6x
J. gen. Virol. 0972), x7, 61-67
Printed in Great Britain
Amino Acid Composition of Polypeptides from
Influenza Virus Particles
By W. G. L A V E R AND N I C O L A B A K E R
Department of Microbiology, The John Curtin School of Medical Research,
Australian National University, Canberra, A.C.T. 26oo, Australia
(Accepted I3 June I972)
SUMMARY
The neuraminidase, the membrane protein, the ribonucleoprotein (nucleocapsid
protein) and the light and heavy polypeptide chains of the haemagglutinin subunits were isolated from particles of influenza virus type A 0 (strain BEL)and type B
(strain LEE).Each of these substances migrated as a single component during electrophoresis on SDS-polyacrylamide gels. The neuraminidase of type A2 influenza was
isolated from the A0-Az recombinant virus, X-7Fv This enzyme contained two polypeptides differing slightly in electrophoretic mobility.
This paper gives the amino acid composition of the individual polypeptides from
the type A0 and type B influenza viruses and the neuraminidase protein from X-7F1
virus.
INTRODUCTION
Particles of influenza viruses have been reported to contain from 5 to 8 different polypeptide chains separable by SDS-polyacrylamide gel electrophoresis (Compans et al. I97o;
Schulze, I97o; Skehel & Schild, I97I; Laver, I971). Reports have also claimed that some
influenza virus polypeptides are relatively rich in arginine, methionine and cysteine (Haslam
et al. 2970, Taylor et al. i97o; White et al. I97o). However, these latter reports were based
on studies of the incorporation of labelled amino acids, which gave no information about
the total amino acid composition of the polypeptides.
In this paper we describe the isolation of polypeptides from particles of type A and type
B influenza viruses and their amino acid analysis using conventional techniques; the isolation
and amino acid composition of the light and heavy polypeptide chains from the haemagglutinin subunits of a type A 0 influenza virus (strain BEE)has already been described (Laver,
I97I). The amino acid composition of particles of influenza types A and B (Knight, I947)
and of three protein fractions released from influenza virus particles by treatment with ether
(Hoyle & Davies, I96 0 have also been reported previously.
METHODS
Viruses. The BEL strain of A0 influenza (Burnet et aI. I942), the LEE strain of influenza
type B (Francis, 194o) and the A0-A~ recombinant virus, X-7F1 (Kilbourne et al. I967) were
grown in the allantoic sac of 11-day-old chick embryos. The virus particles were purified by
adsorption to and elution from chicken erythrocytes followed by differential centrifuging
and sedimentation through a Io to 40 % sucrose gradient in o'I5 M-NaC1 (Laver, I969).
5
VIR I7
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62
W.G.
LAVER AND
N. B A K E R
Isolation of the polypeptide chains. The virus particles were disrupted with 1% SDS at
room temperature (2o °C) and the virus proteins were separated by electrophoresis on
cellulose acetate strips in tris-boric acid-EDTA buffer, pH 9, containing o'4 % SDS (Laver,
I964). The proteins were eluted from the strips into water and filtered to remove pieces of
cellulose acetate. The proteins were then precipitated with cold ( - 2 0 °C) ethanol (3 vol.)
resuspended in water and stored frozen at - 2o °C. The light and heavy polypeptides of the
haemagglutinin subunits were separated by centrifuging on guanidine hydrochloridedithiothreitol density gradients (Laver, I97I).
Acrylamide gel electrophoresis. The proteins were electrophoresed in 7"5 % polyacrylamide
gels containing o.2 % N,N'-bismethylene acrylamide in tris-boric acid-EDTA buffer, pH 9
with o.I o,
/o SDS and o.I mg/ml dithiothreitol (Laver, Wrigley & Pereira, ~969).
Amino acid analyses. Protein samples containing 2oo to 300 #g were hydrolysed with
6 N-HC1 (2.o ml) in sealed evacuated tubes at ~io °C for 22 h and the hydrolysates were
analysed by the method of Moore, Spackman & Stein (I958) using the Model B Beckman
amino acid analyser with an expanded scale (Spackman, 1960). No corrections were made
for losses occurring during hydrolysis.
RESULTS
Isolation of proteins from type Ao influenza virus particles
The proteins of the BEL strain of influenza A0 virus were isolated by electrophoresis on
cellulose acetate strips as previously described (Laver, I970. This paper also described the
separation and amino acid analysis of the light and heavy polypeptide chains of the haemagglutinin subunits of BEL virus. The other proteins which were isolated from the strips, the
neuraminidase, the ribonucleoprotein and the internal (or membrane) protein migrated as
single polypeptides during SDS-polyacrylamide gel electrophoresis (Fig. I; Laver, I97I).
The biological activity of each of the three latter proteins was destroyed during electrophoresis on cellulose acetate; they were identified by their mobilities during SDS-acrylamide
gel electrophoresis (Compans et al. I97O; Schulze, I97o).
Isolation of proteins From type B influenza virus particles
Particles of type B influenza (strain LEE) were disrupted with SDS and the proteins were
separated by electrophoresis on cellulose acetate strips. The pattern of proteins obtained
from LEE virus differed from that obtained with the BEL strain (Fig. I) (Laver, i964, I97I).
The neuraminidase of BEL virus was denatured during electrophoresis whereas the haemagglutinin subunits were not and could be eluted from the strips without loss of biological
activity. By contrast, LEE virus haemagglutinin was denatured during electrophoresis while
its neuraminidase subunits were stable and could be isolated pure and without loss of
biological activity (Laver, 1964).
The proteins of LEE virus, separated by cellulose acetate electrophoresis, were examined
by polyacrylamide gel electrophoresis (Fig. I). Four fractions were isolated from the strips.
NA fraction
This fraction was identified, by its enzyme activity, as the neuraminidase of LEE virus. The
protein in this fraction migrated during SDS-polyacrylamide gel electrophoresis as a single
component with a mol. wt. of about 60 ooo suggesting that the neuraminidase subunit of
LEE virus consists of a single species of polypeptide of this size (Fig. ~).
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63
Amino acids of influenzapolypeptides
Polyacry[amide gel electrophoresis
+ ve
M
HA
Origin
t
t
HA~
HA2
NA =~i
o
B/LEE v i r u s
o
l
M
NA
t
Origin
t
t
HA~
HA:
+ ve
Ao/BEL v i r u s
Fig. I. Stained cellulose acetate strips and polyacrylamide gels showing the electrophoretic separation
of the proteins of the A0/BEL and B/LEE strains of influenza virus. The purified virus particles were
first disrupted with cold (2o °C) SDS and the proteins were separated by electrophoresis on cellulose
acetate strips. The bands of proteins were eluted individually and concentrated by precipitation with
cold ( - 2 o °C) ethanol. Samples of the isolated proteins were dissolved in hot (ioo °C) SDS and
dithiothreitol and electrophoresed on polyacrylamide gels. (HA = haemagglutinin; H A I = haemagglutinin heavy polypeptide; H A z = haemagglutinin light polypeptide; N A = neuraminidase;
N P = ribonucleoprotein antigen; M = membrane protein of virus envelope.) ('Influenza virus
polypeptides and antigens - Summary of influenza virus workshop I ', Journal of bfectious Diseases
x25, 447-455 (I972).)
5-2
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64
W. G. L A V E R A N D N. B A K E R
H A fraction
This had no biological activity but, because of its polypeptide composition, we thought it
contained the haemagglutinin subunits of LEEvirus, denatured by the SDS.
SDS-polyacrylamide gel electrophoresis in the presence of dithiothreitol showed that the
protein in this fraction contained two different polypeptides with tool. wt. of about 6oooo
and 25ooo. These two polypeptides were presumed to be the heavy and light polypeptides
of the haemagglutinin subunits similar to those previously described for Ao/BEL influenza
virus (Laver, I970. The two polypeptides from the HA fraction of LEEvirus were separated
by centrifuging in guanidine hydrochloride-dithiothreitol density gradients (Laver, I971).
SDS-polyacrylamide gel electrophoresis of the separated heavy and light polypeptides
(HA ~ and HA 2) showed that pure preparations were obtained in each case.
NP and M fractions
The mobilities of the polypeptides in these fractions during polyacrylamide gel electrophoresis suggested that fraction NP contained the ribonucleoprotein (nucleocapsid protein)
and fraction M the small mol. wt. internal or membrane protein of LEE virus. Fraction NP
was not completely pure (traces of the other proteins were present) but fraction M (the
membrane protein) was obtained free from all of the other virus proteins except for a trace
of the nucleocapsid protein which remained in the preparation (Fig. I).
Isolation of X-7 F 1 neuraminidase
Particles of the recombinant virus, X-7F1, which contained A0/NWS haemagglutinin
subunits and A~/RI/5 + neuraminidase subunits, were disrupted with SDS and the neuraminidase subunits were isolated by electrophoresis on cellulose acetate strips as described
previously (Webster, Laver & Kilbourne, t968; Laver & Valentine, I969). Neuraminidase
subunits, isolated from X-7F1 virus in this way, completely retained their enzyme activity.
SDS-polyacrylamide gel electrophoresis showed that the neuraminidase subunits of
X-7F 1 virus contained two polypeptides with slightly different mol. wt. This finding was in
agreement with those of others (Skehel & Schild, t97~ ; Webster, I97o).
Amino acid analyses
The individual polypeptides isolated from A0/B~L and B[LEE virus particles and the Az
neuraminidase subunits (consisting of two slightly different size polypeptides) isolated from
X-7F~ virus were hydrolysed with acid and the hydrolysates were analysed for amino acids.
The amino acid composition of the polypeptides is given in Table I. No corrections have
been made for losses of labile amino acids, e.g. serine and threonine, during hydrolysis.
DISCUSSION
This paper reports the amino acid composition of polypeptides isolated from particles of
influenza viruses. Several of the virus proteins (the neuraminidase of LEEand X-7F 1 viruses
and the haemagglutinin of BEL virus) were positively identified by their biological activity.
The other proteins, which were denatured during the isolation procedure, were identified by
their mobilities during SDS-polyaerylamide gel electrophoresis (Compans et aL I97o;
Schulze, I97O).
The neuraminidase subunits of LEEand the haemagglutinin subunits of BELand LEEviruses
were cleanly separated from the other virus proteins during electrophoresis on cellulose
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NA
(2)
NA
(2)
6'8
2.6
4"8
IO.O
8.2
7"6
7"9
4"7
9"6
6.6
2"7
5"o
2"5
56
8.8
3"9
2.8
ND
A m i n o acid
Lysine
Histidine
Arginine
Aspartic acid
Threonine
Serine
G l u t a m i c acid
Proline
Glycine
Alanine
Half-cystine
Valine
Methionine
lsoleucine
Leucine
Tyrosine
Phenylalanine
Tryptophan
6'3
1 '8
3"9
I ~'9
6'I
9'5
lO'5
4'7
9'4
4'5
2'4
6"7
0'9
8'6
6"3
2'5
3'9
ND
BEL
LEE
¢
8'7
2'4
4'3
I 1'4
8'6
6.2
8'4
7'0
Io' I
4'4
I'8
6.8
o'8
6'7
7'3
2'9
2'3
ND
6.2
3'5
3"I
I3'I
5"6
7'6
9"7
~.8
9"o
9"4
1.2
4.2
1. ~
6'5
13"6
t '3
3'2
ND
HA 2
(2)
LEE
2.
5'8
2'o
4'3
1 I'7
7'2
9'2
I r'3
4'6
8'5
5'5
1'5
6.0
0"5
6'6
8'9
3'4
3'3
ND
HA I
(2)
BEL
BEL
8.0
I'6
2.7
I3'9
4"5
7'8
13"3
0"5
I6.6
5"6
1.J
6.0
0.8
6.o
9'6
3'6
4'3
ND
HA 2
(2)
BEL
5'7
1 '3
8"3
8.6
5'6
6'8
15'2
3'9
8'2
8.0
o.8
5"4
I "4
6'3
8'7
x '7
4'3
ND
NP
(I)
E x p r e s s e d as tool of a m i n o acid per loo tool recovered.
Figures in parentheses refer to n u m b e r o f analyses done.
D a t a for BEE H A I a n d H A 2 were taken f r o m Laver (I971).
N D = n o t determined.
4'5
1-2
6"1
14"5
7'1
lO"3
8"7
4"9
9"t
3"8
2"6
7"3
I- t
8"5
4"7
2"4
3'3
ND
HA I
(3)
LEE
Polypeptide
Amino acid composition of influenza virus polypeptides
X-7F~
NA
(2)
T a b l e ~.
LEE
8'8
0"9
5"I
I2.O
6'o
7"3
9'9
3"4
8"9
8"3
o'o
5 .2
1 '9
7"5
9'6
1.8
3'5
ND
NP
(2)
9"5
2' t
5"6
8.2
3"8
6.6
I2.2
1.8
8.8
9"o
I'4
4"8
3"8
5"2
r 2"o
2.o
3' ~
ND
M
(2)
LEE
5'6
I "8
7"2
7"5
6"5
5'8
16.2
3'4
6.6
lO"3
o'5
6"4
2.o
4'8
I 2.o
i "o
2.6
ND
M
(3)
BEL
bt
2"
66
W . G. L A V E R
AND
N. B A K E R
acetate strips (Fig. i). Pure neuraminidase subunits from X-7F1 virus were also obtained in
this way (Webster et al. 1968). The polypeptides of these proteins were obtained pure,
but the other polypeptides, which did not separate so cleanly during electrophoresis on
cellulose acetate (BELneuraminidase and the membrane protein (M) and ribonucleoprotein
( N P ) of BEE and LEE viruses), were not completely pure. However, the amounts of contaminating proteins in the preparations of these polypeptides were small (Fig. I) and
probably did not greatly affect the amino acid analyses reported in Table I.
None of the polypeptides had an unusually large or small quantity of any amino acid,
but some noticeable differences between the polypeptides are worth commenting on.
The heavy polypeptide (HA I) from the haemagglutinin subunits of LEE and BEE viruses
contained much more proline than the light polypeptide (HA 2). The external proteins of
the virus particles, namely the heavy and light polypeptides of the haemagglutinin subunits
and the polypeptides of the neuraminidase subunits, contained more ½-cystine than the
internal proteins (NP and M). These internal polypeptides, on the other hand, were somewhat richer in arginine than the external polypeptides but the differences between the two
groups were small and none of the polypeptides contained sufficient arginine to justify its
description as an ' arginine-rich protein'.
The nucleocapsid (NP) and membrane (M) proteins from LEE (type B) influenza virus had
a different amino acid composition from the nucleocapsid and membrane proteins of BEE
(type A) influenza virus. This is not an unexpected finding since the nucleocapsid proteins
from these two influenza virus types are completely different immunologically, and maps
of the tryptic peptides from the membrane proteins of BEE and LEE viruses have been found
to differ greatly.
The number ofpolypeptides in the neuraminidase subunits of influenza virus is not known.
The neuraminidase of the A0-A 2 recombinant virus, X-7F1, appeared to consist of two
species of polypeptides which migrated with slightly different mobilities during polyacrylamide gel electrophoresis. This confirmed the findings of others (Webster, I97O; Skehel &
Schild, 1971) but it was not known whether the neuraminidase subunits of X-7F~ virus contained two different species of polypeptides or whether there was a single species, some
molecules of which carried a different carbohydrate component from the others. The neuraminidase of BEE (type A) influenza virus appeared to consist of a single species of polypeptide of about 6o ooo mol. wt. (Fig. t) but the enzyme of this virus was inactivated during
its isolation and possibly one of the polypeptides had been lost. The neuraminidase of LEE
(type B) influenza virus, on the other hand, was isolated in pure form without any loss of
enzymic activity and, when examined by polyacrylamide gel electrophoresis, appeared to
consist of only one species of polypeptide (Fig. 1).
Miss Stephanie Day provided excellent technical assistance. The amino acid analyses were
done by Mr L. B. James.
REFERENCES
BURNET, V. M., BEVERIDGE, W. I. B., BULL, D. R. & CLARK, E. (I942). Investigations of a n influenza epidemic in
military c a m p s in Victoria in M a y , I942. Medical Journal of Australia 2, 37t-376.
COMPANS, R. W., KLENK, H. D., CALIGUIRI, L. A. & CHOPPIN, P. W. (I970). Influenza virus proteins. I. Analysis
o f polypeptides o f the virion a n d identification of spike glycoproteins. Virology 42, 88o-889.
FRANCIS, T. (I940). A n e w type o f virus f r o m epidemic influenza. Science, New York 92, 405-4o8.
HASLAM, E. A., HAMPSON, A. W., EGAN, J. A. & WHITE, D. O. (1970). T h e polypeptides o f influenza virus. II.
Interpretation of gel electrophoresis patterns. Virology 42, 555-565.
HOYLE, L. & DAVIES, S. P. (1961). A m i n o acid c o m p o s i t i o n o f the protein c o m p o n e n t s o f influenza A virus.
Virology 13, 53-57.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
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Amino acids of influenza polypeptides
67
KILBOURNE,E. D., LIEF,r. S., SCHULMAN,J. L., JA~tEL,R. L & LAVER,W. G. (1967). Antigenic hybrids in influenza
viruses and their implications. Perspectives in Virology 5, 87-Io6.
I,:NIGHT, C. A. (I947). Amino acid composition of highly purified particles of influenza A and B. Journal o/
Experimental Medicine 86, 125-129.
LAVER, W. G. (I964). Structural studies on the protein subunits from three strains of influenza virus. Journal
of Molecular Biology 9, IO9-t24.
LAVER, W. G. (1969). Purification of influenza virus. In Fundamental Techniques in Virology. Edited by
K. Habel & N. P. Salzman, pp. 82-86. New York: Academic Press.
LAVER, W. G. (I971). Separation of two polypeptide chains from the haemagglutinin subunit of influenza
virus. Virology 45, 275-288Lt~VER, W.G. & VALENTINE,R. C. 0969). Morphology of the isolated haemagglutinin and neuraminidase
subunits of influenza virus. Virology 38, lO5-1 I9.
LAVER,W. G., WRIGLEY,N. 6. & PEREIRA,H. G. (I969). Removal of pentons from particles of adenovirus type
2. Virology 39, 599-6o5.
MOORE,S., SPACKMAN,D. H. & STEIN, W. H. (I958). Chromatography of amino acids on sulfonated polystyrene
resins - an improved system. Analytical Chemistry 30, I 185--1 I90.
SCHULZE,I. T. (I970). The structure of influenza virus. I. The polypeptides of the virion. Virology42, 89o-9o4.
S~ZEHEL,J. J. & SCHILD,G. C. (t 97 I). The polypeptide composition of influenza A viruses. Virology 44, 396-408.
SPACEMAN,D. H. (I960). Instruction manual and handbook. Beckman/Spinco Model i 2o amino acid analyser.
Beckman Instruments Inc., Spinco Division, Palo Alto, California.
TAYLOR, J. M., HAMPSON, A. W . , LAYTON, J. E. & WHITE, D. O. (I970). The polypeptides of influenza virus. IV.
An analysis of nuclear accumulation. Virology 42, 744-752.
WEBSTER, R.G. 097O). Estimation of the molecular weights of the polypeptide chains from the isolated
haemagglutinin and the neuraminidase subunits of influenza viruses. Virology 4o, 643-654.
WEBSTER, R. G., LAVER, W. G. & KILBOURNE, E. D. (t968). Reactions of antibodies with surface antigens of
influenza virus. Journal of General Virology 3, 315-326.
WHITE, D. O., TAYLOR, J. M., HASLAM, E. A. & HAMPSON, A. W. (t97o). The polypeptides of influenza virus and
their biosynthesis. In The Biology of Large RNA Virases. Edited by R. D. Barry & B. W. J. Mahy, pp.
6o2-618. New York: Academic Press.
(Received 2 M a y t972 )
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