Download Effect of Concanavalin A on Cells Infected with Enveloped RNA

J. gen. ViroL (I972), x4, I - 8
I
Printed in Great Britain
Effect of C o n c a n a v a l i n A on Cells Infected with E n v e l o p e d
R N A Viruses
By H. BECHT, R. R O T T AND H.-D. K L E N K
Institut fiir Virologie, Justus Liebig-Universitiit, Giessen, Germany
(Accepted 13 August 1971)
SUMMARY
A variety of cells infected with different enveloped RNA viruses are specifically
agglutinated by Concanavalin A. The production of envelope-components seems
to be essential for the agglutinability of cells infected with myxovirus. A strong
flocculation of purified virus particles by Concanavalin A indicates that the
specific receptor of the host cell membrane is incorporated into the virus envelope.
INTRODUCTION
Transformed cells are known to be agglutinated by some phytagglutinins, while normal
cells are not affected by these reagents (Aub, Tieslan & Lancester, I963; Aub, Sanford &
Cote, 1965; Burger & Goldberg, I967; Inbar & Sachs, I969). Specific receptors present in
a cryptic state in normal cells seem to be exposed at the cell surface after transformation
and to become accessible to phytagglutinins.
Profound changes of the cell membrane take place during maturation of myxoviruses.
Electron microscopic studies have shown that those areas of the membrane where myxoviruses are assembled and extruded during the 'budding' process become thickened (Cornpans et al. I966; Compans & Dimmock, I969) and are devoid of sialic acid (Klenk, Compans
& Choppin, I97o). Infected cells exhibit virus-specific components at the cell surface which
could be detected with ferritin-tagged antibodies (Morgan, Rifkind & Rose, I962) or by
haemadsorption (Vogel & Shelokov, I957). This modification of the cell membrane is also
accompanied by the exposure of similar receptor sites as in transformed cells which results
in the agglutinability of myxovirus-infected cells by Concanavalin A (Con A) (Becht,
Rott & Klenk, I970.
This report describes further studies on the agglutination of cells infected with myxovirus.
Particular interest has been focused on the agglutinability of cells infected with other
enveloped RNA viruses, the specificity of the phenomenon in inhibition studies with the
specific receptor-dominant sugar, and the reaction of virus particles in the presence of
Con A.
METHODS
Viruses. Fowl plague virus (FPV) strain 'ROSTOCK', and a strain adapted to growth on
BHK-cells (Zfivada, I969), and influenza virus A2-Singapore were used as representatives
of the orthomyxovirus group. The Newcastle disease virus (NDV) strains 'ITALIEN' and
'BEAUDETTE' and SV 5 represented the paramyxoviruses, Sindbis and Semliki Forest virus
(SFV) the arboviruses and vesicular stomatitis virus (VSV) the rhabdoviruses. The nonenveloped viruses SV4o (kindly supplied by M. A. Koch) and poliovirus type I were
included as controls.
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H. B E C H T ,
R. R O T T A N D H . - D . K L E N K
Agglutination tests with Con A. The methods were essentially those of Burger (I969) and
Inbar & Sachs (~969). The primary cells and permanent cell lines listed in Table I were
inoculated at a m.o.i, of approximately ~o and incubated in Eagle's medium without serum
after an adsorption period of 30 min. At the periods after infection indicated in Table ~ the
medium was removed, the cells were washed twice with calcium-free phosphate buffered
saline (PBS), detached from the Petri dish with o.o2 ~ versenate, washed once in PBS by
careful centrifugation and suspended in serum-free medium at a ceil density of approximately 2 x io 6 cells/ml. The cells were harvested when virus was produced at an exponential
rate. One mg. of Concanavalin A (Con A, Calbiochem, lot no. oio229) was dissolved in
r ml. PBS, and 0.2 ml. was diluted twofold in a series of cups of a plastic tray. o.2 ml. of
the cell suspension was added to each well. The mixture was rocked gently a few times by
hand at room temperature, and macroscopically visible agglutination patterns were scored
after I5 rain.
Inhibition tests, o.I ml. of a constant amount of Con A (2oo#g./ml.) was mixed with
o.I ml. of a twofold dilution series of c~-methyl-D-glucopyranoside (a-MG), and ~5 min.
later the test cells were added.
Agglutination of virus particles. Virus grown in the allantoic cavity of chick embryos was
concentrated and purified by centrifugation in a sucrose gradient (Laver, I969). One or
several drops of the clear virus suspensions containing between ~ooo and m,ooo haemagglutinating units were mixed with an equal volume of a twofold dilution series of Con A or
of an extract from Dolichos biflorus in the wells of a plastic tray. The reaction mixture was
observed for the appearance of flocculations for 3o min. at room temperature.
RESULTS
Agglutination of infected cells
The results of a series of studies about the agglutinability of various host cells infected
with enveloped R N A viruses are summarized in Table I. These experiments show that
virtually all the viruses t e s t e d - including myxoviruses, arboviruses and r h a b d o v i r u s e s were capable of rendering their host cells agglutinable by Con A. Representatives of the
arbovirus group (Sindbis and Semliki Forest virus) showed an exceptional behaviour.
Although a large amount of virus was produced in chick embryo fibroblasts, these cells were
not clearly and reproducibly agglutinated by 5oo #g./ml. of Con A. If, on the other hand,
the same virus was grown in BHK-cells, aggregates formed in the presence of the
phytagglutinin.
In those systems listed in Table ~, where agglutination of infected cells occurred regularly,
aggregates were obtained by different amounts of Con A, and their appearance varied from
one virus-host system to another. Infection of all cells listed in Table ~ with FPV resulted in
a rapid formation of large floccules almost immediately after the agglutinin had been added.
Cells supporting growth of the paramyxovirus N D V were agglutinated less rapidly and
formed aggregates of a more granular appearance.
The data of Table I show furthermore, that the production of infectious influenza virus
particles was not a prerequisite for the agglutination of host cells. BHK-, HeLa-, or L-cells,
all undergoing an abortive cycle of infection after inoculation with FPV (Franklin & Breitenfeld, ~959; Fraser, I967 ) with the production of haemagglutinating virus material, were all
agglutinated by the same amount of Con A as BHK-cells supporting a productive infection
with the BHK-cell adapted strain.
A similar situation resembling an abortive type of infection could be observed when
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Agglutination by Concanavalin A
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chick fibroblasts inoculated with F P V were i n c u b a t e d at 25 °. U n d e r these c o n d i t i o n s virus
subunits were f o r m e d ( R o t t & Scholtissek, I968), b u t no infectious virus was p r o d u c e d .
These cells were agglutinated too. Poliovirus infected H e L a cells, on the other h a n d ,
r e m a i n e d h o m o g e n e o u s l y in suspension.
Table I. Agglutination of various cell types infected with
different viruses by Con A
Cells
Virus
inoculated
Time of
cell harvest
(hr after
infection)
Chick embryo
fibroblasts
FPV (ROSTOCK)
NDV (1TALIEN)
6
8
N D V (BEAUDETTE)
8
Chick kidney
VSV
Sindbis
SFV
FPV (ROSTOCK)
Influenza A 2
N D V (BEAUDETTE)
Calf kidney
L-cells
BHK
HeLa
8
6
6
24 (25°)
7
8
FPV (ROSTOCK)
NDV (ITALmN)
Influenza A 2
SV5
FPV (ROSTOCK)
6
8
16
24
6
N D V (ITAL1EN)
8
F P V (ROSTOCK)
FPV (BHK)
Sindbis
SFV
7
7
6
6
N D V (BEAUDETTE)
8
FPV (ROSTOCK)
6
7
7
6
4"5
N D V (ITALIEN)
NDV (BeAUDETTE)
Sindbis
Poliovirus type I
Cell associated
virus activity
Cell
agglutinating
titre of Con A
( - logs)
512"
256*
64*
2"5 x IO7~
6
6
5
4
I'O X IO9~
<
I'5 X I09~"
< I
96*
512"
96*
I
384*
512"
20*
32*
32*
I28"
5
4
5
6
6
5
5
7
5
6
5I2.
2"5 × Ios'~
5
5
4"0 x Io~t
8*
64*
6
6
7
I28"
IO*
8
7
512 *
I'O x Iost
6"5 x lost
3
< I
Cells were detached and suspended for agglutination tests at the periods indicated, and their virus production was assayed as * Reciprocal of HA-titre or t p.f.u./ml.
Inhibition tests
c~-MG is k n o w n to inhibit the a g g l u t i n a t i o n o f t r a n s f o r m e d cells by C o n A ( I n b a r &
Sachs, I969). I f the interaction o f C o n A with t r a n s f o r m e d cells o r the virus-infected cells
o f Table I occurs t h r o u g h the same specific b i n d i n g sites, a g g l u t i n a t i o n o f FPV-infected
cells should be inhibited b y this sugar as well. T h e results o f an agglutination-inhibition
test are listed in Table 2 a n d show t h a t c~-MG in a o.or M-concentration c o u l d prevent
aggregation o f these cells by IOO #g./ml. o f C o n A, a b o u t the tenfold m i n i m a l agglutinating
dose.
Agglutinability of cells incubated with inhibitors of viral growth
Since the experiments described so far i n d i c a t e d that p r o d u c t i o n o f infectious virus was
n o t indispensable for r e n d e r i n g the host cells agglutinable by C o n A, a n d t h a t a f o r m a t i o n
o f virus subunits in an a b o r t i v e type o f infection resulted in agglutination, we a t t e m p t e d to
define the c o m p o n e n t responsible for the a g g l u t i n a t i o n o f infected cells. T r e a t m e n t o f F P V -
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H. BECHT, R. ROTT AND H.-D. KLENK
infected fibroblasts with the amino acid analogue p-fluor-phenylalanine (FPA, 300/~g./ml.)
permitted synthesis of an antigenically active RNP-antigen, but inhibited the production o f
virus haemagglutinin (Zimmermann & Sch~fer, I96o ). Cells incubated under such conditions were not agglutinated by Con A. Production of virus haemagglutinin was also prevented, if 2 #g./ml. o f Actinomycin D was added to the medium 9 ° rain. after infection of
chick fibroblasts with F P V (Rott & Scholtissek, I964). Cells treated with this antibiotic
under these conditions were not agglutinated by C o n A. NDV-infected chick fibroblasts,
however, which produced virus in the presence of the antibiotic, showed the agglutination
pattern of untreated ceils. If protein synthesis was cut off with Io #g./ml. o f actidione,
90 rain. after infection, Con A did not cause any clumping o f chick fibroblasts infected
with either FPV or N D V . The results o f these experiments are summarized in Table 3.
Table 2. Inhibition of cell agglutination by o~-methyl-D-glucopyranoside
Concentration of ~-MG
~v
tO -2 M
Plus Con A
Without Con A
.
IO -a M
IO -4 M
0
+
++
++
.
.
.
Chick fibroblasts infected with FPV and harvested 6 hr after infection were added to a mixture of 1oo #g./
ml. of Con A and the indicated concentrations of ~-MG.
(+) Agglutination barely visible; (+ +) total agglutination; ( - ) no agglutination.
Table 3. Agglutinability of infected cells incubated in the
presence of inhibitors of protein synthesis
Virus
Inhibitor
FPV
FPA
Actidione
Actinomycin D
NDV
Cell
Cell associated
agglutinating r
'
titre of Con A Reciprocal of
(- log2)
HA-titre
RNP-titre
6
< 1
<
i
< I
6
512
< z
<
z
< 2
512
32
8
<
Neuraminidase units
1,2oo
8
2
IO
4
4t
172
Actinomycin D
6
256
69
The medium of chick embryo fibroblasts infected with FPV and NDV was supplemented with FPA
(p-fluorophenylalanine, 3oo#g./ml.) immediately after adsorption and with Actidione (to~g./ml.) and
Actinomycin D (z #g./ml.) 9o min. after infection. Cells were harvested as indicated in Table t.
Reactivity of virus particles
Chemical and serological analysis had shown that the host material f o u n d in the virus
envelope reflects the spectrum o f these components detectable in the m e m b r a n e of the host
cell where the virus was grown (Rott et al. I966; Klenk & Choppin, ~969). I f the altered
cell membrane, which had become sensitive to agglutination by C o n A, served indeed as the
matrix for virus-specific components of the virus envelope, one could expect that receptor
sites for Con A could also be detected in the virus particle. W h e n C o n A was added to a suspension o f purified F P V or SV5, large floccules and aggregates formed after a few minutes
in the previously translucent and only slightly opalescent virus preparation (Fig. I). I25/zg./
ml. of C o n A precipitated about ~o,ooo haemagglutinating (HA) units of F P V and 15 pg./ml.
were needed to cause flocculation of a suspension of SV5 (2o00 H A units). A purified suspension of SFV (2 × Io n p.f.u./ml.) was also agglutinated, although the floccules o f this
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Agglutination by Concanavalin A
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virus type appeared less readily than in the two myxovirus preparations tested. Since the
ratio of haemagglutinating or plaque-forming units per number of physical particles has
not been determined for these virus preparations, a comparison of the relative sensitivity
of these virus particles towards Con A was not attempted. No flocculation, however, could
be observed when highly purified SV4o particles over a wide range of concentrations from
o.2 to 26 mg. protein/ml, were in contact with 5oo #g./ml. of Con A.
Fig. i. Flocculation patterns of purified fowl plagne (a), SV5 (b) and Semliki Forest (c) virus
particles after reaction with Con A (mo/xg./ml.). Flocculation is prevented by addition of
c~-MG to FPV (d), SV5 (e), and SFV (f), respectively.
ff the tenfold precipitating dose of Con A was mixed with IO-1 M-MG before it was
added to the virus suspension, flocculation of FPV, SV5 or SFV did not appear (Fig. I).
As for infected cells, the receptor-specific sugar interfered with the reaction of phytagglutinin
with the viral surface.
When the flocculation mixture of FPV and Con A was centrifuged at 3o,ooo g for 3o rain.,
Con A was sedimented with the virus; the supernatant did not agglutinate infected test cells.
A rather firm adsorption of Con A to the virus particles must have taken place.
Binding of Con A to virus and aggregation of virus particles was probably the reason for
a ' neutralizing' effect in the experiment presented in Table 4. Equal volumes of FPV-containing allantoic fluid (HA-titre ~/5Ia) and Con A (zoo #g./ml.) were mixed, left at r o o m
temperature for 20 min. and diluted in logarithmic steps for a plaque assay. A control sample
of the same allantoic fluid was diluted with the same volume of PBS and treated alike. The
plaque numbers of the two series in Table 4 demonstrate that Con A had caused a drastic
reduction of the infectivity of the allantoic fluid.
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H. B E C H T ~
R. R O T T A N D H . - D . K L E N K
Specificity of the phytagglutinin
Since a phytagglutinin from wheat germ has also been shown to agglutinate transformed
cells (Aub et al. I965; Burger & Goldberg, I967), we wished to know more about the
selectivity of the reaction between phytagglutinin and the infected cell surface. We therefore used an aqueous extract from Dolichos biflorus, a phytagglutinin known to react
specifically with blood group A substance (for ref. see Krtipe, I956), which is incorporated
into the virus particle (Springer & Schuster, I964; Rott et al. I966). None of the ceils
agglutinated by Con A showed any reaction with the extract from Dolichos, and no flocculation appeared in a suspension of virus particles when this agglutinin was added (Table 5)Table 4. 'Neutralizing' effect of Con A
p.f.u./ml.
Allantoic fluid + PBS
3"o x 1o s
Allantoic f l u i d + C o n A
3"5 x Io 4
Allantoic fluid containing F P V was mixed with a n equal v o l u m e of Ioo #g./ml. of C o n A in PBS. A control
sample was m i x e d with PBS alone. Residual virus was titrated after 2o rain. at r o o m temperature.
Table 5. Agglutination of infected cells by two different phytagglutinins
Phytagglutinin
Con A
Dolichos bifl.
Agglutinating
titre
( - logs)
Cell
associated
HA-units
7
< I
5 I2
5t2
Test cells were chick fibroblasts infected with FPV. T h e phytagglutinins used were C o n A (5oo #g./ml.)
a n d a n extract f r o m Dolichos biftorus agglutinating blood group A erythrocytes at a dilution o f 2 -8.
DISCUSSION
Cells infected with members of different groups of enveloped R N A viruses show alterations of their surface texture which must resemble those in transformed cells, since both
are agglutinable by Con A. Transformation of cells or maturation of these RNA viruses at
the cell periphery and extrusion of the viruses through the cell membrane causes the exposure
of normally cryptic receptor sites which become accessible to Con A. The determinant group
of the receptor site must be the same as in transformed cells, because agglutination of the
infected cells can be inhibited by the receptor-specific sugar in either case.
Cells infected with different viruses required different amounts of Con A for agglutination just as transformed cells exhibited different degrees of sensitivities towards the agglutinating effect of Con A (Inbar & Sachs, I969). The number of budding areas at the
cell surface varying with the virus used for infection and the degree of exposure of the receptor sites are probably the most important factors determining the minimal cell-agglutinating dose of Con A. This view is supported by the previously reported studies (Becht et
al. I97I) where the appearance of haemagglutinin and agglutinability of the host cells were
compared during the course of the growth cycle of FPV in chick fibroblasts. It was observed
that a smaller dose of Con A was needed for a clear agglutination when the cells were harvested during later periods of the infectious cycle when virus production was reaching its
maximum rate. Since differences among cell types infected with the same virus (NDV) with
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Agglutination by Concanavalin A
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respect to the agglutinating dose of Con A and the quality and speed of aggregate formation
were noted, the surface structure of the host cell also determines the degree of sensitivity of
a given system.
The lack of agglutinability of influenza-infected cells incubated in the presence ofp-fluorphenylalanine shows convincingly that formation of the membrane-bound haemagglutinin
with resultant alteration of the cell surface are prerequisites for agglutination of the infected
cells. The absence of any agglutination of HeLa-cells infected with poliovirus (Table t) or
L-cells infected with ME-virus as previously reported (Becht et at. I97~) is in accord with
this finding and underscores the importance of the budding process for the appropriate
exposure of the Con A receptors. The unchanged reactivity of NDV-infected fibroblasts
incubated in the presence of Actinomycin D indicates that de novo synthesis of cellular
proteins is not essential for the effect.
Since Con A agglutinated cells infected with all the enveloped RNA viruses we have
tested, neuraminidase induced by myxoviruses cannot play an essential part in rendering
the cells agglutinable. Reports (Burger, I969) that treatment of transformed cells with
neuraminidase abolishes their agglutinability for a brief period cannot be evaluated and
compared to the above findings at the present time, since this author employed a wheat
germ phytagglutinin.
The strong ftocculation reactions of Con A with purified enveloped viruses, the specific
inhibition of this reaction with the determinant sugar, and the absence of any fiocculation
of the naked SV4o particles, demonstrates that the specific receptor sites must also be
exposed at the surface of virus envelopes. It can be assumed therefore that those areas of the
cell membrane where the virus particles emerge and derive the matrix of their envelopes
from are the decisive areas of the cell surface where receptor sites become overt and accessible to Con A. The flocculation reaction demonstrates again that defined structural components of the cell membrane become incorporated into the virus envelope. It is conceivable
that the carbohydrate moiety of myxovirus haemagglutinins contains the specific Con A
receptor determinant. This question and other problems concerning the exact location of
membrane components in the virus architecture are the subject of current investigations
employing phytagglutinins as tools for structural studies.
We gratefully acknowledge the excellent assistance of Michaela Orlich. The work was
supported by the Sonderforschungsbereich 47 (Virologic).
Part of this communication was presented at the 2nd International Congress of Virology,
Budapest, I97I.
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(Received 28 June I971 )
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