism underlying variation in peptides, however, destroyed their antithe amino acid sequence and, hence, in the genicity. antigenicity of influenza virus hemagglutiIn this paper we describe the isolation nins is of great biological importance, as of various noncarboxymethylated peptides antibodies directed against this molecule derived from A/Mem/102/72 HA by cyanoneutralize virus infectivity (see Schulman, gen bromide cleavage and compare their 1975). Comparison of primary amino acid antigenicities. We show that antibodies sequences of the hemagglutinins of different raised against virus react specifically with 1 To whom reprint requests should be addressed. the amino-terminal cyanogen bromide pep INTRODUCTION

0042~6822/79/040458-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

458

ANTIGENIC

ACTIVITY

IN THE N-TERMINAL

HALF

459

OF HA1

of Goding (1976). IgG obtained in this way gave a single staining band on SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing condiMATERIALS AND METHODS tions, two discrete bands of approximate Reagents. Protein A-Sepharose CL-4B molecular weights 50,000 and 25,000 were and Sephadex G-100 were obtained from obtained (data not shown). Where necesPharmacia South Seas Pty. Ltd.; cyanogen sary, IgG preparations were adsorbed with bromide from Koch Light Laboratories virus as previously described (Russell and Ltd., Colnbrook, Bucks, U.K.; Nonidet P40 Jackson, 1978; Russell et al., manuscript (NP40) from Shell Chemicals (Australia) submitted). These adsorption protocols Pty. Ltd.; 1251(IMS 30) from the Radio- were carried out in order to produce IgG chemical Centre, Amersham, Bucks, U.K.; preparations specific for the polypeptide diiodoethane from Fluka; and cellulose ace- portion of the hemagglutinin molecule, e.g., tate blocks from Chemetron, Italy. Puri- anti-Memn is derived from anti-(Mem”fied host carbohydrate antigen was a gen- Bel,)IgG by adsorption with S,-Bel, erous gift from Dr. W. G. Laver, A.N.U. virus, which removes all antibodies diViruses and antisera. Influenza viruses rected against neuraminidase and host were grown and purified according to Laver carbohydrate antigen, leaving IgG with (1969). Virus strains used were: A/Be1/42 specificity for the polypeptide moiety of (HONl), referred to as Bel; and the recom- A/Mem/102/72 hemagglutinin. Hemag(HI) titres of these binant viruses A/Memphis/102/72 (H3)- glutination-inhibition A/Be1/42 (Nl), referred to as Mem,-Bel,, antibody preparations are shown in Table 1. Preparation of hemagglutinin and heand A/shearwater/E.Aust/1/72 (HavG)-A/ Be1/42 (Nl), referred to as S,-Bel,. Virus magglutinin heavy chain. Preparation of titres were determined by hemagglutina- HA from the Mem,-Bel, recombinant tion titrations (Fazekas de St. Groth and virus was performed as described by Laver (1964) using cellulose acetate blocks in place Webster, 1966). Outbred New Zealand rabbits were inoe of cellulose acetate strips. Heavy chain was prepared essentially as described by Laver ulated subcutaneously and intramuscularly with purified virus in Freund’s complete (1971) except that whole HA was reduced adjuvant as previously described (Russell and dissociated by incubation at 37” for 2 hr and Jackson, 1978). IgG was obtained from in saturated guanidine hydrochloride conserum by affinity chromatography on pro- taining 0.06 M dithiothreitol and 0.01 M tein A-Sepharose according to the method Tris-Cl (pH 8.5). The guanidine hydro-

tide of the hemagglutinin heavy chain (HA,CNl).

TABLE

1

HEMAGGLUTINATION INHIBITIONTITRESOFIgG PREPARATIONS AGAINSTVARIOUSVIRUSES

Virus Be1 Memn-Bel,

Normal rabbit

Adsorbed anti-Mem,”

<80b ~80

<80 32,000

Unadsorbed anti-(Memn-Be&,) <80 20,700

Unadsorbed anti-Be1 2600 460

Adsorbed anti-Bela 2100 <80

a Anti-Mem, IgG was prepared by adsorption of anti-(Memn-Bel,) IgG with the recombinant virus A/Shearwater/E.Aust./l/72(Hav6)-A/Bel/42(Nl) to remove antineuraminidase and anticarbohydrate antibodies. AntiBe1 IgG was similarly adsorbed. b Results are expressed as the reciprocal of the IgG dilution showing 50% inhibition of 4 HA units of virus. Concentrations of IgG were 1.0 mg/ml.

460

JACKSON

ET ti.

choloride density gradient contained 0.002 M dithiothreitol and 0.008M Tris-Cl (pH 8.5). Sodium dodecyl sulphate -polyacrylamide gel electrophoresis. Electrophoresis

analyses were carried out using the method of Russell and Skehel(19’72) with the modifications previously described (Jackson et al. , 1978). Gels were sliced transversely into 2-mm pieces and radioactivity was determined in a Packard Auto Gamma scintillation spectrometer. Radioiodination. Polypeptides were radioiodinated using a modification of the chloramine T method (Greenwood et al., 1963) according to Russell and Jackson (1978).

.l..i

Detection of antigen-antibody complexes. This was done as previously described (Jackson et al., 1978). Briefly,

radioiodinated peptides were allowed to react with IgG for 72 hr at 37”, and resultant antigen-antibody complexes were detected by passage through a protein A-Sepharose immunoadsorbent gel. Antigen-IgG complexes were eluted with 0.1 M acetic acid in normal saline. Radioactivity present in the eluates was then determined.

10

30 FRKTION

NUMBER

FIG. 1. Fractionation of the cyanogen bromide digest of influenza virus hemagglutinin on a column (1.5 cm* x 120 cm) of Sephadex G-100 in 50% formic acid. The flow rate was 4.6 ml/hr and the fraction size was 2.3 ml. Fractions were pooled as indicated.

acid (Fig. 1). Fractions were pooled as indicated. Dansyl end-group determinations and RESULTS amino acid analysis were used to characPreparation of Disulphide-Linked Cyanoterise the component peptides of the fracgen Bromide Peptides tions (see Fig. 2). Fractions 45-62 contained the two octapeptides, CN4 and CN5, Purified HA obtained from Mem,-Bel, derived from HA, and the two small cyanorecombinant virus [500 nmol in 3.0 ml 70% (v/v) formic acid ] was incubated at 20” for gen bromide peptides, CN4 and CN3, de16 hr with a loo-fold molar excess of cyano- rived from the HA light chain (HA,). Fracgen bromide over the number of methionine tion P2 contained the 92-residue cyanogen residues present (Ward and Dopheide, bromide peptide CN2 from HA, (HA,CNZ) 1976). The digest was fractionated on a and the 98-residue peptide CN2 from HA, column of Sephadex G-100 in 50% formic (HA,CNZ). Fraction Pl, which eluted from ml

HA2

CN2

em

NT-L

cN4

CNZ

II

cu4

v//////////ma//////////n CN3

cN5

CN3

c0a-l CNl

FIG. 2. Schematic representation of the linear arrangement of cyanogen bromide peptides within A/Mem/lOU72 hemagglutinin heavy and light chains based on data of Ward and Dopheide (1979). The figures within the boxes representing individual cyanogen bromide peptides indicate the number of amino acid residues. The peptides represented by cross-hatched boxes are those which are linked together by disulphide bridges and together comprise fragment Pl (for explanation, see text).

ANTIGENIC

ACTIVITY

IN THE N-TERMINAL TABLE

HALF

OF HA,

461

2

SUMMARYOF ELUTION PROFILESOBTAINED BY ELUTION OF VARIOUS COMBINATIONSOF IgG AND HA-DERIVED PEPTIDES FROMA PROTEIN A-SEPHAROSE AFFINITY COLUMN 1gG*sC ‘Y-labeled HA fragment” HA, Pl (HA,CNl-HA,CNl-HA,CN3) P2 (HA,CNZ + HA,CNZ)

Reoxidised HA&N1 Reoxidised HA,CN3 Reoxidised HA,CNl + HA,CN3

Total cps added

Normal rabbit

Anti-Mem,

2,300 1,891 48 768

75,387 90,235 6,962

48,954 19,290

24,082 16,691 32 3,154

132

143

961

64,742

2,960

n Equimolar amounts of iodinated peptide were treated with 25 pg of IgG. b Anti-Mem, IgG is prepared from anti-(Mem,-BelN) IgG by adsorption with SH-Bel, recombinant virus. r Results are expressed as the cps eluted from the protein A-Sepharose with 0.1 M acetic acid in normal saline.

the column in a highly aggregated form, contained the disulphide-linked cyanogen bromide peptides, CNl and CN3, derived from HA, (HA,CNl and HA1CN3, respectively) and the C-terminal cyanogen bromide peptide, CNl, derived from HA, (HA,CNl) (see Ward and Dopheide, 1979). Samples of Pl and P2 were radioiodinated and tested for antigenic activity as described in Materials and Methods. Fractions 45-62 were not investigated further, as each of the peptides eluting in this position lacks tyrosine (see Ward and Dopheide, 1979) and would not therefore be susceptible to radioiodination. Only fragment PI demonstrated significant antigenicity (Table 2). Experiments were next carried out in order to separate the three component polypeptides present in fraction PI and to establish which of them carried antigenic determinant(s). In a previous paper (Jackson et al., 1978) we reported that reduction and carboxymethylation of cyanogen bromide peptides obtained from HA, resulted in destruction of antigenic activity, whereas cyanogen bromide peptides prepared with disulphide bridges intact exhibited substantial antigenicity. For this reason, the components of PI were fractionated under reducing conditions and the separated peptides allowed to reoxidise before assaying antigenie activity.

Reduction of Cyanogen Bromide Fragment Pl and Separation of Components Cyanogen bromide fraction Pl was reduced for 16 hr at 50” under nitrogen in 6 M guanidine hydrochloride/500 mM TrisHCl (pH 8.4), with a 50-fold molar excess 2

FRKTION

NUMBER

FIG. 3. Fractionation of cyanogen bromide peptides, derived by reduction of complex Pl (Fig. 1) on a column (1.5 cm* x 120 cm) of G-100 Sephadex in 50% formic acid, 1 mM in dithiothreitol. The flow rate was 4.6 ml/hr, and fractions were collected every 30 min.

462

JACKSON ET AL.

of dithiothreitol. The reaction mixture was then fractionated on Sephadex G-100 in 50% formic acid containing 1 mM dithiothreitol (Fig. 3). Fractions SH-1, SH-2, and SH-3 were pooled as indicated. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fragment Pl (Fig. 4) and its components, SH-1 (gel profile not shown), SH-2, and SH-3 (Fig. 5), gave peaks with identical electrophoretic behaviour to authentic samples of HA,CNl, HA,CN3, and HA,CNl. The large amount of material which did not enter the gel shown in Fig. 4 represents the highly aggregated HA,CNl. These results, together with dansyl end-group and amino acid analyses, confumed the identity of each of the reduced peptides (see Ward and Dopheide, 1979): SH-1 is the carboxyl-terminal cyanogen bromide fragment from HA2 (HA,CNl), 100

n*l li

1

DYE 1

800

1OOL

80.

20.

FRPCTlON

I’HJMBER

FIG. 5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis profiles of HA&N1 (A A) and HA,CN3(A - - - A). Purified peptides were obtained by reduction of fragment Pl (Fig. 1) and subsequent separation of component peptides on G-100 (Fig. 3). Reoxidised SH-2 (HA,CNl) and SH-3 (HA,CN3) were radiolabeled and introduced to 10 cm, 10% polyacrylamide gels. Slices (2 mm) of each gel were taken and radioactivity was determined. Electrophoresis is from left to right, and the arrow indicates the position of HA,. The number of cps for each gel has been normalised; the 100% peak count value for HA,CNl is 2143 cps, and that for HA,CN3,4106 cps.

SH-2 is the amino-terminal cyanogen bromide peptide from HA, (HA,CNl), and SH-3 is the fourth cyanogen bromide fragment in the HA heavy-chain sequence (HA,CN3). The yield for each peptide was approximately 50%. FRACTION

NUMBER

FIG. 4. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic profile of fragment Pl. Fragment Pl, obtained by cyanogen bromide cleavage of hemagglutinin and subsequent isolation by gel chromatography on G-100 (Fig. I), was radiolabeled and subjected to electrophoresis on a lo-cm 10% polyacrylamide gel. Slices (2 mm) of the gel were then counted for Y. Electrophoresis is from left to right. The arrow indicates the position of HA, assumed under identical electrophoresis conditions, and the 100% peak count has a value of 1073 cps.

Reoxidution of Reduced Cyanogen Bromide Peptides

Although fractions SH-1, SH-2, and SH-3 were isolated under reducing conditions, complete reduction was ensured prior to reoxidation under highly dilute conditions. Derivatives were dissolved in 1.2 ml of 6 M guanadine hydrochloride1500 mM Tris-Cl (pH 8.4) in small vials and flushed with N, for 60 min. Dithiothreitol(200 pmol) was then added, and capped vials were incu-

ANTIGENIC

ACTIVITY

IN THE N-TERMINAL

HALF OF HA,

463

bated for 5 hr at 50”. Solutions were de- over, if the antigenic activity of HA, is salted on a column (2 x 30 cm) of Sephadex attributed to carbohydrate, it would not G-25 superfine in 30% acetic acid. The pep- be expected that reduction and carboxytide fractions were diluted with 500 ml of methylation of cyanogen bromide peptides N,-saturated water and adjusted to pH derived from HA, would affect antigenicity. To confirm the premise that the antigenie 8.5 with concentrated ammonia solution. Reoxidation was carried out by adding activity of HA, detected in these studies to each diluted fraction a solution of diiodo- is dependent only on the protein portion of ethane in methanol (15 mg/lO ml) dropwise the molecule, the ability of purified host with stirring under nitrogen over a period antigen to inhibit the formation of immune of 10 min. The amount of diiodoethane complexes between lz51-labeled HA, and added represents a 50- to loo-fold molar various adsorbed and unadsorbed antiviral excess over the expected amount of peptide IgG preparations was investigated. The thiol. After standing overnight, the solu- results (Table 3) show that the formation of immune complexes between adsorbed tions were freeze-dried. Peptides HA,CNl and HA,CN3 were anti-Mem, IgG and HA, is not due to the dissolved in PBS, and samples were radio- involvement of carbohydrate present on the iodinated and examined for antigenic activ- polypeptide, as purified host carbohydrate antigen added to the system had no signifiity as described above. Peptide HA&N1 was insoluble in aqueous medium and con- cant inhibitory effect. Only a slight inhibisequently could not be examined for anti- tion was observed when unadsorbed antiIgG was used, indicating genie activity by this method. As shown (Mem,-Bel,) in Table 2, HA,CNl was the only compo- that this antibody preparation contained nent of fragment Pl which demonstrated little anticarbohydrate activity. This is supported by the observation (Table 1) significant antigenie activity. that unadsorbed anti-(Mem,-Bel,) IgG Effect of Host Antigen on Formation HA,-IgG Complexes

of

The possibility exists that part of the antigenicity exhibited by HA, and its fragments is due to the presence of host carbohydrate antigen (Harboe, 1963; Laver and Webster, 1966) on these polypeptides. Antisera raised against chicken egg-grown viruses contain anticarbohydrate antibodies in addition to antibodies directed against the polypeptide regions of the HA (Gerhard et al., 1975). Each of the AJMem/102/72 HA-derived fragments, HA,, Pl, and HA,CNl, possesses carbohydrate components (Ward and Dopheide, 1976; Dopheide and Ward, 1978; Ward and Dopheide, 1979) and might be expected to react with antihost antibodies if these are present. However, no antihost antibodies should be present in the IgG preparations used in our studies because all IgG preparations were exhaustively adsorbed with chicken egggrown recombinant virus to produce antibody preparations specific for the protein portion of the hemagglutinin only. More-

TABLE 3 EFFECTOFPURIFIEDHOSTCARBOHYDRATEANTIGEN ON FORMATIONOF~YMUNE COMPLEXES BETWEEN HA, AND IgG PREPARATIONS

IgG” Anti-Memnd Anti-(Memn-Be&,) (unadsorbed) Anti-Be1 (unadsorbed) Normal rabbit

Total CPs added

Host carbohydrate antigen”sr Present

Absent

79,000

31,893

32,657

79,000

11,596

13,670

79,000 79,000

1,928 1,125

13,293 1,625

UTwenty-five micrograms of IgG preparation was incubated in each case with 20 pmol of ‘?-labeled HA,. b Host antigen was present at 100 to 200-fold molar excess over the amount of HA, present. c Results are expressed as cps lzsI eluted from protein A-Sepharose immunoadsorbent. d Anti-Mem, IgG is obtained from anti-(MemnBel,) (unadsorbed) IgG by adsorption with S,-Bel, recombinant virus.

464

JACKSON

has little HI activity against Be1 virus. In contrast, when unadsorbed IgG directed against the serologically unrelated virus A/Bel/42 was incubated with Mem/72 HAI, a significant number of immune complexes was formed which were due almost entirely to antibodies directed against the host antigen, as purified host carbohydrate almost completely inhibited the formation of these complexes. Prior adsorption of anti-AlBeV42 IgG with S,-Bel, recombinant virus successfully removed these anticarbohydrate antibodies and abolished the ability of this antibody preparation to interact with A/Mem/ 102/72 HA, (Jackson et al., 1978).

ET AL.

son, unpublished observation) and that the interaction of antiviral IgG with pure HA is inhibited by HA, (Jackson et al., 1978). The preparation of the disulphide-linked complex Pl (HA,CNl-HA,CNl-HA,CN3) did not involve the breakage of any disulphide bridges, but a removal of peptide chain pieces from the whole HA molecule by means of cyanogen bromide cleavage. This left the three half-cystine-containing cyanogen bromide peptides bound together by S-S bonds, a property which would help the preservation of secondary structure and also the reattainment of a favourable tertiary structure. The third derivative showing antigenic DISCUSSION activity, the cyanogen bromide peptide Investigations into the relationship be- HAICN1, is perhaps the most interesting. tween antigenicity and protein structure It was prepared by reductive cleavage of are made difficult by the fact that antigenic the disulphide-linked peptide complex Pl activity depends upon protein conformation. followed by controlled reoxidation at high Because of this requirement for conforma- dilution to minimise the chances of intertional integrity, exposure of a native protein molecular disulphide interchange. The relato denaturants, fragmentation by proteoly- tively lower antigenic activity of this derivsis, or chemical modification carries the risk ative compared with that of HA, and Pl that the ability of the resulting derivative is not surprising when we consider that its to bind to antibody, raised against the disulphide-linked peptide progenitor Pl native protein, will be affected (for review, (HA,CNl-HA,CNl-HA,CN3) benefited see Habeeb, 1977). Any study into the na- from a high molecular weight and relatively ture of the antigenic sites present on pro- intact secondary structure. Both of these teins must take this consideration into ac- properties should greatly aid the maintecount in the final interpretation of results. nance of a nonrandom conformation within In this study we have described proce- the Pl fragment. In addition, the highly dures for the preparation of fragments of aggregated state of Pl, due to the compoinfluenza virus hemagglutinin and exam- nent HA&N1 (Fig. 4), could lead to multiined the ability of these derivatives to com- molecular complexes which might be antibine with IgG raised against whole virus. genically multivalent and form highly avid Of these fragments, HA, and the disulphide- complexes with IgG. linked complex Pl (HAICN1-HA&NlThe lower antigenicity of HA,CNl could HA,CN3) demonstrated substantial anti- also be due in part to a lack of conformagenie activity, while the cyanogen bromide tional stability brought about by incomplete peptide HAICN1, in a reoxidised form, and/or mismatched S-S bond formation showed significant antigenic activity. during reoxidation. Nevertheless, HA,CNl The preparation of HA, involved the was found to be significantly antigenic, reduction of disulphide bonds, but this whereas none of the other cyanogen broreduced polypeptide presumably reanneals mide peptides (HA,CNZ, HA,CN3, and to an antigenically active form. This is sup- HA,CNB) tested demonstrated activity. Although it is tempting to speculate that ported by observations that it is able to elicit production of antibodies capable of HA,CNl carries the antigenic determinant(s) of the hemagglutinin, the results reacting with whole HA from SDS-disrupted virus (Brand and Skehel, 1972;Jack- do not preclude the possibility that other

ANTIGENIC

ACTIVITY

IN THE N-TERMINAL

HALF

OF HA,

465

HARBOE, A. (1963). The influenza virus hemagglutinacyanogen bromide peptides contributed tion-inhibition by antibody to host material. Acta. directly or indirectly to the antigenic acPathol. Microbial. Stand. 57, 317-330. tivity of HA,CNl or that they bear additional antigenic determinants which are JACKSON, D. C., RUSSELL, R. J., WARD, C. W., and DOPHEIDE, T. A. (1978). Antigenic determilost as a result of conformational change nants of influenza virus hemagglutinin. I. Cyanogen during fragmentation of the intact hemagbromide peptides derived from A/Memphis/72 glutinin. hemagglutinin possess antigenic activity. Virology ACKNOWLEDGMENT

The suggestion to investigate the effect of purified host antigen on the detection of antigenic activity of the various peptides was made by Dr. W. G. Laver.

REFERENCES

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FAZEKAS DE ST. GROTH, S., and WEBSTER, R. G. (1966). Disquisitions on original antigenic sin. I. Evidence in man. J. Ezp. Med. 124, 331-345. GERHARD,W., BRACIALE, T. J., and KLINMAN, N. R. (1975). The analysis of the monoclonal immune response to influenza. I. Production of monoclonal anti-viral antibodies in vitro. Eur. J. Immunol. 5, 729-725. GODING, J. W. (1976). Conjugation of antibodies with fluorochromes: Modifications to the standard methods. J. Immunol. Methods 13, 215-226. GREENWOOD,F. C., HUNTER, W. H., and GLOVER, J. S. (1963). The preparation of 1311-labelledhuman growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. HABEEB, A. F. S. A. (1977). In “Immunochemistry of Proteins” (M. Z. Atassi, ed.), Vol. I, pp. 163-229. Plenum Press, New York and London.

89, 199-205. LAVER, W. G. (1964). Structural studies on the protein subunits from three strains of influenza virus. J. Mol. Biol. 9, 109-124. LAVER, W. G. (1969). In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 66-82. Academic Press, New York. LAVER, W. G. (1971). Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus. Virology 45, 275-288. LAVER, W. G., and WEBSTER, R. G. (1966). The structure of influenza viruses. IV. Chemical studies of the host antigen. Virology 30, 104-115. RUSSELL, R. J., and JACKSON, D. C. (1978). Direct solid phase radioimmunoassay for measuring antigenie differences between the hemagglutins of influenza viruses. J. Immuwl. Methods 22,201-209. RUSSELL, W. C., and SKEHEL, J. J. (1972). The polypeptides of adenovirus-infected cells. J. Gen. Viral. 15, 45-57. SCHILD, G. C., and DOWDLE, W. R. (1975). In “The Influenza Viruses and Influenza” (E. D. Kilbourne, ed.), pp. 316-372. Academic Press, New York. SCHULMAN, J. L. (1975). In “The Influenza Viruses and Influenza” (E. D. Kilbourne, ed.), pp. 373-393. Academic Press, New York. WARD, C. W., and DOPHEIDE, T. A. (1976). Size and chemical composition of influenza virus hemagglutinin chains. FEBS Lett. 65, 365-368. WARD, C. W., and DOPHEIDE, T. A. (1979). Primary structure of the Hong Kong (H3) hemagglutinin. Brit. Med. Bull. 35, 51-56. WEBSTER, R. G., and LAVER, W. G. (1975). In “The Influenza Viruses and Influenza” (E. D. Kilbourne, ed.), pp. 269-314. Academic Press, New York.