Increased Induction of Apoptosis by a Sendai Virus Mutant Is Associated with Attenuation of Mouse Pathogenicity

Copyright © 1998, American Society for Microbiology

Increased Induction of Apoptosis by a Sendai Virus Mutant Is

Associated with Attenuation of Mouse Pathogenicity

MASAE ITOH,

_{* HAK HOTTA,}

_{MORIO HOMMA}

Department of Microbiology, Kobe University School of Medicine, Chuo-ku, Kobe 650,

_{and Faculty of Home}

Economics, Kobe Women’s University, Suma-ku, Kobe 654,

_Japan

Received 2 September 1997/Accepted 12 December 1997

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-MK

cell cultures (M. Itoh, Y. Isegawa, H. Hotta,

and M. Homma, J. Gen. Virol. 78:3207-3215, 1997). In LLC-MK

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

pro-duced 20 times more progeny virus than M1 at 1 day postinfection, inpro-duced 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.

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.

Experimen-tal infection in mice has been studied as a model for

respira-tory 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

corre-lates with virus replication in the mouse lung (35). We then

showed that susceptibility of the fusion (F) envelope

glycopro-tein 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

rep-lication 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.

When infected with a virus, the host attempts to suppress

virus replication in infected cells and viral spread to

neighbor-ing cells by means of host defense mechanisms such as

induc-tion of immune responses, interferon producinduc-tion, 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

find-ings that many viruses, such as poxviruses (2, 13), adenovirus

(30, 31), Epstein-Barr virus (9–11), hepatitis C virus (7, 32),

* Corresponding author. Mailing address: Department of

Microbi-ology, 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@med.kobe-u.ac.jp.

FIG. 1. Progeny virus production in and viability of LLC-MK2cells infected

with M1 or MVC11. LLC-MK2cells 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-MK2cells infected with M1 or MVC11 as

described above was collected every 24 h, and viability of the cells was deter-mined by the trypan blue dye exclusion test.

2927

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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

pul-monary epithelial cells, as well as in LLC-MK

cells, in order to

elucidate the mechanism of restricted replication of MVC11 in

mouse lungs. We found that whereas M1 maintained to

pro-duce 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

viru-lence through restricting virus spread in mouse lungs.

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-MK2cells 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 Sci-ence).

Primary culture of mouse pulmonary epithelial cells.Tracheotomy was per-formed 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

FIG. 2. Apoptosis induced by SeV. (a) Condensation of chromatin. LLC-MK2cells 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-MK2cells (MOI of 10) at 24, 36,

and 48 h p.i. and electrophoresed in a 1% agarose gel.

FIG. 3. Replication of M1 or MVC11 in primary cultures of mouse pulmo-nary epithelial cells. (a) Progeny virus production. Primary cultures of mouse pulmonary epithelial cells were infected with M1 (F_{) or MVC11 (}E_{) 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 infec-tion, 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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centrifugation, suspended in Dulbecco’s MEM supplemented with 10% FBS, and cultivated at 38°C.

Determination of apoptotic cell death.Occurrence of apoptosis was deter-mined 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 10mg of Hoechst 33342 per ml in PBS for 10 min at room temperature.

For detection of DNA fragmentation, cells (53105_{) 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 10,0003g for 5 min at 4°C. The supernatants were treated with

50mg of proteinase K per ml and 10mg 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 electro-phoresis 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 10,0003g 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 SeV rabbit anti-serum and subsequently with peroxidase-conjugated goat anti-rabbit immu-noglobulin 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 instruc-tions.

In situ terminal end-labeling.The terminal deoxynucleotidyl transferase-me-diated dUTP nick end-labeling (TUNEL) technique was used to label DNA

strand breaks in apoptotic cells. Paraffin-embedded lung sections were deparaf-finized 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 50mg 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 frag-mented DNA with digoxigenin-11-dUTP and peroxidase-conjugated antidigoxi-genin 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 phos-phatase-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 al-kaline 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 C9

(nt 81) proteins, were inserted into the unique BamHI site of the pSG5 mam-malian 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 (C170}S_{), 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 Univer-sity).

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 (aSeV) 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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RESULTS

Detection of apoptosis in SeV-infected LLC-MK

cells.

We

previously showed that progeny virus production of MVC11 in

LLC-MK

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

rep-lication in LLC-MK

cells for a longer period of time. The

culture media of M1- and MVC11-infected LLC-MK

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-MK

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-MK

cells throughout the observation

pe-riod without killing the host cells. On the other hand,

MVC11-infected LLC-MK

cells underwent injury and died.

Accord-ingly, MVC11 progeny virus production diminished rapidly

after 24 h p.i. (Fig. 1b).

Since SeV was reported to induce apoptosis (38), we

exam-ined whether the strong cytopathic effect of MVC11 was

caused by apoptosis. Staining of nuclei with Hoechst 33342

clearly demonstrated condensed chromatin in

MVC11-in-fected, but not M1-inMVC11-in-fected, LLC-MK

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).

Restricted production of progeny virus as a result of

apo-ptosis 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-MK

cells. In a primary culture of mouse

pulmonary epithelial cells, M1 virus growth took place more

slowly than in LLC-MK

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

FIG. 5. Detection of apoptosis in lungs of mice infected with SeV. Three-week-old male ICR mice were inoculated intranasally with 1.253105_{CIU of either M1}

(a and b) or MVC11 (c and d) in 25ml of PBS or with 25ml 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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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

oc-curred more rapidly to a maximum level at 1 day p.i. and was

strongly suppressed thereafter.

SeV-infected mouse pulmonary epithelial cells were

exam-ined 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

demon-strated 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.

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

sig-nals. 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

pro-duced 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).

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

demon-strate that MVC11 triggered apoptosis as a result of virus

infection, while M1 did not.

Induction of apoptosis by the C protein.

MVC11 possesses

two amino acid mutations; one is in the C protein at position

170 (Phe

3

Ser), and the other is in the L protein at position

2050 (Glu

3

Ala) (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 (C

_{) (Fig. 7b), MVC11 (C}

_{) (Fig. 7d), and Z (Fig. 7f).}

It was unlikely that the apoptosis was induced simply by

over-expression of a protein, since strong over-expression of the NP

protein did not induce apoptosis in the cells (Fig. 7h).

Induc-tion of apoptosis by C

_{and C}

_{, but not by the NP protein,}

was confirmed also in HeLa and L929 cells (data not shown).

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

cells, the C protein accumulated gradually until 96 h p.i. (Fig.

8a). In MVC11-infected LLC-MK

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,

prob-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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ably due to the limited number of cells, whereas that of MVC11

was detected at 1 day p.i. and diminished thereafter (Fig. 8b).

DISCUSSION

Apoptosis is a built-in cell suicide program required for

normal embryonic development, tissue homeostasis, and

sev-eral 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

apo-ptosis 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

apopto-sis 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

prob-ably 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

pro-FIG. 7. Induction of apoptosis by the C protein. The C proteins of M1 (C170F_{), MVC11 (C}170S_{), and Z (C}Z_{) 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_{, C}170S_{, or C}Z_{exhibit chromatin condensation and/or nuclear fragmentation, whereas NP-positive cells do not.}

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duction of progeny virus were strongly suppressed.

Consider-ing that about 10

_{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

ap-optosis. 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

pathoge-nicity of the virus.

There is increasing evidence that many viruses encode

teins that interact with the cellular pathways regulating

pro-grammed 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

tran-siently 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

pro-tein.

Despite the marked difference between M1 and MVC11 in

the capacity to induce apoptosis through viral infection, the C

protein of M1 (C

_{) induced apoptosis to practically the}

same extent as the C protein of MVC11 (C

_{) 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-MK

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

consid-eration is that another SeV protein(s) is involved in the

induc-tion of apoptosis. Further analysis to elucidate the mechanism

of SeV-induced apoptosis is in progress.

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

Min-istry of Health and Welfare, Japan, and by a research grant from

Yakult Co., Ltd.

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