J. gen. Virol. (1984), 65, 799-802. Printed in Great Britain
799
Key words: influenza virus/mass determination~composition~STEM
Characterization of Three Highly Purified Influenza Virus Strains by
Electron Microscopy
By R. W. H, R U I G R O K , * P. J. A N D R E E , R. A. M. H O O F T V A N
H U Y S D U Y N E N AND J. E. M E L L E M A
Department of Biochemistry, State University of Leiden, Wassenaarseweg 64, 2333 A L Leiden,
The Netherlands
(Accepted 26 January 1984)
SUMMARY
Mass determinations on highly purified influenza virus preparations were performed
using the technique of scanning transmission electron microscopy. The masses of the
three strains, X49, B/Singapore/222/79 and B/Hong Kong/8/73 were determined. The
average value was 174 × 106 daltons with only small differences between the three
strains. The mass of virus particles after removal of the protruding spike proteins,
haemagglutinin and neuraminidase by bromelain treatment was determined to be 86 ×
106 daltons. From the mass difference and the known molecular weight of the spike
proteins the number of spikes was estimated to lie in the range 400 to 500.
Influenza virus is a complex virus belonging to the Orthomyxoviridae. The virus is composed
of protein, lipid, carbohydrate and RNA; there are four major proteins involved in the virus
architecture. The outer virus envelope consists of a lipid bilayer from which the so-called spikes
project externally. These spikes consist of trimers and tetramers of haemagglutinin (HA) and
neuraminidase (NA) respectively. The inner core of the virus consists of M protein,
nucleoprotein and RNA. The molecular biology of the virus was reviewed recently (Webster et
al., 1982).
There is no general agreement on the size, shape and exact composition of the virus. Most of
the information on its structure was obtained from biochemical studies (Schulze, 1973; Laver,
1973), electron microscopic results (Wrigley, 1979; Nermut & Frank, 1971), X-ray
crystallographic work (Wilson et al., 1981 ; Varghese et al., 1983) and solution scattering data
(Mellema et al., 1981). Here we describe the determination of particle weight and spike content
by scanning transmission electron microscopy (STEM). This method consists of scanning a
focused electron beam across the specimen and obtaining a digital signal from the scattered
electrons that can be computer-processed. Under certain well defined conditions the scattering
intensity is proportional to the scattering mass (see Freeman & Leonard, 1981).
As proposed by Freeman & Leonard, we determined particle masses by using an internal
standard. Influenza particle masses were determined by comparison with tobacco mosaic virus
which has a very well known mass per unit length. A linear relationship was assumed between
the scattering mass and the scattering intensity; this assumption holds under our specific
measurement conditions (acceleration voltage, carbon film thickness, etc.) up to a thickness of
approximately 60 nm. From shadowing experiments the thickness of the unstained, flattened,
influenza particles was found to be 48 + 4 nm, which is in the linear range. The validity of the
mass determination method was indicated by the very good reproduction of published data on
masses of a number of plant viruses with well known molecular weights. A full description of our
implementation of the STEM mass determination method will be published elsewhere (R. W.
H. Ruigrok et al., unpublished results).
Influenza virus preparations were obtained from D U P H A R (Weesp, The Netherlands).
Three strains were investigated: X49/H3N2 (a recombinant strain of A/England/864/75/H3N2
and A/PR/8/34/HIN1) and B/Hong Kong/8/73 which were inactivated with 0.019/o flDownloaded from www.microbiologyresearch.org by
0022-1317/84/0000-5949$02.00
© 1984 SGM
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:49:03
800
Short communication
40
O
20
N 0
o
6 20
Z
I
I I I
I
I I
I
i
I
40
(a)
'U
40
I I
20
I I I I
I I I I I I
40
(b)
I I I I I I I I
80
20
I
I
I
I I
I
I
I
I
I
I
I
I
I
I
I
I
1--
20
0
01
20
20
401
140 200 260
I
(c)
I I I I [ I
20
I I I I~
80 140 200 260
Mass (daltons × 10-6)
401 -I I
20
I
80
I
140 200
260
Fig. 1. The mass of influenza virus particles of strains (a) X49, (b) B/Singapore/222/79 and (c) B/Hong
Kong/8/73. The histograms show the mass of intact (upper panels) as well as bromelain-treated (lower
panels) particles. The preparation of the particles was carried out as outlined in the text. The mean
values of the mass are given in Table 1.
Table 1. Molecular mass o f various influenza virus strains
Mass (daltons × 10 -6)
F
Strain
X49
B/Singapore/222/79
B/Hong Kong/8/73
Intact virus
161 +_ 17
182 _+ 19
178 _+22
Spikeless virus
75 + 17
87 ± 17
95 __24
* For each determination about 200 virus particles were measured.
propiolactone, and B/Singapore/222/79 which was not inactivated. The inactivation procedure
did not lead to an observable change in molecular mass. The virus was purified to a
monodisperse preparation (Mellema et al., 1981). The preparations a p p e a r e d to be stable, as
mass determinations performed immediately after isolation and on suspensions which had been
kept in PBS buffer (140 mM-NaC1, 3 mM-KCI, 10 mM-phosphate buffer p H 7.0) with 0.001 ~o
azide for more than 1 year yielded identical results.
The spike proteins were removed by incubating the virus with bromelain (Sigma) as described
by Skehel et al. (1982) and the resulting virus was purified by sucrose gradient centrifugation.
The homogeneity of the virus preparations to be used for mass determinations was verified by
obtaining electron microscopic images of the virus preparations using the negative staining
method. The measured diameters of the virus, negatively stained with uranyl acetate, were 140
+ 12 and 115 + 12 nm for intact and spikeless particles of the X49 strain. Similar results were
obtained for the other two strains.
The average diameters found are in good agreement with earlier electron microscopy work on
negatively stained preparations (Wrigley, 1979; N e r m u t & Frank, 1971). Neutron scattering
studies on the virus in solution (Mellema et al., 1981) resulted in diameters of 116 nm and 89 nm
for intact and spikeless particles; these values agree very well with electron microscopic d a t a on
freeze-dried virus (Nermut & F r a n k , 1971). The diameters determined from negatively stained
specimens are somewhat larger, presumably as a result of flattening during specimen
preparation.
Specimens of the unstained virus to be used for mass determinations were prepared on
ultrathin carbon film (2.5 to 5.0 nm) after extensive dialysis against a m m o n i u m acetate to
remove all non-volatile salts. The results of the mass determinations are presented in Table 1
which summarizes the average mass values and in Fig. 1 where histograms of the particle mass
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:49:03
Short communication
801
distributions are shown. The average values obtained for the masses of intact and spikeless virus
are 174 x 106 and 86 × 106 daltons respectively.
In a previous study, a value of 100 x 106 daltons was obtained for the molecular mass of
influenza A virus from transmission electron microscopic images with latex spheres as reference
(Silverman et al., 1967). In this study no particular attention was given to the homogeneity of the
virus preparation. Another mass estimate was given by Landsberger et al. (1971) who
calculated the mass of a spikeless particle of influenza A/WSN to be 147 x 106 daltons from the
particle diameters measured from electron microscopic images, but flattening, a likely
preparation artefact, was not taken into consideration and this value is possibly too high. From
neutron scattering data obtained on solutions of influenza B/Hong Kong (Mellema et al., 1981) a
mass of 200 × 106 daltons was derived. This value might be somewhat too high in view of the
inaccuracies in the values used for the partial specific volume and the absolute particle
concentration (Jacrot & Zaccai, 1981).
The standard deviations of the populations, as given in Table 1, are about twice as large as the
standard deviations we determined for populations of other systems, including adenovirus
which is similar in size to influenza virus (R. W. H. Ruigrok et al., unpublished results).
Therefore, we are led to believe that roughly half of the standard deviation represents a real
distribution of masses in the virus preparation while the remainder is contributed by
measurement errors. The fact that the absolute values of the standard deviations in intact and
spikeless particles are very similar can be taken as an indication that the mass variation of the
virus stems from the core and not from the spikes. Finally, we note that the standard deviation in
the average mass can be obtained by dividing the standard deviation as measured by the square
root of the number of particles included in the measurement (approx. 200). The resulting
standard deviations in the average are about 2 × t06 daltons.
The number of spike protein molecules can be calculated using the known molecular weights
for HA (208000; Wilson et al., 1981) and NA (192000; Webster et al., 1982) after protease
digestion. Assuming that HA is five times more abundant than NA, the average spike molecular
weight is 205000. The calculated numbers are 420, 463 and 405 for X49, B/Singapore and
B/Hong Kong respectively. It is reassuring that similar values were found for three different
strains, which in fact were used to make three independent measurements. Our conclusion is
that there are 400 to 500 spike structures in the intact virus. This conclusion would be wrong only
if systematic errors exist which act differently on intact and spikeless virus molecules. This can
be compared with other electron microscopic studies in which Wrigley (1979) estimated that
there were about 800 spikes per particle and Tiffany & Blough (1970) estimated 550. With
biochemical methods, Schulze (1972) found a total number of 550 spikes per particle. In the
neutron scattering solution study on B/Hong Kong (Mellema et al., 1981) the number of spikes
was estimated to lie between 350 and 450.
From Table 1 we calculate that the total weight of HA and NA amounts to about 5 0 ~ of the
particle weight while another 2 8 ~ can be attributed to lipid (Mellema et al., 1981). The R N A
content amounts to more than 2 ~ of the virus weight (McGeoch et al., 1976) which implies
that more than 10~ of the virus weight is R N P (Duesberg, 1969). This implies that only 12~
can be left for the other structural protein, the M protein. As the molecular weight of the M
protein is 25 000 there could not be more than 800 copies of this protein per virus particle. Earlier
reports stated that the M protein accounts for 30 to 5 0 ~ of the virus protein (Compans et al.,
1970; Kendal et aL, 1977; Schulze, 1970). Nermut calculated that there are 1500 to 2000
molecules of M protein per particle from estimates of the dimensions of viral substructures
obtained from electron microscopic images (Nermut, 1972). The results reported here are,
however, supported by neutron scattering data which indicate that the M protein accounts for
13 ~ of the viral mass (Cusack, 1982). The results obtained by our group are not necessarily in
conflict with those obtained by Nermut, as the possibility exists that the M protein does not form
a continuous layer underneath the virus membrane as was assumed in the calculations, of
Nermut.
Our conclusions differ from some of the earlier published estimates for the amounts of spike
protein and M protein. A possible explanation is that we are comparing data obtained for virus
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:49:03
802
Short communication
s u s p e n s i o n s w i t h d i f f e r e n t d e g r e e s o f h o m o g e n e i t y . T h e virus p u r i f i c a t i o n m e t h o d used in t h e
w o r k r e p o r t e d here, m a y by e x c l u d i n g all f i l a m e n t o u s a n d o t h e r a b e r r a n t particles, alter t h e
p r o t e i n c o m p o s i t i o n in solution. I n fact, t h e larger virus p a r t i c l e s e x i s t i n g in less p u r i f i e d
p r e p a r a t i o n s could well c o n t a i n less s p i k e p r o t e i n , as t h e a v a i l a b l e s u r f a c e a r e a p e r u n i t v o l u m e
will generally be s m a l l e r for larger particles. K e n d a l a n d c o w o r k e r s r e p o r t e d t w o s u b p o p u l a t i o n s
a f t e r p u r i f i c a t i o n w h i c h h a d q u i t e d i f f e r e n t p r o t e i n c o m p o s i t i o n s ( K e n d a l et al., 1977).
F u r t h e r e x p e r i m e n t s to c h a r a c t e r i z e the virus a n d to s t u d y t h e s t o i c h i o m e t r y o f t h e v a r i o u s
c o m p o n e n t s , by c o n t r o l l e d s t r i p p i n g o f s u c c e s s i v e layers o f t h e virus, are i n p r o g r e s s .
We are indebted to Drs J. Jacobs and F. W. van Voorthuizen (DUPHAR, Weesp, Holland) for their generous
donation of the influenza viruses used in the experiments. We thank Dr H. Blanksma for his help with some
statistical problems, Mr Theo de Mooy for his excellent technical assistance, Mr Jos van der Voort for help with
the software, and Mr Peter Krijgsman for purifying the virus preparations. This work was supported by grants
from the Netherlands Foundation for Chemicai Research (SON) and the Netherlands Organization for the
Advancement of Pure Research (ZWO).
REFERENCES
COMPANS,R. W., KLENK,H-D., CALIGUIRI,L. A. & CHOPPIN, P. W. (1970). Influenza virus proteins. I. Analysis of
polypeptides of the virion and identification of spike glycoproteins. Virology 42, 880-889.
CUSACK,S. (1982). Neutron scattering studies of virus structure. In Proceedings of the Brookhaven Symposium on
Neutrons in Biology. Edited by B. P. Schoenborn. New York: Plenum Press (in press).
DUESBERG,P. H. (1969). Distinct subunits of the ribonucleoprotein of influenza virus. Journal of Molecular Biology
42, 485-499.
FREEMAN,R. & LEONARD,K. R. (1981). Comparative mass measurements of biological macromolecules by scanning.
transmission electron microscopy. Journal of Microscopy 122, 275-286.
JACROT, B. & ZACCAI,G. (1981). Determination of molecular weight by neutron scattering. Biopolymers 20, 2413
2426.
KENDAL, A. P., GALPHIN, J. C. & PALMER,E. L. (1977). Replication of influenza virus at elevated temperatures:
production of virus-like particles with reduced matrix protein content. Virology 76, 186-195.
LANDSBERGER,F. R., LENARD,J., PAXTON,J. &COMPANS,R. W. (1971). Spin-label electron spin resonance study of the
lipid-containing membrane of influenza virus. Proceedings of the National Academy of Sciences, U.S.A. 68,
2579-2583.
LAVER, W. G. (1973). The polypeptides of influenza virus. Advances in Virus Research 18, 57-103.
McGEOCH, D., FELLNER,P. & NEWTON,C. (1976). Influenza virus genome consists of eight distinct RNA species.
Proceedings of the National Academy of Science, U,S.A. 73, 3045-3049.
MELLEMA,J. E., ANDREE,P. J., KRIJGSMAN,P. C. J., KROON,C., RUIGROK,R. W. H., CUSACK,S., MILLER,A. & ZULAUF,M.
(1981). Structural investigation of influenza B virus. Journal of Molecular Biology 151, 329-336.
NERMUT,i . V. (1972). Further investigation on the fine structure of influenza virus. Journalof General Virology 17,
317 331.
NERMUT, M. V. & FRANK,H. (1971). Fine structure of influenza A2 (Singapore) as revealed by negative staining,
freeze-drying and freeze-etching. Journal of General Virology 10, 37-51.
SCHULZE, I. T. (1970). The structure of influenza virus. I. The polypeptides of the virion. Virology 42, 890-904.
SCHULZE,I. T. (1972). The structure of influenza virus. II. A model based on the morphology and composition of
subviral particles. Virology 47, 181-196.
SCHULZE, I. T. (1973). Structure of the influenza virion. Advances in Virus Research 18, 1-55.
SILVERMAN,L., FROMMHAGEN,L. H. & GLICK, D. (1967). Measurements of influenza virus-antibody reaction by
quantitative electron microscopy. Journal of Cell Biology 35, 61-67.
SKEHEL,J. J., BAYLEY,P. M. BROWN,E. B., MARTIN,S. R., WATERFIELD,M. D., WHITE,J. M., WILSON,I. A. & WILEY,D. C.
(1982). Changes in the conformation of the influenza virus hemagglutinin at the pH-optimum of virusmediated membrane fusion. Proceedings of the National Academy of Sciences, U.S.A. 79, 968-972.
TIFFANY,L M. & BLOUGPI,H. A. (1970). Estimations of the number of surface projections on myxo- and paramyxoviruses. Virology 41, 392 394.
VARGHESE, J. N., LAVER, W. G. & COLMAN, P. M. (1983). Structure of the influenza virus glycoprotein antigen
neuraminidase at 2.9 ,~. resolution. Nature, London 303, 35-40.
WEBSTER,R. G., LAVER,W. G., AIR, G. M. & SCHILD,G. C. (1982). Molecular mechanisms of variation in influenza
virus. Nature, London 296, 115-121.
WILSON, I. A., SKEHEL,J. J. & WILEY,D. C. (1981). Structure of the haemagglutinin membrane protein of influenza
virus at 3 A resolution. Nature, London 289, 366-373.
WRIGLEY, N. G. (1979). Electron microscopy of influenza virus. British Medical Bulletin 35, 35-38.
(Received 4 October 1983)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 22:49:03