Download HN proteins of human parainfluenza type 4A virus expressed in cell

Journal of General Virology (1994), 75, 567-572.
567
Printed in Great Britain
H N proteins of human parainfluenza type 4A virus expressed in cell lines
transfected with a cloned cDNA have an ability to induce interferon in
mouse spleen cells
Yasuhiko Ito,* Hisanori Bando, Hiroshi Komada, Masato Tsurudome, Machiko Nishio,
Mitsuo Kawano, Haruo Matsumura, Shigeru Kusagawa, Tetsuya Yuasa, Hisataka Ohta,
Morihisa Ikemura and Noriko Watanabe
Department of Microbiology, Mie University School of Medicine, 2-174 Edobashi, Tsu-Shi, Mie Prefecture,
514 Japan
Primary monkey kidney cells infected with human
parainfluenza type 4A virus (HPIV-4A) were treated
with various concentrations of formaldehyde. Formaldehyde (0-275%) treatment completely blocked virus
production. However, when mouse spleen cells were
cocultured with the fixed virus-infected cells, interferon
was produced in the culture fluid. On the other hand,
when mouse spleen cells were incubated with the fixed
virus-infected cells in the presence of anti-HPIV-4A
antiserum or a mixture of anti-HN protein monoclonal
antibodies, interferon activity could scarcely be detected
in the culture fluid. These findings indicated that the
fixed virus-infected cells had an ability to induce
interferon in mouse spleen cells and that the HN protein
was related to interferon induction. Subsequently, a
recombinant plasmid was constructed by inserting the
cDNA of the HN gene of HPIV-4A into a pcDL-SR~
expression vector. Mouse spleen cells produced interferon when cocultured with COS7 cells transfected
with the recombinant plasmid, but did not when
cocultured with COS7 cells transfected with the vector
alone. Furthermore, we established HeLa cells constitutively expressing HPIV-4A HN (HeLa-4aHN cells)
or F protein (HeLa-4aF cells). Type I (~/fl) interferon
was detected in culture fluids of mouse spleen cells with
HeLa-4aHN cells, but was not detected in those with
HeLa-4aF cells. Therefore, it was concluded that the
HN glycoproteins on the cell surface were sufficient for
interferon induction to occur.
Introduction
glycoproteins and the cell surface was sufficient for
interferon induction in mouse spleen ceils, suggesting
that the actual inducers of interferon in mouse spleen
cells are the viral glycoproteins, probably HN proteins.
Our hypothesis, however, is not universally accepted.
In our previous study (Ito et al., 1974), interferon was
produced by mouse spleen cells through contact with
BHK cells persistently infected with Sendai virus. Mouse
spleen cells attached to the virus-infected cells, recognized
virus antigen(s) present on the surface of infected cells
and produced interferon. It was inferred that the
interferon production was initiated by membranemembrane interaction between lymphoid cells and virusinfected cells.
In this study, the interferon-inducing abilities of fixed
cells infected with human parainfluenza type 4A virus
(HPIV-4A) and of the HN protein expressed in cell lines
transfected with a recombinant plasmid in mouse spleen
ceils were investigated. The results clearly indicate that
HN protein is the active component of the paramyxovirus responsible for interferon induction in mouse
Interferons were first identified as biological agents
interfering with virus replication, but more recently have
been found to have antiviral, antiprotozoal, immunomodulatory and cell growth regulatory activities,
indicating that the interferons are a family of potent
multifunctional cytokines (Sen & Lengyel, 1992). The
most important interferon inducer is infection with
viruses. In general, RNA-containing viruses are good
interferon inducers, whereas DNA-containing viruses,
with the exception of poxviruses, are rather poor
interferon inducers (Joklik, 1990). Attempts have been
made to determine whether the induction of interferon
formation can be ascribed to any specific viral function,
but the molecular basis of the induction by virus infection
remains to be discovered (Joklik, 1990). Our previous
study (Ito et al., 1978a; Ito & Hosaka, 1983) showed that
the penetration of paramyxovirus and functional RNA
were needed for interferon induction in mouse fibroblasts. On the other hand, the interaction between HN
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568
Y. Ito and others
spleen cells and the biological significance of this
observation is discussed.
Methods
Mice. Male C57BL/6 mice weighing 25 to 30 g were used in the
present study.
Virus and cells. HPIV-4A strain M-25 was propagated in monolayer
cultures of primary monkey kidney (PMK) cells. PMK, COS7, HeLa
and mouse L929 cells were grown in Eagle's MEM supplemented with
5 % fetal calf serum. The method used for preparation of mouse spleen
cells was described previously (Ito et al., 1973). The spleen cells were
cultured in MEM with 10% fetal calf serum. Infectivity titration of
HPIV-4A in PMK cells was carried out by the haemadsorption
endpoint method.
Antisera. Anti-HPIV-4A sera were raised in monkeys by repeated
intramuscular injections of HPIV-4A-infected PMK cells. Monoclonal
antibodies directed against HPIV-4A HN and F proteins were
described previously (Komada et al., 1989). Anti interferon c~/fl rabbit
serum and anti-interferon 7 rabbit serum were kindly donated by Drs
S. Kohno and M. Kohase (National Institute of Health, Japan)
respectively.
Haemadsorption and leukoadsorption assay. Tests to assess the
capability of cells to adsorb guinea pig erythrocytes (Had) or mouse
spleen cells (Lad) into their surfaces were performed as follows. Either
1 ml of a 0.5% suspension of guinea-pig erythrocytes or a 0.2%
suspension of mouse spleen cells in MEM was added into the
monolayers, and the cultures were left at room temperature for 30 min.
Unadsorbed erythrocytes or spleen cells were removed by washing with
MEM, and the extent of cell adsorption was observed microscopically.
The degree of haemadsorption was also measured with a photometer as
A40~Interferon titration. Interferon was assayed by the c.p.e, inhibition
method using mouse L929 cells with vesicular stomatitis virus being
used as the challenge virus (Ito & Montagnier, 1977). The reciprocal of
the highest dilution of the sample causing 50 % protection was taken to
be the interferon titre. One unit of interferon was found to be
equivalent to 2-6 reference units of mouse interferon. Possible effects of
infectious virus contaminating the interferon samples were eliminated
by treating the sample with anti-HPIV-4A serum.
Construction of recombinant plasmids. A cDNA clone of the HPIV4A F gene inserted in pcDL-SRc~ 296 plasmid between the PstI site and
the KpnI site (pDS-4aE5) was cut with Clal and SaPl. The cDNA
fragment was inserted into the ClaI and SalI sites of the pkan-2 vector
(4aF-pkan2). The pkan-2 plasmid contains the gene for resistance to
G418 (Geneticin; Gibco). The pcDL-SRe 296 and pkan-2 plasmids
were kindly donated by Dr Y. Takebe (NIH, Japan) (Takebe et al.,
1988).
A cDNA clone of the HPIV-4A HN gene inserted in pcDL-SRc~ 296
plasmid between the PstI site and the KpnI site (pDR-4aH5) was cut
with SalI. The cDNA fragment was inserted into the SalI site of the
pkan-2 vector to construct the HN clone 4aHN-pkan2.
Transient expression o f the HPIV-4A H N prote#l. The recombinant
plasmid pS4AHN was constructed by inserting the coding sequence of
the HPIV-4A HN gene cDNA into a site downstream of the SR~
promoter in the expression vector pcDL-SRe and the plasmid was
introduced into COS7 cells by the DEAE-dextran method.
Constitutive expression o f the HPIV-4A H N or F protein. Transfection
experiments. HeLa cells were grown in 90 mm dishes at 37 °C in MEM
containing 5 % calf serum. The cells were washed twice with warm
MEM, and then transfected with the plasmids 4aF-pkan2 and/or
4aHN-pkan2 with lipofectin (Gibco) in 4 ml of MEM. After incubation
for 8 h at 37 °C, MEM with 10% calf serum was added. After 2 days
of further incubation the culture medium was changed to MEM
containing 10% calf serum, 1 mg/ml Geneticin and 0.1% agarose, and
the cells were then cultured for about 3 weeks.
HeLa-4aF cell line constitutive expression of the HPIV-4A F
protein. HeLa cells transfected with the plasmid 4aF-pkan2 were
purified by the colony isolation procedure. The expression of the
HPIV-4A F protein was detected by ELISA using the F-specific
monoclonal antibody (MAb) A161 (Kamada et al., 1989). The surface
expression of the F protein was confirmed by indirect immunofluorescence staining of paraformaldehyde-fixed cells. HeLa-4aF cells
showed surface fluorescence at a level similar to that of HeLa cells
infected with HPIV-4A.
HeLa-4aHN cell line constitutive expression of the HPIV-4A HN
protein. We also isolated HeLa-4aHN cell lines after transfection with
4aHN-pkan2. The expression of the HN protein was detected by
ELISA using the HN-specific MAb A147 (Komada et al., 1989). The
surface expression of the HN protein was examined by the haemadsorption assay. Almost all the HeLa-4aHN cells were positive for
haemadsorption.
Results
Effects of formaldehyde treatment on the biological
activities of HPIV-4A-#fected P M K cells
PMK cells were grown to monolayers in 12-well plates
for 2 days; the monolayers were infected with HPIV-4A
at an m.o.i, of 1 and incubated for a further 2 days. The
virus-infected cells (HPIV-4A-PMK cells) were fixed
with various concentrations of formaldehyde for 5 min
at room temperature and washed four times with MEM.
Tests were performed to assess the capability of the
formaldehyde-treated cells to adsorb erythrocytes or
mouse spleen cells. In addition, the formaldehyde-treated
cells were incubated at 37 °C for 12 h and then virus
infectivity in culture fluids was titrated. Formaldehyde
(2.2%) treatment abolished the haemadsorbing and
leukadsorbing activities of the virus-infected cells, and
0.275% formaldehyde completely blocked virus production (Table 1).
Subsequently, virus-infected cells (approximately 2 x
10~) treated with formaldehyde were incubated at 37 °C
with 1 x 107 mouse spleen cells in 2 ml of growth medium.
Twenty h later, the supernatant of the culture fluid was
obtained and assayed for interferon activity. Formaldehyde (1.1%)-treated cells induced interferon to the
same extent as untreated cells (Table 1). This finding
indicated that the fixed virus-infected cell preserved a
capability to induce interferon in mouse spleen cells and
that its production might be initiated by membranemembrane interaction between mouse spleen cells and
HPIV-4A-PMK cells. However, there was a discrepancy
between haemadsorbing and interferon inducing activities, that is concentrations of formaldehyde that
reduced haemadsorption and lymphocyte adsorption
from highly positive to weakly positive levels had no
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HPIV-4A HN protein and interferon induction
569
Table 1. Effects of formaldehyde treatment on the biological activities of
HPIV-4A-infected P M K cells
Formaldehyde
concentration
(%)
Degree of Had (A40s)
36
18
9
4.5
2-2
1.1
0.55
0.275
0.14
None
-+
+ + +
+ + + +
+ + + +
+ + + +
(0.014)
(0.364)
(1.244)
(1.499.)
(1.592)
(1.5271
Degree
of Lad
-+
+ +
+ + +
+ + + +
+ + + +
Virus
yield
Interferon
titre*
< 10
< 10
<10
< 10
< 10
< 10
< 10
< 10
10-"
106.3
<4
< 4
<4
64
128
256
256
256
256
256
* I n t e r f e r o n titres are t h o s e i n d u c e d b y c o c u l t i v a t i o n w i t h m o u s e spleen cells.
Table 2. buerferon-inducing activity of fixed HPIV-4A-
P M K cells in mouse spleen cells
Fixed cells
None
U n i n f e c t e d P M K cells
U n i n f e c t e d P M K cells
H P I V - 4 A - P M K cells
H P I V - 4 A - P M K cells
H P I V - 4 A - P M K cells
Mouse
spleen Anti-HPIV-4A Interferon
cells
serum
titre
+
None
+
None
+
+
None
None
None
None
None
+ *
< 2
< 2
< 2
< 2
256
< 2
* Initial titre o f a n t i - H P I V - 4 A m o n k e y s e r u m w a s 1024 h a e m a g g l u t i n a t i o n i n h i b i t i o n u n i t s a n d final titre w a s 10 units.
effect on the ability to induce interferon production, and
even the absence of adsorption was associated with 50 %
interferon production. The discrepancy is partially due
to a difference in experimental conditions, namely
tests to assess the capability of the cells to adsorb
erythrocytes or spleen cells were performed at room
temperature (about 20 °C) for 30 min whereas those to
examine interferon inducibility were conducted at 37 °C
for 20 h. We have reported that although Sendai virus
heated at 56°C for 5 m i n had no haemolytic or
haemagglutinating activity, it induced a considerable
amount of interferon in mouse spleen cell cultures (Ito &
Hosaka, 1983). Mouse spleen cells can react more
actively to the fixed virus-infected cells at 37 °C than at
20 °C. In the following experiments 0.36 % formaldehyde
was used.
Blockage of interferon induction by treatment of fixed
HPIV-4A-h~fected cells with anti-HPIV-4A antiserum
To confirm the speculation made above, various combinations of HPIV-4A-infected and uninfected P M K cells
were tested for their ability to induce interferon in mouse
spleen cells (Table 2). Culture fluids of HPIV-4A-PMK
cells without or with formaldehyde treatment showed no
mouse interferon activity. Mouse spleen cells without
virus-infected cells did not produce detectable amounts
of interferon. When mouse spleen cells were incubated
with the virus-infected cells, interferon was produced in
the culture fluid. On the other hand, when mouse spleen
cells were incubated with virus-infected cells in the
presence of anti-HPIV-4A serum (final HI titre 10 units),
interferon activity could not be detected in the culture
fluid. Therefore, it was inferred that mouse spleen cells
recognized virus-specific antigen(s) present on the surface
of infected cells and produced interferon. The interferon
activity was not neutralized by anti-HPIV-4A serum, but
was blocked by anti-interferon c~/fl serum (data not
shown), indicating that interferon induced in this system
is murine type I interferon.
Kinetics of interferon production
In the next experiment, the kinetics of interferon
production was investigated. Mouse spleen cells were
cocultivated with fixed HPIV-4A-PMK cells, and then
the culture fluid was harvested at appropriate periods
and assayed for the interferon activity. As shown in Fig.
I, interferon production begins between 6 and 8 h,
reaching a maximal level by 8 to 24 h of cocultivation.
Effects of monoclonal antibodies against HPIV-4A H N
and F proteins on interferon induction
Virus-specific antigens on the surface of HPIV-4A-PMK
cells are composed of H N and F proteins. To determine
whether the H N or F protein was involved in interferon
induction, the effects of monoclonal antibodies directed
against the H N or F protein on interferon induction were
studied. As shown in Table 3, mixtures of anti-HN
protein monoclonal antibodies suppressed the interferon
induction, although each antibody showed a weak effect
on induction. Anti-F protein monoclonal antibodies
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Y. Ito and others
570
.
.
.
.
.
.
.
.
.
.
.
256
"l
w
~
Table 4. Induction of interferon by COS7 cells
transiently expressing HPIV-4A H N protein
128
Cells
64
.3
COS7
COS7
COS/HN*
COS/HN
COS/vectort
None
32
,.a
16
8
4
<4
I
I
I
2
4
6
1
I
1
8 1; 112 114 1; 18 20 22
Time after infection (h)
24
Fig. 1. Kinetics of interferon production. Mouse spleen cells were
cocultivated with formaldehyde-fixed HPIV-4A-PMK ceils and at
appropriate intervals culture fluid was harvested and assayed for
interferon activity.
against the H N protein of HPIV-4A on the #Tterferoninducing activiO, of fixed HPIV-4A-PMK cells"
Antibody
(-)
Anti-HPIV-4A
monkey serum
MAb A138
A141
A140
A158
A138+140+158
A138+141+158
A140+ 141 + 158
A138 + 140 + 141
A138+ 140+ 141 + 158
Epitope
HI
titre
10
I
I
II
IV
I+II+IV
I+IV
I + II + I V
I + II
I+II+IV
40
10
< 1
< 1
Interferon
titre
192
4
64
64-128
64-128
64-128
32
24
16
3~64
4-8
showed no effect on interferon induction (data not
shown). These results showed that the HN protein was
related to interferon induction and that a blockage of
multiple epitopes by monoclonal antibodies was required
for inhibition of the interferon induction.
Interferon induction by HPIV-4A H N protein
transiently expressed from a recomb#lant plasmid DNA
To determine whether the actual inducer of interferon in
mouse spleen cells was HN glycoprotein on the surface of
HPIV-4A-PMK cells, a recombinant plasmid was constructed by inserting cDNA of the HN gene of HPIV-4A
into a pcDL-SR~ expression vector. COS7 cells transfected with the recombinant plasmid (COS/HN cells)
showed strong haemadsorption and the HN proteins in
the COS7 cells were strongly immunostained with anti-
None
+
None
+
+
+
Interferon
titre
< 2
3
< 2
256
8
< 2
* COS7 cells transfected with pS4AHN DNA, which was constructed by inserting the coding sequence of the HPIV-4A HN gene
cDNA into a site downstream of the SRe promoter in the expression
vector pcDL-SRc~.
t COS7 cells transfected with pcDL-SRc~ vector D N A alone.
Table 5. Induction of interferon by HeLa cells
constitutively expressing HPIV-4A HN protein
Cells
Table 3. Effects of monoclonal antibodies directed
Mouse
spleen
cells
HeLa-4aHN
HeLa-4aHN
HeLa-4aF
HeLa-4aF
None
Mouse
spleen
cells
Interferon
titre
None
+
None
+
+
< 2
92
< 2
< 2
< 2
HN monoclonal antibody (data not shown). Interferon
activity could not be detected in culture fluids of COS7
cells (approximately 5 x 105 cells) cocultured without or
with mouse spleen cells, C O S / H N cells alone or mouse
spleen cells alone (Table 4). Mouse spleen cells produced
interferon when cocultured with C O S / H N cells, but did
not produce it when cocultured with COS7 cells
transfected with the vector alone (Table 4).
Interferon induction by HPIV-4A H N protein
constitutively expressed from a recombinant plasmid
DNA
To obtain more stable and reproducible conditions, we
tried to establish HeLa cell lines constitutively expressing
HPIV-4A HN or F protein. This system has several
advantages: more stability and reproducibility can be
obtained compared with transient expression systems;
glycoproteins are expressed in all the cells analysed,
whereas in transient expression systems other than
virus-vector, some cells express the proteins and the
expression rate varies in every experiment; the transfection procedure causes a variable degree of cell damage,
whereas established cells constitutively expressing the
glycoproteins are stable and the growth of these cells is
indistinguishable from that of untreated cells. As shown
in Table 5, no interferon activity was found in the culture
fluids of HeLa-4aHN cells (2 x l0 s cells), HeLa-4aF cells
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HPIV-4A H N protein and interferon induction
Table 6. Antigenic typing off interferon induced by HeLa
cells constitutively expressing HPIV-4A H N protein
Interferon titre
Neutralization with
anti-interferon serum
Anti-IFN
Anti-IFN
Interferon
MEM
~/fl*
Yt
HN-IFN~
Recombinant IFN- 7
IFN-~/fl§
92
128
64
< 2
92
< 2
128
< 2
64
* Rabbit antiserum directed against interferon o~/fl produced by
Newcastle disease virus (NDV) infected L929 cells was used at a
dilution of 1 : 100.
t Rabbit antiserum directed against interferon y produced by
concanavalin A-stimulated mouse spleen cells was used at a dilution of
1:100.
Interferon produced by cocultivation of HeLa-4aHN cells and
mouse spleen cells.
§ Interferon produced by NDV-infected L929 cells.
(2 x 106 cells) or mouse spleen cells (3 x 107 cells) alone.
Mouse spleen cells produced interferon when cocultured
with HeLa-4aHN cells, but did not produce it when
cocultured with HeLa-4aF cells. Interferon induced in
this experiment was neutralized by anti-interferon 7/fl
antibody, but not by anti-interferon y antibody, indicating that the interferon detected in culture fluids of
mouse spleen cells with HeLa-4aHN cells is type I (c~/]3)
interferon (Table 6). Therefore, it was concluded that the
HN glycoproteins on the cell surface were sufficient for
the induction of interferon.
Discussion
This study clearly indicated that the interaction between
HN glycoproteins and receptors on the cell surface
triggered production of interferon in mouse spleen cells.
The HN glycoprotein of paramyxoviruses is a carbohydrate (sialic acid)-binding protein and shows haemagglntination. Mumps virus glycoprotein(s) selectively
inhibits cellular RNA synthesis (Yamada et al., 1984).
Furthermore, the isolated H N glycoprotein of Sendai
virus was reported to be mitogenic for mouse spleen cells
(Kizaka et aI., 1983), indicating that the paramyxovirus
H N protein has mitogenic activity under some conditions. These properties are very similar to those of
lectins (Goldstein et al., 1980), and hence the HN protein
can be thought of as being a viral lectin. Plant lectins are
classified into mitogenic and non-mitogenic types (Ito et
al., 1984). In our previous study (Ito et al., 1984), 22
species of plant lectin were tested for their ability to
induce interferon in mouse spleen cells. All of the
mitogenic lectins tested, that is, concanavalin A, succinylated concanavalin A, Lens culinaris lectin types A and B,
and pokeweed mitogen, induced mainly interferon y. In
571
addition, five of 17 non-mitogenic lectins, lotus Tetragonolobus purpureas seed lectin, crude and type II
lectins of Ulex europaeus, Bandeiraea sirnplicifolia type II
and Solanum tuberosum lectins, and wheatgerm agglutinin, induced type I interferon. Therefore, not only
mitogenic lectins, but also some non-mitogenic lectins
can induce interferon in mouse spleen cells. Accordingly,
it is no surprise that viral glycoproteins capable of
binding to cellular receptors induce interferon in lymphoid cells via their lectin-like activity. The interferon titres
induced in the present study are almost equivalent to
those induced in spleen cells exposed to plant lectins. The
isolated viral glycoprotein(s) of Sendai virus had an
interferon-inducing ability in mice (Ito et al., 1978 b) and
a non-mitogenic lectin, wheatgerm agglutinin, also
induced interferon in the circulation when injected
intraperitoneally in mice (Ito et al., 1984). Taken
together, a lectin-like activity of viral glycoprotein(s)
could also trigger interferon induction in vivo.
Although interferon was first discovered as an antiviral
substance (Isaacs & Lindenmann, 1957), it has since been
shown to affect a wide variety of cellular functions such
as cell multiplication-inhibitory activity, immune regulatory function, and the enhancing activity of multiple
cellular genes (Kizaka et al., 1983). Therefore, interferon
can be considered to be a cytokine. Lymphocytes and/or
macrophages have been reported to produce a variety of
cytokines including interferon, tumour necrosis factor
and interleukin 6, through cocultivation with cells
infected with various viruses. The molecular basis of the
induction of these cytokines by virusqnfected cells
remains to be determined. One possible mechanism is
that the induction of cytokines is also triggered by the
interaction between virus glycoproteins on the surface of
virus-infected cells and the virus receptor on lymphoid
cells, i.e. the lectin-like activity of viral glycoprotein(s).
Interestingly, both gpl20 and gpl60 of human immunodeficiency virus (HIV) were reported to be able to induce
interleukin 6 in peripheral blood mononuclear cells in
vitro and induction of the cytokine was considered to be
related to B cell polyclonal activation by HIV (Oyaizu et
al., 1991). Therefore, the lectin-like activity of viral
glycoproteins may modify immune reaction and cytokine
networks.
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(Received 8 September 1993; Accepted 11 October 1993)
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