J. gem Virol. (198o), 48, 213-217
Printed in Great Britain
2I 3
The Polypeptide Composition
of Isolated Surface Projections
of Avian Infectious Bronchitis Virus
( A c c e p t e d 20 N o v e m b e r I979)
SUMMARY
Disruption of avian infectious bronchitis virus (IBV) particles with 4 % Triton
X-Ioo and I.O M-KCI and centrifugation through a sucrose gradient containing
o. 1% Triton X-too and I.o M-KC1 enabled separation of the petal-shaped surface
projections. By negative-contrast electron microscopy the separated projections
appeared mainly as rosettes containing 3 to I2 projections radiating from a central
core, although single projections and rosettes containing up to t6 projections were
seen. SDS-PAGE of these preparations revealed two polypeptides of 86ooo and
66ooo tool. wt. The larger polypeptide was glycosylated.
Studies aimed at identifying the structural polypeptides associated with the surface
projections (spikes or peplomers) of coronaviruses have generally relied on specific digestion
of these structures from the intact virus particles by proteases and the association of polypeptides by their absence after such treatment (Bingham, I975; Garwes & Pocock, I975;
Hierholzer, I976; Hierholzer et al. I972; Macnaughton et al. I977; Sturman, I977). However, in studies with other enveloped viruses such as avian myeloblastosis virus (Bolognesi
et al. I972) Semliki Forest virus (Simons et al. I973) and Newcastle disease virus (Scheid &
Choppin, t973), the use of non-ionic detergent or non-ionic detergent and KC1 at high
ionic strength enabled the isolation of functional undigested surface projections from virus
particles. Using the latter method, we have isolated the surface projections of avian infectious bronchitis virus (IBV) and determined the associated polypeptides by polyacrylamide
gel electrophoresis (PAGE).
IBV strain Massachusetts 4~ was grown in embryonated fowls' eggs and purified by
sucrose density gradient centrifugation as described by Alexander & Collins 0977). Concentrated purified virus (o.8 ml) containing I mg protein, estimated by the method of
Lowry et al. 095D, was dissociated byadding 0-2 m120% (V/V)Triton X-Ioo in o'ot M-trisHC1 buffer, pH 7"0 and KCI to a concentration of ~.o M. The mixture was left at room
temperature for 2 h before applying to a 4 ml 20 to 65% (w/w) sucrose gradient in o.ot Mtris-HC1 buffer, pH 7"0 containing o.I% Triton X-too and t.o M-KCl and centrifuged at
io4ooo g for ~8 h. After centrifugation, 3o five-drop fractions were taken from the gradient.
Absorbance at 260 nm indicated three peaks, one at the top of the gradient with density r'I 4
g/ml, a smaller peak with density I.r8 g/ml and spread of r'I7 to I'I9 g/ml and a very
broad peak with density greater than ~.I9 g/ml and including some pelleted material. The
fractions containing the three peaks were pooled, dialysed against o.ot M-tris-HCl buffer,
pH 7"0, to remove sucrose and KCI and then concentrated by dialysis against polyethylene
glycol. Negative-contrast electron microscopy of the three peaks revealed amorphous material in the lightest and densest peaks but the small peak of I'I7 to I't9 g/ml consisted of
distinct rosette-like structures formed from typical petal-shaped surface projections of the
virus. Although single projections or rosettes having as many as I6 projections were seen,
more usually rosettes were made up of 3 to 12 projections (Fig. I a). The diameters of the
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Fig. J. Negative-contrast electron micrographs of preparations of IBV surface projections. Staining was with 2 % phosphotungstate at pH 6'6.
The projections were dissociated with 4~, Triton X-Ioo and l.o M-KCI and separated by sucrose density gradient centrifugation. (a)Typical
rosette structures formed from the petal-shaped projections; (b) higher magnification of a single rosette; (c) aggregate of rosettes seen in
concentrated preparations.
c,o
~o
i,o
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(a)
(b)
(c)
(d)
215
(e)
(f)
180
107
86~
~gp86
75m
66--
54~
47~
40 1
Fig. 2. Analysis of IBV structural polypeptides on 1o% acrylamide gels by discontinuous SDSPAGE. (a to c) Whole virus; (d to f ) isolated petal-shaped surface projections. (a), (c), (d)and (f)
stained with Coomassie brilliant blue; (b) and (e) stained with Schiff's reagent for glycopolypeptides.
Figures represent apparent tool. wt. x to -3 estimated by comparison with standards of known
tool. wt.
rosettes were in the range of 40 to 47 nm, i.e. slightly longer than twice the length of the projections on intact virus particles. The petal-shaped projections, with wide end outermost,
radiated from a dense central core to form the rosettes (Fig. ~a, b). This arrangement gave
the impression that each projection ended in a small knob which made up the central core in
the rosettes but would be concealed in the envelope of intact viruses. In the concentrated
preparations large aggregates or clumps of rosettes were frequently seen (Fig. I c).
Preparations of separated IBV projections were subjected to SDS-PAGE as described
(Alexander et al. I979) and staining with Coomassie brilliant blue revealed two polypeptides
of tool. wt. 86ooo and 660o0. Both corresponded to polypeptides present in whole virus
preparations (Fig. 2) although both the polypeptides associated with the projections repreDownloaded from www.microbiologyresearch.org by
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216
Short communications
sented relatively minor bands of the whole virus polypeptide profile. S D S - P A G E and
staining with Schiff's reagent (Alexander & Collins, I977) indicated that the 860oo tool. wt.
polypeptide was strongly glycosylated and, on some gels, a band of faint Schiff's staining
corresponded to the 66ooo tool. wt. polypeptide (Fig. 2).
In other studies with IBV virus particles have been treated with bromelain to remove
surface projections and polypeptides associated with the surface projections by their
absence from the polypeptide profiles after treatment. This method led to the association
with the projections of five polypeptides of tool. wt. I8oooo, t3oooo, IO6OOO, 83ooo and
7oooo of which 18oooo and 83ooo were glycosylated (Bingham, I975) and three glycopolypeptides of tool. wt. t3oooo, Io5ooo and 74o00 (Macnaughton et al. I977)- Although
the polypeptide profiles of the whole virus reported by Bingham 0975), Macnaughton
et al. 0977) and ourselves are not absolutely comparable, our present results would suggest
that the protease treatment used in their studies removed more than just the petal-shaped
surface projections. Whether or not the additional material is present as another surface
projection, morphologically distinct from the petal-shaped species, as suggested by Macnaughton et al. (i977) has not been determined.
It would be expected that the surface projections would play an important role in the
immunogenicity of IBV. It is therefore of interest that polypeptide analysis of the structural
polypeptides of different serotypes of IBV has indicated differences in the migration of the
glycopolypeptide corresponding to the 86ooo mol. wt. polypeptide of Massachusetts 4 I.
However, variation in the migration of the glycopolypeptide corresponding to the 3IOOO
tool. wt. polypeptide of Massachusetts 4x was also seen in the different serotypes (Collins &
Alexander, ~98o).
Work is in progress on the separation and comparison of the surface projections of
serologically a n d / o r morphologically distinct IBV strains.
We thank Miss Ruth Hull for her excellent assistance.
M. S. COLLINS
D. J. ALEXANDER
Poultry Department
Central Veterinary Laboratory
New Haw, Weybridge,
Surrey KTI 5 3NB, U.K.
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Short communications
217
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(Received 24 August I 9 7 9 )
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