Biochem. J. (1981) 193,953-962Printed in Great Britain

953

Amino acid sequence and oligosaccharide distribution of the haemagglutininfrom an early Hong Kong influenza virus variant A/Aichi/2/68 (X-3 1)

Colin W. WARD and Theo A. DOPHEIDECSIRO, Division ofProtein Chemistry, 343 Royal Parade, Parkville, Victoria 3052, Australia

(Received 8 July 1980/Accepted 19 November 1980)

The amino acid sequence and oligosaccharide distribution for the haemagglutinin fromthe early Hong Kong influenza virus A/Aichi/2/68 (X-3 1) was investigated. The twopolypeptide chains, HA 1 and HA2, were fragmented by CNBr and enzymic digestion,and the amino acid sequence of each small peptide was deduced by comparing itschromatographic behaviour, electrophoretic mobility, amino acid composition andN-terminus with that of the corresponding peptide of the haemagglutinin of knownstructure from the influenza-virus variant A/Memphis/102/72. Those peptides in whichchanges were detected were sequenced fully. The complete amino acid sequence of thehaemagglutinin HA1 chain (328 residues) and 188 of the 221 residues of the HA2chain were established by this approach, and revealed only twelve differences betweenthe amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins.These occurred at positions 2, 3, 122, 144, 155, 158, 188, 207, 242 and 275 in the HAlchain and 150 and 216 in the HA2 chain. The highly aggregated hydrophobic region(residues 180-121) near the C-terminal end of the HA2 chain was not resolved bypeptide sequencing. The oligosaccharide distribution in variant-A/Aichi/68 haemag-glutinin was identical with that found in that of A/Memphis/72, with sugar unitsattached at asparagine residues 8, 22, 38, 81, 165 and 285 in the HAl chain and 154 on

the HA2 chain. The monosaccharide compositions of the individual carbohydrate unitson variant-A/Aichi/68 haemagglutinin differed from those of the corresponding units invariant-A/Memphis/72 haemagglutinin, and evidence was found for heterogeneity in theoligosaccharide units attached at single glycosylation sites.

Influenza remains a poorly controlled infectionthat frequently affects a large proportion of thepopulation, largely because of the ability of the virusto undergo frequent and often dramatic antigenicchange. Although both viral coat proteins (haemag-glutinin and neuraminidase) can change, thehaemagglutinin is considered the more importantantigen because of its central role in the infectionprocess (Drzeniek, et al., 1966; Laver & Kilbourne,1966; Klenk et al., 1975; Lazarowitz & Choppin,1975).Two kinds of antigenic variation have been

observed in influenza virus: major changes, termed'antigenic shift', and minor changes, termed 'anti-genic drift'. Since the first influenza virus wasisolated from man in 1933 (Smith et al., 1933),antigenic shifts have occurred in 1957 (Asianinfluenza), 1968 (Hong Kong influenza) and 1977(Russian influenza). Since the appearance of theHong Kong sub-type of influenza type A virus inman in 1968, rapid antigenic drift has occurred,

Vol. 193

resulting in the appearance of new variants eachyear (Schild et al., 1974; Pereira, 1976). Peptide'maps' have shown that antigenic shift and drift areassociated with major and minor differences respec-tively in the amino acid sequences of the haem-agglutinin (Laver & Webster, 1968, 1972).

Previous reports have described the amino acidsequence of the haemagglutinin from the HongKong variant A/Memphis/102/72 as determined byprotein (Dopheide & Ward, 1978, 1979, 1980;Ward & Dopheide, 1979, 1980; Ward et al., 1980a)and nucleic acid (Sleigh et al., 1980) procedures, andthese data have been used to determine the partialamino acid sequences of several natural Hong Kongfield strains (Laver et al., 1980), and variantsselected with monoclonal hybridoma antibodies(Laver et al., 1979). In the present paper we describeour investigations of the amino acid sequence of thehaemagglutinin from X-3 1, a recombinant viruscontaining the haemagglutinin from the early HongKong strain A/Aichi/68. We have determined the

0306-3275/81/030953-10$01.50/1 © 1981 The Biochemical Society

C. W. Ward and T. A. Dopheide

structure to assist the characterization of the extentof chemical and antigenic change that has occurredin the Hong Kong sub-type during antigenic drift,and to provide the sequence data required for theinterpretation of the 0.3 nm (3 A)-resolution electron-density map derived from X-ray-crystallographicanalysis of X-31 haemagglutinin crystals (Wiley &Skehel, 1977; Wilson et al., 1980).

Materials and methodsVirusThe primary inoculum of the X-31 virus was

kindly supplied by Dr. W. G. Laver, AustralianNational University, Canberra, Australia. Virus wasgrown in the allantoic sac of 11 day-old-chickembryos and purified as described by Laver (1969).A total of 4000 chick embryos yielded 600mg ofvirus.

Haemagglutinin isolation and peptide fragmen-tationThe procedures for haemagglutinin isolation by

electrophoresis on cellulose acetate blocks, theseparation of the heavy (HAl) and light (HA2)chains by centrifugation on guanidine hydro-chloride density gradients, reduction and S-carboxy-methylation with iodo[2-"4C]acetate, CNBr cleavageand the separation of the CNBr peptides have beenfully described, as have the procedures for pyro-glutamyl aminopeptidase (EC 3.4.11.8), trypsin (EC3.4.21.4), thermolysin (EC 3.4.24.4), pepsin (EC3.4.23.1) and Staphylococcus aureus proteinase (EC3.4.21.19) digestion (Dopheide & Ward, 1978,1979, 1980; Ward & Dopheide, 1979, 1980; Wardet al., 1980a). The digestion of peptide HA2 CN1(for nomenclature, see below) with trypsin (1% w/w)was extended for 48h at 370C in 0.1M-N-ethyl-morpholine/acetate, pH8.0, with stirring; a furtheraddition of enzyme was made after 24h. Solubletryptic peptides were intially fractionated on twocoupled Sephadex G-50 (Fine grade) columns(150cmxO.9cm each, in series) in 0.O1M-NH4HCO3/10% (v/v) propan-2-ol. Pooled fractionswere further purified by high-voltage electrophoresisand, where necessary, chromatography in butanol/acetic acid/water/pyridine (15 :3 :12: 10, by vol.) onWhatman 3 MM paper.

AnalyticalAmino acid analyses were performed after hydro-

lysis in 5.7 M-HCl/0.004 M-thioglycollic acid at1080C in vacuo for 24h. Tryptophan-containingpeptides were identified by staining side strips withErhlich reagent (Easley, 1965), and were hydrolysedin 4M-methanesulphonic acid containing 0.2% tryp-tamine at 1150C for 24h (Simpson et al., 1976).

C-Terminal amino acids were detected by selective3H-labelling (Holcomb et al., 1968). Automatedsequence degradation of the deblocked HA1 chainwas performed with a protein program (Inglis et al.,1979), and manual dansyl (5-dimethylaminonaph-thalene-l-sulphonyl)-Edman degradations were per-formed as described previously (Ward & Dopheide,1979). Glucosamine was determined on an aminoacid analyser after hydrolysis in 3M-p-toluenesul-phonic acid at 1000C for 24h (Allen & Neuberger,1975). Neutral sugars were determined as alditolacetates by g.l.c. (Albersheim et al., 1967). Allsamples (12-70nmol) were hydrolysed in 2ml of2.5 M-trifluoroacetic acid at 1000C for 2h in a sealedtube under N2. After hydrolysis, the acid wasremoved by rotary evaporation and the hydrolysesreduced with NaBH4 and acetylated (Albersheim etal., 1967). The resulting alditol acetate derivativeswere separated by g.l.c. on a column (1.5m x2mm) of 3% SDP 2340 on Supelcoport 100/120.

Peptide nomenclaturePeptides have been assigned a prefix according to

the digest from which they were recovered (CN,CNBr; T, tryptic; C, chymotryptic; Th, thermo-lytic; P, peptic; S.a., Staphylococcus aureus pro-teinase) and a number (1, 2, 3) according to theorder in which they occur in the final sequence.Peptides resulting from partial cleavages, sharingcommon regions of sequence, are denoted la, lb, Icetc.

Results

In the present study we have fragmented the twochains (HA 1 and HA2) of variant-X-31haemagglutinin by CNBr cleavage and enzymicdigestion and deduced the amino acid sequence ofeach peptide by comparing its chromatographicbehaviour, electrophoretic mobility, amino acidcomposition and N-terminus with that of thecorresponding peptide from the haemagglutinin ofvariant A/Memphis/102/72 (A/Mem/72), the struc-ture of which is already known (see the introductionfor references). For those peptides in which changeswere detected, the amino acid sequence was deter-mined directly.

Structure ofthe HAI chainCNBr cleavage of S-carboxymethylated HA 1

(700nmol) gave five peptides, CN1-CN5, whichcould be readily separated by gel filtration (Fig. 1).The large fragments CN1 (residues 1-168), CN2(residues 169-260), and CN3 (residues 269-320)were digested with trypsin and the digests fraction-ated as shown in Figs. 2(a), 2(b) and 2(c). The largesoluble tryptic peptides, CN1.Tlb, CN1.Tlc,CN1.T3 and CN3.T2, as well as the large insoluble

1981

954

Primary structure of X-31 influenza haemagglutinin

2.5-6000

CU

2.0 0-5000

1.5 - 4000

I~~ ~~~~-3000.1.0

2000~

0.5 I/ ~~~~~~-1000~

S4[2-14C Icarboxymethylated variant-X-31 haemag-glutinin chain HA I

The digest was fractionated on a column (150cm x1.Ocm) of Sephadex G-100 in 50% (v/v) formicacid. Peptides were identified by A280 ( ) andradioactivity in lOl samples (----). The fractionsize was 2.1 ml, and the flow rate 4.2 ml/h.

tryptic peptides, CN1.T5, CN2.T2a and CN2.T10,were further digested with either thermolysin, S.aureus proteinase or pepsin.

The complete sequence of X-31 HA1 is shown inFig. 3, along with the known sequence of A/Mem/72 HA1 and the location of the peptides usedto deduce the sequence. Only ten sequence changeswere found between X-31 and A/Mem/72 HA 1polypeptides. These occurred at positions 2, 3, 122,144, 155 and 158 in peptide CN1, 188, 207 and 242in peptide CN2 and 275 in peptide CN3. Theamino acid compositions, electrophoretic mobilitiesand amino acid sequences for the peptides con-taining these changes are shown in Table 1. Directsequenator analysis (40 cycles) of carboxymethy-lated HA 1 (150 nmol), after the N-terminal pyro-glutamic acid blocking group has been removed withcalf-liver pyroglutamyl aminopeptidase, confirmedthe suspected sequence changes at residues 2 and 3in the blocked peptide CN1.Tlb.Thla (see Fig. 3).

The nucleic acid sequence for the haemagglutiningene from A/Mem/72 (Sleigh et al., 1980) showedthat the 'Asx' residues at positions 137 and 250 inHA1 were asparagine not aspartic acid as found inthe protein sequence (Ward & Dopheide, 1980;Ward et al., 1980a) and that the sequence at residues.290-293 was Asn-Asp-Lys-Pro not Lys-Pro-Asp-Asp (Dopheide & Ward, 1978). As shown in Fig. 3,the X-31 sequence agrees with the A/Mem/72nucleic-acid-sequence data. Peptides CNI.T5.Th5,CN2.TIO.P3 and CN3.T2.Th4 were each recoveredas both neutral and acidic (deamidated) versions,supporting the assignment of residues 137, 250 and290 or 291 as asparagine. Manual sequence analysis

1400_

1200 EEcc

10000

800 -E

600 ci

400 *>C.g

200 o

CU0 X4

'-7~ ' ' . " .' " .' '- '.'7' I l.

0.5 _ 40 50 60 70 80 90 100 110 12Q 2000

0.4 150-1 0

0.341000

0.2~~~~~~~~~~~~~~~~~0

0i.2.Crmtgah oftytcdgss f CNBr

0.1

- A T5 T3c CU0 T4~~~~~~~~~~~

40 50 60 70 80 90 100 110 120Fraction no.

Fig. 2. Chromatography of tryptic digests of CNBrpeptides (0.6,umol, 18h digestion) from variant-X-31

haemagglutinin chain HAITwo columns (each 150cm x 0.9 cm) of SephadexG-50 (Fine grade) connected in series were used,and the columns eluted with 0.01 M-NH4HCO3/10%(v/v) propan-2-ol at a flow rate 4.4 ml/h andmonitored by A230 ( ) and radioactivity in 10 or20,ul samples (----). Fractions (2.2 ml) werepooled as shown, and the peptides present at eachfraction are indicated. (a) Tryptic peptides from S-[2-'4C]-carboxymethylated peptide CN1; (b) trypticpeptides from peptide CN2; (c) tryptic peptidesfrom S-[2-'4C]carboxymethylated peptide CN3.

of peptide CN3.T2.Th4 (100mol) confirmed thesequence of residues 290-293 as Asn-Asp-Lys-Pro.All other peptides isolated from X-31 HA 1 andindicated in Fig. 2 were identical with the corres-ponding peptides from A/Mem/72 in end group,composition and chromatographic behaviour (seeDopheide & Ward, 1978; Ward & Dopheide, 1980;Ward et al., 1980a). Oligosaccharide units were

Vol. 193

955

956 C. W. Ward and T. A. Dopheide

.4em/72 Gix ASP Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cvs Leu (ly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr lie ThrX-31 Clx Tyr. eu Pro Cly Asn A Asn G;y.TAy.Y .Ly8.Tt.Hlv.Tbtr

30

------------------Thla---------------- --Thlb-4 l--Th2--1 1-----.Th3--- ---Th4--------- ,---Th5---- 3---------CNI I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~CNI1

Mem/72 Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr Cly Lys Ile Cys Asn Asn Pro His Arg Ile Leu AspX-31 Asn.AVp.1Cji .(4V. .Yqj Thr As*n Ala Thr Clu Leu Val Gln Ser Ser Ser Thr (fly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp

60Tlc- 1 T2 11 6

----Sal .----------- 4 .---------Sa2.--------- 4 --------------. Sa3 -------------- --------ThlCN1 CNM

Mem/72 Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro ais Cys Asp Gly Phe Gln Ain Glu Thr Trp Asp Leu Phe Val Glu ArgX-31 Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln Asn Glu Thr Trp Asp Leu Phe Val Glu Arg

90-T3

-- 14-----Th2a---- A -----Th3a--- +- Th-4 4---------Th4d ---------------- 4 ---------Th5a------------ A i--Th6-1 ---Th7----CN1 4b CN1

Mem/72 Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu PheX-31 Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe

120

4T4b--1 T4c 1i--Th1- i----------------- Th2-------------i

CN1 CN1I

Mem/72 Ile Asn Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro s Ser Gly Phe Phe Ser ArgX-31 Ile Glu.Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro ASer GlyPhePe r g

IT 1050i5 T-i- b ---6b

,-----Th3--4A---------Th4---4-------------------Th5--------------------- 6aCN1 CNI

Mem/72 Leu Asn Trp Leu Lys SerG Ser Thr Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile TrpX-31 Leu Asn Trp Leu Ty Lys Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp............ ......... .... ^....... ...... ...,---.18

1T7T 1 4 - T1 l - T2 ---Th1-4 4----Th2

4---P2------CN 1 CN2

Mem/72 Gly Val His His Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser GlnX-31 Gly Val His His Pro Ser Thr AsGln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser.Thr Arg Ar Ser Gln

$ . * .. - * ..* ss ........................................ . ...... . ....21.T- 210

- ~~~~~~~~~ ~~~~~T21 5a @ir---44 -------------Th3 ------------------------- 4 1--Th4--A 4-----T5--

c

- P2 ------------------------------ - - ---4

CN2 CN2

Mem/72 Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro GlyX-31 Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg lie Ser Ile Tyr TreThr l

240T6 T7a a- -T-I TIO

---Pi- -----.P2.------1 ---------P3----------------Plb----------4

CN2 CN2Mem/72 Asp Tii Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser

X-31 Aspl Vl! Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser270

T10lI T1 T IlFTlt1. __ _ ______ .__---------p4.---------------- .

A _____P5_----- 12

CN2 CN4 CN3

Mem/72 Asp Ala Pro lie Gly Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys IleX-31 Asp Ala Pro Ile Asp Thr Cys Ile Ser ;lu Cys lie Thr Pro Asn Gly Ser Ile Pro Asn As Lys Pro Phe Gin Asn Val Asn Lys le

300T2

--ThIT--4 --M----- 4 ----Th3------4 4----------------------Th4--------------------- 41-----Th5--4 ----Th6-- |-CN3 CN3

Mem/72 Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln ThrX-31 Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr

T3b - 1t- T4----I I- T5 - - -T-- -IT3c-1T-3a- --4

CN 3 CM5

Fig. 3. Amino acid sequence ofvariant-X-31 haemagglutinin chain HAIThe alignment of the tryptic and appropriate thermolytic, peptic and S. aureus-proteinase peptides used to derive thesequences is shown and is based on comparison with the known amino acid sequence of A/Mem/102/72haemagglutinin. The N-terminal 'Glx' residue is pyroglutamic acid; methionine residues occur in both strains atpositions 168, 260, 268 and 320. Carbohydrate groups are attached in both strains at asparagine residues 8, 22, 35,81, 165 and 285. The regions sequenced directly are underlined with dots. The residues that differ between the twohaemagglutinins are enclosed in boxes.

1981

Primary structure of X-3 1 influenza haemagglutinin

Table 1. Amino acid composition and sequence of variant-X-31 haemagglutinin peptides that diferedfrom the corre-spondingpeptides in varientA/Mem/72

For the location of these peptides and their comparison with A/Mem/72, see Figs. 3 and 6. Abbreviations used:<Glu, pyroglutamic acid; Hse, homoserine; CmCys, carboxymethylcysteine.

Peptide N-terminus Mobility Composition Sequence

<Glu -0.30 Asp2.7 Thrl. SerO.8 Glul. Glx-Tyr-Leu-Pro-Gly-Asn-Pro,., Glyl.1 LeuO.9 Tyro.8 Asp-AsnGlcNAc2.1

Ile Thro09 Glul.1 Glyl. Ileo.9 Ile-Thr-Glu-GlyGly +0.32 Serl.7 Prol.0 Gly2.9 Phe2.2 Gly-Pro-Gly-Ser-Gly-Phe-

Arg1.O Phe-Ser-ArgLeu +0.32 Aspl0 Thrl.0 Leu2.0 Lysl.Trp Leu-Asn-Trp-Leu-Thr-LysSer -0.13 Asp.2 Thr2.1 Serl.8 Glul.0 Ser-Glu-Ser-Thr-Tyr-Pro-Val-

Hseo.9 Pro1.1 Val1.9 Leu1.1 Leu-Asn-Val-Thr-MetTyr1.0 GlcNAc1.2

Val +0.22 Asp1.1 Thr2.0 Ser2.0 Glu3.2 Pro,., Val-His-His-Pro-Ser-Thr-Asn-ValO.7 His1.7 Gln-Glu-Gln-Thr-Ser

Val +0.39 Thr2.0 Serl.0 Val1.9 Arg1.1 Val-Thr-Val-Ser-Thr-ArgIle 0 Asp1.1 Pro1.1 Glyl.0 Val,.8 Ile-Val-Lys-Pro-Gly-Asp-Val-

leo.8 Leu1.2 LysO.9 LeuIle -0.89 CmCys1 0 As-p1. Thuro.9 Ile1.0 Ile-Asp-Thr-Cys

CmCys -0.6 CmCys1.2 Asp2.0 Sero.9 Glul.Alal.0 1el.8 Argo.9

Gly +0.45 Aspl0 Glyl.0 Ileo.8 Argo.9

Position

1-10

121-124142-150

151-156157-168

182-183

202-207236-243

274-277

Cys-Asp-Asn-Ala-Cys-Ile-Glu- 144-153Ser-Ile-Arg

Gly-Asn-Ile-Arg 213-216

1.0

0.8

0.6

0.4

0.2

0

0

500a

400 >

cI200

100 :

0

;t_0C

Fraction no.

Fig. 4. Separation of CNBr peptides CN1-CN4 fromS-[2-14C]carboxymethylated haemagglutinin chain HA2

of variant X-31For details, see the legend to Fig. 1.

found at asparagine residues 8, 22, 38, 81, 165 and285.

Structure ofHA2CNBr cleavage of S-carboxymethylated HA2

(800nmol) yields four CNBr peptides, CN1-CN4,which can be separated by gel filtration as shown inFig. 4. The N-terminal peptide CN4 was notinvestigated further as its sequence has already beenpublished by Waterfield et al. (1979) and shown to

Vol. 193

500 E0

300 E

200 <'

100

CLd

a:40'Ul

30 40 50 60 70 80 90 100 110

Fraction no.

Fig. 5. Chromatography of tryptic digests of peptides(0.6.pmol, 18 h digestion) from variant-X-31 haemag-

glutinin chain HA2For details, see the legend to Fig. 2. (a) Trypticpeptides from S-[2-'4C]carboxymethylated peptideCN1; (b) tryptic peptides from peptide CN2.

ChainHAI

HA2

CNI.Tlb.Thla

CN I.T5.Th3bCN I.T6b

CN1.T7CN1.T8

CN2.T2.Th3

CN2.T5aCN2.TIO.P3

CN3.T2-Th2

CN1.T3

CN1.T8

_'I- o__

, CN CN2, CN3/4I I 40 50 6

10 20 30 40 50 60

957

19

0 0

C. W. Ward and T. A. Dopheide

be identical with that of A/Mem/72. The otherCNBr peptides, CN1, CN2 and CN3, were digestedwith trypsin and the digests of the large peptides(CN1 and CN2) fractionated by gel filtration as

shown in Figs. 5(a) and 5(b). The 27-residue peptideCN2.T11 was digested further with S. aureusproteinase.

The amino acid sequence of X-31 HA2 and thelocation of the peptides used to establish thestructure are shown in Fig. 6, along with the knownsequence for the corresponding regions of A/Mem/72 light chain. The only differences found were theasparagine for aspartic acid at residue 49, theglutamic acid for glycine at position 150 and thearginine for glutamic acid at residue 216. PeptideCN2.T3 was isolated as both a neutral and acidic

(deamidated) form in the present study, but only inthe deamidated form in the study of A/Mem/72(Ward & Dopheide, 1979). The nucleic acidsequence shows that residue 49 in A/Mem/72 isasparagine (Sleigh et al., 1980). Carbohydrate wasfound attached to asparagine residue 154.

Oligosaccharide distribution and compositionThe monosaccharide compositions for the seven

carbohydrate side chains on X-31 haemagglutininare shown in Table 2. As found for A/Mem/72haemagglutinin (Ward et al., 1980b), the carbo-hydrate units on residues 8, 22 and 81 of HA1 and154 of HA2 are of the N-acetyl-lactosamine type,whereas those on residues 165 and 285 are of theoligomannoside type. In A/Mem/72 haemag-

Mem/72 Gly Leti Phe Cly Ala Ile Ala Cly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser GluX-31 Gly Leu Phe Gly Ala lie Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu

i_ T1 _T_2 30

CN4 CN2

Mem/72 Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn

X-31 Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn

T2 ii T3 T4 I -T- 1 60

CN2 CN2

Mem/72 Glu Lys Pie His Gin lie Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp

X-31 (:u Lys Phe His Gln lie Clu Lys nlu Phe Ser Giu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp

TC, T7 IT8 1i T9 -4 -T10 -J4 90

CN2 CN2

Mem/72 Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Clu Asn Gln His Thr Ile Asp Leu Thr Asp Ser ilu Met Asn Lys Leu Phe GluX-31 Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Clu

Ti 1 i-T I-1 F-T2----------Sal-------------l I---------Sa2.---------- ---------------------Sa3---------------------.I

CN2 CN2 CN3

Mem/ 72 Lys Thr Arg Arg Gln Leu Arg Clu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile GyiX-31 Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys (:jjeAs AaCy le u|

11 T3 -T T41i T I |~ ~ T1 2 T3CN3 CNI

Mem/72 Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys SerX-31 Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys

180I ~~ ~~T4 - T5 ~TS6 _T7

CNI CN1

Mem/72 Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala CysX-31 insolubl- 210

CN1 CNI

Mem/72 Gln Lys Gly Asn Ile Cys Asn Ile Cys IleX-31 Gy Asn Ie Cys Asn Ile Cys Ile

CNI

Fig. 6. Amino acid sequence ofX-31 HA2The alignment of the tryptic and S. aureus-proteinase peptides used to derive the sequence is shown and is based on

comparison with the amino acid sequence of variant A/Mem/102/72. Methionine residues occur at positions 17, 115,

133 and in the insoluble hydrophobic tail region. Carbohydrate is attached at residue 154. For other details, see thelegend to Fig. 3.

1981

958

Primary structure of X-31 influenza haemagglutinin

Table 2. Carbohydrate composition ofthe seven oligosaccharide units on X-31 haemagglutininThe values in parentheses are the numbers of residues per mol of peptide. Glucosamine was determined on anamino acid analyser and is assumed to be N-acetylated. Neutral sugars were determined by g.l.c. as their alditolacetates. The two different forms of peptide CN1.T4 from HA2 had the same amino acid composition, butdiffered in electrophoretic mobility at pH 6.5. The location of the peptides in the sequence of HAl and HA2 isshown in Figs. 3 and 6.

Sugar composition (nmol/30nmol of peptide)

Chain PeptideHAl

CNI.Tlb.ThlaCN 1.Tlb.Th4CNI.Tlc

CN I.T3.Th5aCN1.T8CN3.T2.Th4

Asparagine ,residue Glucosamine

8 129 (4.3)22 129 (4.3)38 63 (2.1)

81 115 (3.8)165 56 (1.9)285 58 (1.9)

Mannose Galactose

160 (5.3)98 (3.3)187 (6.2)

102 (3.4)258 (8.6)132 (4.4)

77 (2.6)63 (2.3)17 (0.6)

93 (3.1)

154 117 (3.9) 113 (3.8) 69 (2.3)154 96 (3.2) 86 (2.9) 45 (1.5)

Fucose-N Carbohydrate

type

51 (1.7) Complex57 (1.9) Complex7.7 (0.3) Simple and

complex27 (0.9) Complex

SimpleSimple

44 (1.5) Complex26 (0.9) Complex

glutinin the carbohydrate unit as asparagine-38 wasalso of the N-acetyl-lactosamine type, whereas inX-31 it closely resembles the oligomannoside type incomposition, with less than molar ratios of galactoseand fucose. The tryptic peptide containing the singleN-acetyl-lactosamine type oligosaccharide unit onHA2 was isolated in two different forms, T4a andT4b. T4b contains significantly less (one residueeach) of glucosamine, mannose, galactose andfucose than T4a (Table 2).

Discussion

There has been considerable speculation over themechanisms of antigenic shift and drift in influenzavirus [see Fazekas de St. Groth (1978) and Webster& Laver (1975) for reviews] and the number andnature of antigenic determinants on the influenza-virus haemagglutinin (Laver et al., 1974; Virelezieret al., 1974; Gerhard, 1978; Russell et al., 1979).Consequently, several laboratories have beenengaged in establishing the chemical basis ofantigenic variation in influenza virus. The currentavailability of monoclonal hybridoma antibodiesagainst the influenza haemagglutinin (Gerhard,1976) and the recent advances in the elucidation ofthe primary (Ward & Dopheide, 1980; Dopheide &Ward, 1980; Waterfield et al., 1979) and tertiary(Wilson et al., 1980) structures of the differenthaemagglutinins and the genes that code for them(Porter et al., 1979; Gething et al., 1980; Min-Jou etal., 1980; Sleigh et al., 1980; Both et al., 1980; Air,1980) are beginning to establish the chemicalchanges associated with antigenic variation.

Partial sequence analysis of several Hong Kong(H3) variants selected with monoclonal hybridoma

antibodies (Laver et al., 1979; Webster et al., 1980)and several natural field strains isolated between1968 and 1977 (Laver et al., 1980) showed that thehaemagglutinins from these antigenically distinctviruses could differ by as few as one or two aminoacid residues. They also showed that the peptidesthat changed in the variants selected with mono-clonal antibodies also changed in the natural fieldstrains, although in almost all cases the changes thatoccurred were different. However, in these studiesnot all of the sequence was accounted for, and not allchanges observed could be established with cer-tainty. The insoluble peptides that comprise residues110-141 and 229-255 in HAl were not recoveredand the large soluble peptides that account forresidues 1-50, 58-90, 156-201 and 270-299 inHA1 were not reliably analysed by single hydro-lyses. Similar gaps also occurred in the analysis ofHA2. To fully characterize these variants, completeamino acid sequences are required.

Here we present the complete amino acidsequence (328 residues) of the haemagglutinin heavychain (HA 1) from the early Hong Kong variantX-31 and the sequence of 188 of the 221 residuesfrom the light chain (HA2). The highly aggregatedhydrophobic region (residues 180-212) near theC-terminal end of HA2 was not resolved by peptidesequencing. The sequence was found to be almostidentical with that of A/Mem/72, including thelocation of the seven oligosaccharide units atasparagine residues 8, 22, 38, 81, 165 and 285 inHAl and 154 in HA2. The results show that thereare only ten sequence differences (at residues 2, 3,122, 144, 155, 158, 188, 207, 242 and 275) in theHAl polypeptides of X-31 and A/Mem-72 and twodifferences (residues 150 and 216) in HA2 (see Figs.

Vol. 193

HA2CNI.T4aCNI.T4b

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Primary structure of X-3 1 influenza haemagglutinin 961

3 and 6). Of the two differences found in HA2,that at position 216 does not occur in any otherHong Kong strain examined (Both et al., 1980;Min-Jou et al., 1980). These strains, in commonwith X-31, contain arginine at this position. Withregard to the ten heavy-chain differences, three (atpositions 144, 155 and 207) were detected in thecomparative peptide-'map' analyses of Laver et al.(1980) and one (at residue 275) in the partialsequence analysis of Waterfield et al. (1979). Thethreonine/serine substitutions at positions 270 and279 found by Waterfield et al. (1979) were notpresent in our isolate of X-3 1.

The results also show that although the distri-bution of oligosaccharide units on X-31 HA1 andHA2 and the amino acid sequences around theseglycosylation sites are identical with that found inthe haemagglutinin from A/Memphis/102/72, thesugar compositions for the homologous oligo-saccharide units are not (see Table 2 and Ward etal., 1980b). Similar heterogeneity of carbohydrategroups has been reported for many glycoproteins(see Gottschalk, 1969) and is illustrated in thepresent paper by the two different forms of theN-acetyl-lactosamine-type oligosaccharide on X-31HA2. The nature of the attached carbohydrategroups at each position on X-31 HAl and HA2 arethe same as those on A/Memphis/102/72 except forthat at asparagine-38. In A/Mem/72 this oligo-saccharide was of the N-acetyl-lactosamine typewith the composition Fuc0.6 Gal2.5 Man54 GlcNAc4,whereas in X-31 it has the composition FucO3 GalO.6Man6G1cNAc2. Since both N-acetyl-lactosamine-and oligomannoside-type carbohydrates are synthe-sized via a common mannose-rich intermediate (seeSchacter et al., 1979), this suggests that only partialprocessing of the precursor may have occurred.Alternative possibilities are that both N-acetyl-lactosamine and oligomannoside units are attachedto the same locus on different haemagglutininmolecules, which seems unlikely (see Rosner et al.,1980), or that the peptide containing this carbo-hydrate unit was contaminated with another N-acetyl-lactosamine-type glycopeptide (although thiswas not apparent from the end-group or amino acidanalysis).

Antigenic analysis of Hong Kong field strainswith a panel of monoclonal hybridoma antibodies(Webster & Laver, 1980) indicates that there weretwo antigenic variants of H3N2 viruses, Hong Kong/1/68 and Aichi/2/68, co-circulating in 1968, andboth are referred to as supplying the haemagglutiningene for the recombinant virus X-31 (Kilbourne,1969; Min-Jou et al., 1980). The antigenic relation-ships suggested that Hong Kong/1/68 gave rise tothe early England strains (Eng/878/69, England/187/70), whereas A/Aichi/2/68 gave rise to theAmerican isolate A/Mem/1/71. The comparative

sequences summarized in Fig. 7, in conjunction withthe partial sequence data of Laver et al. (1980),confirm this suggestion. The X-31 haemagglutininsequence described in the present paper is identicalwith the partial sequence data for A/Mem/1/71(Air, 1980), except for residue- 150 in HA2. It differsfrom the haemagglutinin-sequence data for both theX-31 strain examined by Verhoeyen et al. (1980),and the early Australian isolate A/NT/60/68 (Bothet al., 1980) at positions 2, 31, 78 and 158 in HAl,and from the X-31 strain of Verhoeyen et al. (1980),at position 132 in HA2.The sequence homologies in conjunction with the

antigenic data of Webster & Laver (1980) suggestthat the X-31 haemagglutinin described in thepresent paper is that of A/Aichi/2/68, whereas thatdescribed by Verhoeyen et al. (1980) may beA/Hong Kong/1/68.

The data in Table 7 also show that antigenic driftdoes not involve the sequential replacement, at asingle locus, of hydrophobic amino acid residues ofincreasing side-chain surface area (Fazekas de St.Groth, 1978). Instead, as first reported by Laver etal. (1980), once an amino acid residue changedduring antigenic drift, it did not change again insubsequent mutant generations. Rather, amino acidsubstitutions accumulated at other loci as antigenicdrift proceeded.

We thank Mr. R. W. O'Brien for technical assistance,Dr. A. S. Inglis for the automated sequenator run and Dr.C. M. Roxburgh for the carbohydrate analyses and helpwith some amino acid analyses.

References

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Albersheim, P., Nevins, D. J., English, P. D. & Karr, A.(1967) Carbohydr. Res. 5, 340-345

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1981