or. gen. Virol. (I97D, II, 53-58
53
Printed in Great Britain
Parainfluenza Virus Surface Projections: Glycoproteins with
Haemagglutinin and Neuraminidase Activities
( A c c e p t e d 28 D e c e m b e r I97o)
The parainfluenza virus SV5 has been found to contain six proteins with mol. wts
ranging from about 4I,ooo to 76,ooo (Caliguiri, Klenk & Choppin, x969; Mountcastle,
Compans & Choppin, I97~). The nucleocapsid of the virus particle is composed of a single
polypeptide chain with a mol. wt. of about 6i,ooo (Mountcastle et aL I97o); the other
five proteins are presumably associated with the virus envelope. Two of the proteins are
covalently linked to carbohydrate (Klenk, Caliguiri & Choppin, ~97o). We report here
that these glycoproteins comprise the spikes or projections on the surface of the SV 5 virus
particle.
The w3 strain of SV5 (Choppin, I964) was grown in M D B K cells (Choppin, I969) in
reinforced Eagle's medium (Bablanian, Eggers & Tamm, I965) with 2 % calf serum. Cells
were inoculated at a multiplicity of about IO p.f.u./cell. For labelling of virus proteins,
3 parts of reinforced Eagle's medium without amino acids and ~ part of standard reinforced
Eagle's medium were used, and I/zc/ml. of [14C]amino acids, 5/zc/ml. of [3H]amino acids
or 5 #c/ml. of [3H]glucosamine were added as indicated. Glucosamine has been shown to
be specifically incorporated into the carbohydrate of SV5 (Klenk et al. t97o ) and Sindbis
virus (Strauss, Burge & Darnell, ~97o). After about 48 hr, the supernatant medium was
harvested, centrifuged for 2o min. at 3ooo g to remove cell debris, and mixed with an equal
volume of saturated ammonium sulphate. After stirring at 4 ° for 30 min. the precipitate was
pelleted at 3ooog for 3o min., resuspended in Eagle's medium, layered over a I5 to 4o %
(w/w) potassium tartrate gradient, and centrifuged at 23,ooo rev./min, for 2 hr in a Spinco
SW 25-I rotor. The virus formed a visible band which was collected and dialysed against
o. I M-tris + HC1 buffer, pH 7"o, before enzyme treatment.
A solution of a protease from S t r e p t o m y c e s griseus (Protease Type VI, Sigma Chemical
Co, St Louis, Missouri) in o.I ~i-tris buffer, pH 7"o was added to the purified virus suspension to give a final concentration of 5o0 haemagglutinating units (HAU) of virus and 2 mg.
of enzyme/ml. The mixture was incubated at 37 ° for about 2 hr, and the digestion of spikes
was monitored in the electron microscope. Control virus preparations were incubated at 37 °
without the enzyme. When the removal of spikes was judged to be essentially complete, the
enzyme-treated virus was separated from enzyme and digestion products in a potassium
tartrate gradient as described above, and control preparations were also recentrifuged in a
second gradient. For measurement of infectivity and haemagglutinin, virus preparations were
dialysed overnight against reinforced Eagle's medium; for determinations of protein and
neuraminidase assay, preparations were dialysed against water. Neuraminidase activity was
determined by the method of Aminoff(I96I) as described previously (Compans et al. I97O).
Treatment with Protease Type VI resulted in an essentially complete removal of the spikes
from the surface of the virus particle. Fig. 1 to 3 show untreated virus particles which have
well-defined surface spikes about Ioo ~ in length, and, by contrast, the smooth-surfaced
particles obtained after protease treatment. Papain, subtilopeptidase A, bromelain, and ficin
were found to be less satisfactory, in that the spikes were relatively resistant to these enzymes.
The spikeless particles are sufficiently stable to withstand banding in a potassium tartrate
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g r a d i e n t w i t h o u t a p p a r e n t m o r p h o l o g i c a l damage. The biological activities o f spikeless virus
particles were c o m p a r e d with those o f c o n t r o l p r e p a r a t i o n s (Table I). The infectivity o f
p r o t e a s e - t r e a t e d virus was reduced a b o u t a millionffold, a n d h a e m a g g l u t i n i n a n d neuraminidase activities were abolished. Thus, these activities a p p e a r to involve the virus spikes.
T h e loss o f infectivity is p r e s u m a b l y secondary to failure o f a d s o r p t i o n after loss o f
haemagglutinin. It was shown previously t h a t treatment o f Newcastle disease virus with
Fig. I to 3. SV 5 particles negatively stained with sodium phosphotungstate.
Fig. L Untreated SV 5 virus particles showing the layer of surface projections or spikes characteristic of paramyxoviruses. The particle on the right, which is penetrated by the stain, shows an
electron-lucent membrane beneath the spike layer. The helical nucleocapsid is partially extruded
from the particle.
Fig. 2, 3. SV 5 particles after treatment with protease showing smooth-surfaced spikeless particles,
The particles which have been penetrated by the stain show an electron-lucent membrane surrounding the nucleocapsid. This membrane often shows outpocketing, giving the particles bizarre
shapes.
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pronase removed surface projections with loss of haemagglutinin activity (Calberg-Bacq,
Reginster & Rigo, ~967).
The polypeptides present in control and protease-treated SV5 virus were compared by
polyacrylamide gel electrophoresis. The procedures employed in the preparation of polyacrylamide gels, dissociation of the virus with sodium dodecyl sulphate and mercaptoethanol,
electrophoresis of proteins, and processing of gels for determination of radioactivity have
been described previously in detail (Caliguiri et al. 1969; Mountcastle et aL I97o). Fig. 4
compares the proteins of enzyme-treated particles with those of a control sample from the
same SV5 preparation which was labelled with [14C]amino acids and [3H]glucosamine. The
electrophoretic pattern of the control virus preparation (Fig. 4a) corresponds to the pattern
described previously (Caliguiri et al. i969; Klenk et al. 197o); the glucosamine label is
associated with proteins 2 and 4, indicating that these are glycoproteins. These two glycoproteins are completely absent in the enzyme-treated particles, indicating that they are
components of the virus spikes (Fig. 4b). The small peak following protein 6 probably
represents the product of incomplete degradation of spike proteins in this experiment. Such
a peak is not always present after enzyme treatment (Fig. 5).
Table I. Comparison of biological activities of S V5 virus
before and after removal o f spikes
Activity/rag.protein
Virus
Control
Protease-treated
p.f.u.
4"4x Io~°
9"8× Io3
HAU
5,632
< 48
Neuraminidase
1-63
< o'o5
The neuraminidase and haemagglutinin of influenza virus appear to be two kinds of
spikes present on the surface of the particle (Laver & Valentine, I969; Webster & Darlington,
1969), and the present results indicate that SV5 haemagglutinin and neuraminidase are
activities of the virus spike proteins. It has also been reported recently that these activities
are associated with the spikes on haemagglutinating virus of Japan (HVJ) (Maeno et al.
197o). It is not yet certain which of the two SV5 glycoproteins is associated with which
biological activity.
Fig. 4 b shows that, although the spike glycoproteins were removed, three non-glycoproteins, corresponding in mobility to proteins 3, 5 and 6, remain in the enzyme-treated
particles. Co-electrophoresis of a control virus preparation labelled with [3H]amino acids
and enzyme-treated virus labelled with [l~C]amino acids (Fig. 5) shows that the electrophoretic mobilities of the proteins remaining in the enzyme-treated virus correspond
precisely with those of the control virus proteins. This indicates that the proteins 3, 5 and 6
which remain in the enzyme-treated particles have not been altered by the protease, probably
because they are protected by the lipid in the virus membrane. Protein 3 is the subunit of the
nucleocapsid (Mountcastle et al. I97O), and although the precise location in the virus
envelope of proteins 5 and 6 has not yet been determined, it appears that protein 6 with a
mol. wt of about 41,ooo is the major protein in the smooth-surfaced membrane which
surrounds the spikeless particles. Proteins with similar mol. wts (38,ooo to 4I,OOO) which
contain no carbohydrate have also been found in Newcastle disease and Sendai virus
particles (Mountcastle et al. x971), indicating that a protein in this size range is a common
feature of paramyxovirus envelopes.
The present demonstration that the two SV 5 glycoproteins are spike proteins and previous
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studies indicating that the spikes of influenza (Compans et al. ~97o; Schulze, [97o) and
Sindbis virus (Compans, I97[) are glycoproteins, suggest that spikes composed of glycoproteins are a characteristic feature of enveloped viruses in general. This conclusion is also
supported by preliminary observations with Sendai virus (W. E. Mountcastle, R.W.
Compans & R. W. Choppin, unpublished results) and vesicular stomatitis virus (J. McSharry,
R. W. Compans & P. W. Choppin, unpublished results) which suggest that, in these two
(a)
2
3
4
5
6
9000
1200
.m
E
~'~ 6000
E
g
800 .9
"'o
u
,~ 3000
400
i
40
2O
(b)
3
60
80
S
6000
800
o
'N
3000
-
20
40
Fraction
60
400 :~
u
80
no.
Fig. 4. (a) Polyacrylamide gel electrophoresis o f the polypeptides of untreated SV 5 virus particles
labelled with both [3H]glucosamine (© - - - ©) and [14Clamino acid mixture (@----@). The glycoproteins, 2 and 4, are labelled with glucosamine. Protein I, which is a very minor component, is not
seen in the gel. (b) SV 5 virus particles after protease treatment. The two glycoproteins have been
removed, and proteins 3, 5 and 6 remain.
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viruses, the surface spikes also consist of glycoproteins. The spike proteins of all of these
viruses are incorporated into the plasma membrane of the infected cell before virus maturation, and the presence of carbohydrate bound to these proteins may be a requirement for
this mode of assembly. It has also been found that spikeless virus particles clump together,
suggesting that one fnnction of the carbohydrate in the spikes is to provide a hydrophilic
surface which permits the virus to be freely dispersed in an aqueous environment (Compans
et al. 197o; Compans, 197I ).
2
3
4
5
6
2000:
C
0
L-
"N
loo
1000
?
i
o
#O,
~:
b° 6
I
20
40
60
80
Fraction no.
Fig. 5. Co-electrophoresis of the polypeptides of untreated SV 5 virus particles labelled with
pH]amino acid mixture and protease-treated particles labelled with [l~C]amino acid mixture.
The electrophoretic mobility and the relative amounts of proteins 3, 5 and 6, which remain after
protease treatment, were unaltered by the enzyme.
Supported by Contract AT (3o--I)--3983 from the United States Atomic Energy Commission and Research Grant AI--o56oo from the National Institute of Allergy and Infectious
Diseases, United States Public Health Service.
C. CHEN
R . W . COMPANS
P.W. CHOPPIN
The Rockefeller University
New York, New York IOO2I
U.S.A.
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