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J Virol, April 1998, p. 2927-2934, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Increased Induction of Apoptosis by a Sendai Virus
Mutant Is Associated with Attenuation of Mouse Pathogenicity
Masae
Itoh,1,*
Hak
Hotta,1 and
Morio
Homma2
Department of Microbiology, Kobe University
School of Medicine, Chuo-ku, Kobe 650,1 and
Faculty of Home Economics, Kobe Women's University, Suma-ku,
Kobe 654,2 Japan
Received 2 September 1997/Accepted 12 December 1997
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ABSTRACT |
An avirulent mutant of Sendai virus, Ohita-MVC11 (MVC11), was
generated from a highly virulent field strain, Ohita-M1 (M1), through
successive passages in LLC-MK2 cell cultures (M. Itoh, Y. Isegawa, H. Hotta, and M. Homma, J. Gen. Virol. 78:3207-3215, 1997). In LLC-MK2 cells, MVC11 induced a high degree of
apoptotic cell death that was demonstrated by chromatin condensation of the nucleus and DNA fragmentation, and production of MVC11 declined markedly after prolonged culture. On the other hand, M1 did not induce
prominent apoptosis and maintained high virus titers. In primary mouse
pulmonary epithelial cell cultures, M1 replicated rather slowly to
reach maximum level of virus production at 3 days postinfection, and
high levels of virus production were maintained thereafter without
causing apoptosis. In contrast, MVC11, which produced 20 times more
progeny virus than M1 at 1 day postinfection, induced a high degree of
apoptotic cell death before the virus replication cycle was completed.
Accordingly, the production of progeny virus was strongly inhibited
thereafter. In the lungs of mice infected with MVC11, virus antigens
and signals of DNA fragmentation detected by the in situ terminal
deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique
colocalized in bronchial epithelial cells, clearly demonstrating that
infection by MVC11 triggered apoptosis in vivo as well as in vitro.
These results suggest the possibility that induction of apoptosis by
MVC11 plays an important role in attenuation of mouse pathogenicity by
restricting progeny virus production in the lung. The C protein was
shown to have the capacity to induce apoptosis, and the increased level
of the C protein in MVC11-infected cells was considered to account
partly, if not entirely, for the induction of apoptosis.
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INTRODUCTION |
Sendai virus (SeV), a member of the
paramyxoviruses, is also called murine parainfluenza virus type 1 and
often causes outbreaks of lethal pneumonia in mouse
colonies. Experimental infection in mice
has been studied as a model for respiratory viral infection. Although
there are SeV strains differing remarkably in pathogenicity to mice,
the determinants of their pneumovirulence are largely unknown. In our
previous work, we demonstrated that the pathogenicity of SeV closely
correlates with virus replication in the mouse lung (35). We
then showed that susceptibility of the fusion (F) envelope glycoprotein
to trypsin and to the activating proteases in the mouse respiratory
tract determines the efficiency of virus replication, and therefore the
virulence, by supporting multiple-cycle replication through cleavage
and activation of the F protein (16). Kato et al.
(20) reported that an SeV mutant lacking the V protein replicated less efficiently in the lungs of mice and was strongly attenuated. However, the mechanism by which the lack of the V protein
leads to the decreased production of progeny virus is not known.
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FIG. 1.
Progeny virus production in and viability of
LLC-MK2 cells infected with M1 or MVC11.
LLC-MK2 cells infected with M1 (a) or MVC11 (b) at an MOI
of 10 were incubated in MEM supplemented with 8% FBS in the absence of
trypsin at 38°C. Culture medium was taken and cells were refed with
fresh medium every 6 or 12 h after infection as indicated. Virus
released into the culture medium within 6 or 12 h before the
sampling was assayed after activation with trypsin (15).
Another set of LLC-MK2 cells infected with M1 or MVC11 as
described above was collected every 24 h, and viability of the
cells was determined by the trypan blue dye exclusion test.
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When infected with a virus, the host attempts to suppress virus
replication in infected cells and viral spread to neighboring cells by
means of host defense mechanisms such as induction of immune responses,
interferon production, and suicidal cell death (apoptosis). By turning
on the switch for apoptosis before the virus has completed the
replication cycle, the host cells prevent the virus from producing
progeny virions that infect neighboring cells. This idea is supported
by recent findings that many viruses, such as poxviruses (2,
13), adenovirus (30, 31), Epstein-Barr virus
(9-11), hepatitis C virus (7, 32), and
baculovirus (3), contain genes whose function appears to
interfere with the apoptotic process, presumably to allow cell survival
and continued virus replication (36).
Recently, we reported the characterization of an avirulent mutant of
SeV, the Ohita-MVC11 (MVC11) strain, which was derived from a highly
virulent field strain, the Ohita-M1 (M1) strain, and possessed two
amino acid mutations, one in the C protein and the other in the L
protein (17). MVC11 exhibited strongly suppressed virus
replication in mouse lungs and had almost entirely lost pathogenicity
to mice. In this work, we studied replication of M1 and MVC11 in
cultured mouse pulmonary epithelial cells, as well as in
LLC-MK2 cells, in order to elucidate the mechanism of
restricted replication of MVC11 in mouse lungs. We found that whereas
M1 maintained to produce progeny virus for a long period of time
without killing the host cells, MVC11 induced apoptotic cell death that
interfered with the following progeny virus production. We propose a
hypothesis that the increased capacity of MVC11 to induce apoptosis may
play an important role in attenuation of virulence through restricting
virus spread in mouse lungs.
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MATERIALS AND METHODS |
Viruses, cells, and antibodies.
The M1 strain of SeV, a
fresh isolate from an outbreak of SeV in experimental animal
facilities, and the MVC11 strain, a laboratory-adapted mutant of M1
obtained through passaging in rhesus monkey (LLC-MK2) cells, were described elsewhere (17). Infective virus titers were determined as described previously and expressed as PFU or cell-infecting units (CIU) (19). CIU were essentially
equivalent to PFU. LLC-MK2 cells were grown in Eagle's
minimum essential medium (MEM) supplemented with 8% fetal bovine serum
(FBS). Polyclonal anti-SeV antibodies were made by immunizing rabbits
with purified SeV strain Fushimi. Polyclonal anti-C guinea pig serum
(29) was a kind gift from K. Iwasaki (Tokyo Metropolitan
Institute of Medical Science).
Primary culture of mouse pulmonary epithelial cells.
Tracheotomy was performed on 6-week-old male ICR/CRJ (CD-1) mice under
ether anesthesia, and 1 ml of protease type X (2 mg/ml; Sigma) was
infused into the lung with a syringe. After incubation for 10 min at
room temperature, the lung was taken and minced in phosphate-buffered
saline (PBS). Blocks of the tissues were removed by filtration through
four layers of sterilized gauze, and single cells were collected by
centrifugation, suspended in Dulbecco's MEM supplemented with 10%
FBS, and cultivated at 38°C.
Determination of apoptotic cell death.
Occurrence of
apoptosis was determined by the following two methods.
For detection of chromatin condensation of the nuclei, cells
grown on coverslips were fixed with ethanol for 20 min at room temperature and stained with 10 µg of Hoechst 33342 per ml in PBS for
10 min at room temperature.
For detection of DNA fragmentation, cells (5 × 10
5)
were lysed in 0.5% Triton X-100-10 mM Tris-HCl (pH 7.5)-10 mM EDTA,
and
nuclei were removed by centrifugation at 10,000 ×
g for
5 min
at 4°C. The supernatants were treated with 50 µg of
proteinase
K per ml and 10 µg of RNase A per ml for 1 h at
37°C, and DNAs
were extracted with phenol-chloroform and precipitated
with ethanol.
The pellets were dissolved in Tris-EDTA (pH 7.5) and
separated
by electrophoresis in 1% agarose gels.
Western blot analysis.
Cells were lysed by treatment with
0.5% Triton X-100 in 10 mM Tris-HCl (pH 7.5) for 15 min on ice, and
nuclei were removed by centrifugation at 10,000 × g for 5 min at 4°C. Supernatants were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (8 or 16% acrylamide for
detection of the C protein) under reducing conditions, and the proteins
were electrotransferred onto nitrocellulose membranes. After
blocking with 3% skim milk in PBS, the membranes were incubated with
anti-SeV rabbit antiserum and subsequently with peroxidase-conjugated
goat anti-rabbit immunoglobulin G (IgG). To detect the C protein, the
membranes were treated with anti-C guinea pig antiserum and then with
peroxidase-conjugated goat anti-guinea pig IgG. Virus-specific proteins
were visualized with an ECL chemiluminescence kit (Amersham) according
to the manufacturer's instructions.
In situ terminal end-labeling.
The terminal deoxynucleotidyl
transferase-mediated dUTP nick end-labeling (TUNEL) technique was used
to label DNA strand breaks in apoptotic cells. Paraffin-embedded lung
sections were deparaffinized with absolute and 95, 75, and 50% ethanol
solutions and then washed with PBS. After endogenous peroxidase was
inactivated in 3% hydrogen peroxide, slide preparations were treated
with 50 µg of proteinase K per ml for 30 min at room temperature.
Subsequent steps were performed with a kit (Apop Tag; Oncor), according
to the manufacturer's instructions, to end label the fragmented DNA
with digoxigenin-11-dUTP and peroxidase-conjugated antidigoxigenin
antibody. Color development was achieved with diaminobenzidine as the
substrate.
Double labeling of tissue sections for detection of SeV antigens
and TUNEL signals.
The TUNEL reaction was completed as far as the
wash steps after the labeling reaction. The sections were then
incubated sequentially with anti-SeV rabbit antiserum,
biotin-conjugated goat anti-rabbit IgG, and alkaline
phosphatase-conjugated streptavidin. Finally, color reactions were
performed with diaminobenzidine to detect the peroxidase-labeled DNA
fragmentation and then with 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium to detect alkaline phosphatase-labeled SeV antigens.
Expression of the C protein.
Sau3AI-BglII
fragments (nucleotides [nt] 107 to 812) of cDNAs of the P genes of M1
and MVC11, both containing the start codon for the C protein (nt 114)
but lacking those for the P (nt 104) and the C' (nt 81) proteins, were
inserted into the unique BamHI site of the pSG5 mammalian
expression vector (8). EcoRI linker-ligated NP
cDNA (nt 26 to 1674) derived from M1 was inserted into the EcoRI site of pSG5. The resulting plasmids, pSG-CM,
pSG-CMVC, and pSG-NP, which express the C protein of M1
(C170F), that of MVC11 (C170S), and the NP
protein, respectively, under the control of the simian virus 40 early
promoter, were introduced into cells by calcium phosphate-mediated
transfection or by using Lipofectin (GIBCO BRL). We also used pSV2-C,
which expresses the C protein of SeV strain Z (kindly supplied by H. Taira, Faculty of Agriculture, Iwate University).
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RESULTS |
Detection of apoptosis in SeV-infected LLC-MK2
cells.
We previously showed that progeny virus production of MVC11
in LLC-MK2 cells within 24 h postinfection (p.i.) was
higher than that of M1 through increased mRNA synthesis
(17). We also reported that, although it was higher than
that of M1 within 1 day p.i., replication of MVC11 in mouse lungs was
strongly restricted at 2 days, p.i. and thereafter (17). In
the present study, therefore, we first examined M1 and MVC11 virus
replication in LLC-MK2 cells for a longer period of time.
The culture media of M1- and MVC11-infected LLC-MK2 cells
were replaced every 6 h (0 to 48 h p.i.) or every 12 h
(48 to 96 h p.i.), and the virus titers were assayed. M1-infected LLC-MK2 cells continued to release high titers of progeny
virus until 96 h p.i., with approximately 90% of infected cells
being alive (Fig. 1a). All the living cells were confirmed to be
infected with M1 by immunofluorescence analysis with anti-SeV antiserum (data not shown). Since the cells were infected at a multiplicity of
infection (MOI) of 10 and cultivation was performed in the absence of
trypsin, multiple-cycle replication would not take place. Thus, a high
degree of virus replication was maintained in M1-infected
LLC-MK2 cells throughout the observation period without
killing the host cells. On the other hand, MVC11-infected LLC-MK2 cells underwent injury and died. Accordingly, MVC11
progeny virus production diminished rapidly after 24 h p.i. (Fig.
1b).
Since SeV was reported to induce apoptosis (
38), we examined
whether the strong cytopathic effect of MVC11 was caused by
apoptosis.
Staining of nuclei with Hoechst 33342 clearly demonstrated
condensed
chromatin in MVC11-infected, but not M1-infected, LLC-MK
2 cells (Fig.
2a). Also, strong DNA
fragmentation was detected at
36 h p.i. and later in
MVC11-infected cells, whereas slight DNA
fragmentation was observed in
M1-infected cells only at 48 h p.i.
(Fig.
2b).
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FIG. 2.
Apoptosis induced by SeV. (a) Condensation of chromatin.
LLC-MK2 cells infected with M1 or MVC11 at an MOI of 10 were fixed with ethanol at 48 h p.i. and stained with Hoechst
33342. (b) DNA fragmentation. DNAs were extracted from M1- or
MVC11-infected LLC-MK2 cells (MOI of 10) at 24, 36, and
48 h p.i. and electrophoresed in a 1% agarose gel.
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Restricted production of progeny virus as a result of apoptosis in
MVC11-infected mouse pulmonary epithelial cells.
MVC11 has almost
completely lost lethality against mice, with its replication being
extremely suppressed in the mouse lungs (17). To get
information on the mechanism(s) of suppression of progeny virus
production, we investigated whether MVC11 could trigger apoptosis to
interrupt virus production in mouse pulmonary epithelial cells, the
target cells of SeV in vivo, as observed with LLC-MK2
cells. In a primary culture of mouse pulmonary epithelial cells, M1
virus growth took place more slowly than in LLC-MK2 cells
and reached a maximum titer at 3 days p.i., which was maintained at
least until 11 days p.i. (Fig. 3a). On
the other hand, virus titers in MVC11-infected cells reached a maximum
level at 1 day p.i., and diminished rapidly thereafter. Figure 3b shows
synthesis of SeV-specific proteins in the primary culture of epithelial cells. In accordance with the time course of virus production (Fig.
3a), synthesis of viral proteins in M1-infected mouse pulmonary epithelial cells took place relatively slowly, reaching a maximum level
at 3 days p.i., which was maintained throughout the observation period.
On the other hand, protein synthesis in MVC11-infected cells occurred
more rapidly to a maximum level at 1 day p.i. and was strongly
suppressed thereafter.
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FIG. 3.
Replication of M1 or MVC11 in primary cultures of mouse
pulmonary epithelial cells. (a) Progeny virus production. Primary
cultures of mouse pulmonary epithelial cells were infected with M1
( ) or MVC11 ( ) at an MOI of 10 and incubated in Dulbecco's MEM
supplemented with 10% FBS in the absence of trypsin. Culture medium
was harvested and cells were refed with fresh medium at the indicated
days after infection, and virus titers were determined as PFU per
milliliter. (b) SeV-specific proteins. On the indicated days after
infection, lysates prepared from cells infected with M1 or MVC11 as
described for panel a were subjected to Western blot analysis to detect
virus-specific proteins with anti-SeV polyclonal rabbit antiserum.
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SeV-infected mouse pulmonary epithelial cells were examined for
apoptosis by staining the nuclei with Hoechst 33342. The nuclei
of
M1-infected cells appeared to be intact at both 2 and 6 days
p.i. (Fig.
4b and d). Some of the M1-infected cells
were even
found to proliferate (Fig.
4d), suggesting that M1 caused
persistent
infection without killing the host cells. On the other hand,
the
nuclei of MVC11-infected cells demonstrated condensed chromatin
at
both 2 and 6 days p.i. (Fig.
4f and h), with large numbers
of cells
having detached from the bottom of the plastic dish on
day 6. These
results indicate that MVC11 could induce strong apoptosis
of the
infected cells, which interrupted the following synthesis
of viral
proteins and progeny virus production. On the other hand,
M1 possessed
a very limited capacity, if any, to trigger apoptosis,
which allowed M1
to replicate for a prolonged period of time.
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FIG. 4.
Chromatin condensation in primary cultures of mouse
pulmonary epithelial cells infected with SeV. Primary cultures of mouse
pulmonary epithelial cells were infected with either M1 (a to d) or
MVC11 (e to h). At 2 or 6 days p.i., cells were fixed with ethanol and
subjected to immunofluorescence staining with anti-SeV rabbit antiserum
( SeV) as the first antibody and fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG as the second antibody
for detection of virus antigens (a, c, e, and g), followed by staining
with Hoechst 33342 for detection of chromatin condensation (b, d, f,
and h). The arrows in panel d show the nuclei of dividing cells
infected with M1, and the arrowheads in panels f and h depict chromatin
condensation in the nuclei.
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Induction of apoptosis in the lungs of mice infected with
MVC11.
To examine whether MVC11 could induce apoptotic cell death
in mouse lungs in vivo, lung sections obtained from M1- and
MVC11-infected mice were stained for TUNEL signals. TUNEL signals were
detected only slightly in the lungs of mice infected with M1 at 2 days p.i. (Fig. 5a). Although M1 infection
spread over the lung, even to the alveoli, and produced high titers of
virus on day 6 (17), only a few TUNEL signal-positive cells
were detected (Fig. 5b). On the other hand, nuclei of bronchial
epithelial cells in MVC11-infected mice were strongly stained by TUNEL
at 2 days p.i. (Fig. 5c). On day 6, the number of cells with
TUNEL-positive nuclei decreased and the intensity of the signals
diminished (Fig. 5d), which was associated with both the elimination of
MVC11 antigen-positive cells and the sharp decline of virus titers in
the lung (17).
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FIG. 5.
Detection of apoptosis in lungs of mice infected with
SeV. Three-week-old male ICR mice were inoculated intranasally with
1.25 × 105 CIU of either M1 (a and b) or MVC11 (c and
d) in 25 µl of PBS or with 25 µl of PBS as a control (e). Tissue
sections from 2 days p.i. (a, c, and e) and 6 days p.i. (b and d) were
labeled by the TUNEL reaction for detection of DNA fragmentation.
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Lung sections prepared from the mice described above were then dually
stained for SeV antigens and DNA fragmentation to confirm
that the
cells dying by apoptosis were infected with MVC11. Although
bronchial
epithelial cells of mice inoculated with M1 were stained
dark purple,
which verified the infection by SeV, brown TUNEL
signals were not
detected in those cells (Fig.
6a). In
MVC11-inoculated
mice, bronchial epithelial cells with DNA
fragmentation (brown)
were shown to be positive for SeV antigens
(purple) (Fig.
6b).
The lungs of control mice inoculated with PBS alone
demonstrated
neither SeV antigens nor TUNEL signals (Fig.
6c). These
results
clearly demonstrate that MVC11 triggered apoptosis as a result
of virus infection, while M1 did not.
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FIG. 6.
Colocalization of viral antigens and TUNEL signals in
SeV-infected mouse lungs. Tissue sections were prepared on day 2 from
the same mice as in Fig. 5 inoculated with M1 (a), MVC11 (b), or PBS
(c). Double labeling was performed with anti-SeV antiserum (purple) and
the TUNEL reaction (brown) to demonstrate colocalization of DNA
fragmentation and SeV antigens.
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Induction of apoptosis by the C protein.
MVC11 possesses two
amino acid mutations; one is in the C protein at position 170 (Phe
Ser), and the other is in the L protein at position 2050 (GluAla) (17). We tested possible involvement of the C
protein in the induction of apoptosis. As demonstrated in Fig.
7, COS-7 cells transiently expressing the
C protein exhibited condensation of chromatin. There was no apparent
difference in apoptosis-inducing capacity among the C proteins of
strains M1 (C170F) (Fig. 7b), MVC11 (C170S)
(Fig. 7d), and Z (Fig. 7f). It was unlikely that the apoptosis was
induced simply by overexpression of a protein, since strong expression
of the NP protein did not induce apoptosis in the cells (Fig. 7h).
Induction of apoptosis by C170F and C170S, but
not by the NP protein, was confirmed also in HeLa and L929 cells (data
not shown).
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FIG. 7.
Induction of apoptosis by the C protein. The C proteins
of M1 (C170F), MVC11 (C170S), and Z
(CZ) and the NP protein of M1 were expressed transiently in
COS-7 cells. At 60 h after transfection, COS-7 cells transfected
with pSG-CM (a and b), pSG-CMVC (c and d), or pSV2-C (e and f) were
stained with anti-C guinea pig serum and fluorescein
isothiocyanate-conjugated goat anti-guinea pig IgG (a, c, and e), and
cells transfected with pSG-NP were stained with anti-SeV polyclonal
rabbit antiserum and fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (g). The cells were then stained with Hoechst 33342 to
detect chromatin condensation (b, d, f, and h). Cells positive for
C170F, C170S, or CZ exhibit
chromatin condensation and/or nuclear fragmentation, whereas
NP-positive cells do not.
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Since the C proteins of M1 and MVC11 induced apoptosis equally, we then
examined the synthesis of the C protein in M1- and
MVC11-infected
cells. In M1-infected LLC-MK
2 cells, the C protein
accumulated gradually until 96 h p.i. (Fig.
8a). In MVC11-infected
LLC-MK
2 cells, on the other hand, synthesis of the C
protein took
place more rapidly and to a larger extent than with M1
until 24
h p.i. but decreased rapidly thereafter. In mouse
pulmonary epithelial
cells, the C protein of M1 was not detected
throughout the course
of infection, probably due to the limited number
of cells, whereas
that of MVC11 was detected at 1 day p.i. and
diminished thereafter
(Fig.
8b).
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FIG. 8.
Synthesis of the C protein in M1- and MVC11-infected
cells. At the indicated times after infection, lysates prepared from
LLC-MK2 cells (a) or mouse pulmonary epithelial cells (b)
infected with M1 or MVC11 (MOI of 10) were subjected to Western blot
analysis with anti-C polyclonal guinea pig antiserum to detect the C
protein. For panel b, the same cell lysates as for Fig. 3b were used.
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DISCUSSION |
Apoptosis is a built-in cell suicide program required for normal
embryonic development, tissue homeostasis, and several immunological processes. Infection of cultured cells with a wide variety of viruses,
including herpesviruses (14, 18, 21, 22, 25), parvoviruses
(27), retroviruses (5, 23, 26, 28, 33), paramyxoviruses (4), myxoviruses (6, 12, 34),
alphaviruses (24, 39), and picornaviruses (37),
results in activation of the apoptosis pathway. It has been shown that
virus-induced activation of programmed cell death in certain cell
populations, such as neurons and immune cells, may be directly
associated with viral pathogenicity (1). In such cases,
virulent strains cause apoptosis more strongly than avirulent strains.
On the other hand, data have accumulated that host cells trigger
apoptosis when infected with viruses, which interferes with virus
production, offering an important host defense mechanism to combat
virus infection (36). Suppression of virus production by
apoptosis was reported with some viruses, such as poliovirus
(37) and vaccinia virus (13).
In this study, we demonstrated that MVC11, an avirulent mutant of SeV
derived from the virulent wild-type isolate M1, induced apoptosis in
mouse pulmonary epithelial cells within 2 days p.i. (Fig. 4f). As shown
in the one-step growth experiment (Fig. 3a), replication of SeV appears
to take place relatively slowly in primary cultures of mouse epithelial
cells, and probably in vivo as well, reaching a maximum titer at 3 days
p.i. Apoptosis triggered by MVC11 therefore could have caused cell
death before the virus replication cycle was completed and, as a
result, the following synthesis of virus proteins and production of
progeny virus were strongly suppressed. Considering that about
104 mouse pulmonary epithelial cells were used for the
virus growth experiment of Fig. 3a, a single MVC11-infected cell
produced approximately 100 virus particles on the first day of
infection and much less thereafter. On the other hand, an M1-infected
cell released 1,000 virus particles every day throughout the
cultivation period without undergoing apoptosis. Therefore, it is clear
that apoptosis induced by MVC11 inhibited virus production in the
culture. The same concept could be applied to the in vivo experiments,
where apoptosis interfered with the replication and spread of MVC11 in
mouse lungs. Thus, it is likely that induction of apoptosis by MVC11
plays an important role in attenuation of the mouse pathogenicity of
the virus.
There is increasing evidence that many viruses encode proteins that
interact with the cellular pathways regulating programmed death
(36). However, the molecular mechanism by which SeV
infection activates the death pathway is unknown. Tropea et al.
(38) reported that alpha interferon had no effect on
SeV-induced apoptosis. We demonstrated in the present study that the C
protein of SeV induces apoptosis when transiently expressed in COS-7
cells (Fig. 7) and in HeLa and L929 cells (data not shown). To our
knowledge, this is the first study that pinpoints an SeV protein as an
apoptosis-inducing protein.
Despite the marked difference between M1 and MVC11 in the capacity to
induce apoptosis through viral infection, the C protein of M1
(C170F) induced apoptosis to practically the same extent as
the C protein of MVC11 (C170S) in transient-expression
experiments (Fig. 7). A possible explanation for this discrepancy is
that apoptosis is triggered by the increased level of C protein
expression in MVC11-infected cells: the amounts of the C protein in
MVC11-infected cells in the early stage of infection (12 to 24 h
p.i. in LLC-MK2 cells and 1 day p.i. in mouse pulmonary
epithelial cells) were significantly larger than those in M1-infected
cells throughout the infection (Fig. 8). Another possibility that
should also be taken into consideration is that another SeV protein(s)
is involved in the induction of apoptosis. Further analysis to
elucidate the mechanism of SeV-induced apoptosis is in progress.
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ACKNOWLEDGMENTS |
We thank K. Iwasaki and H. Taira for their kind gifts of anti-C
guinea pig antiserum and pSV2-C, respectively.
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture,
by a Research Program for Slow Virus Infection grant from the Ministry
of Health and Welfare, Japan, and by a research grant from Yakult Co.,
Ltd.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Phone: 81-78-341-7451, ext. 3302. Fax: 81-78-351-6347. E-mail: masae{at}med.kobe-u.ac.jp.
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J Virol, April 1998, p. 2927-2934, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
