Department of Virology1 and Department of Ophthalmology3, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan
Department of Pathology, Teikyo University School of Medicine, Kaga, Itabashi-ku, Tokyo 173-8650, Japan2
Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyou-ku, Tokyo 113-0033, Japan4
Author for correspondence: A. Hajime Koyama. Fax +81 886 33 7080. e-mail koyama{at}basic.med.tokushima-u.ac.jp
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Abstract |
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Introduction |
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In contrast to these DNA viruses, RNA viruses in general are thought not to carry an antiapoptosis gene. Under permissive conditions, they usually induce apoptosis in the infected cells but escape a deleterious effect of apoptosis by rapid multiplication (Koyama et al., 1995 , 1998c
; Kurokawa et al., 1999
; for review see Koyama et al., 1998a
). The only exception among these orthodox RNA viruses is poliovirus (PV) (HIV is the other exception among RNA viruses; Fukumori et al., 1998
). Tolskaya et al. (1995)
reported that, although PV induces apoptosis of the infected HeLa cells under non-permissive conditions for virus multiplication, the virus does not induce apoptosis in the productive infection and, by characterizing the effects of PV infection on apoptosis induced by the inhibitors of protein synthesis or RNA synthesis, they concluded that PV has an antiapoptosis function. However, the use of those inhibitors (1) blocks the progress of the normal virus infection process and (2) takes longer (approximately 45 h by actinomycin D) to induce apoptosis than sorbitol-induced apoptosis (within 2 h). In addition, we previously demonstrated that Sendai virus does not induce apoptosis in most of the infected cells, but this virus does not have an antiapoptosis gene, indicating that the lack of apoptosis in the virus-infected cells is not always accompanied by the presence of an antiapoptosis gene in the virus (Koyama et al., 2001
). Furthermore, PV-induced apoptosis has been observed even under permissive conditions: in a mouse central nerve system (Girard et al., 1999
), in a human enterocyte-like cell line (Ammendolia et al., 1999
) and in a human promonocytic cell line (Lopez-Guerrero et al., 2000
). These observations are inconsistent with the presence of an antiapoptosis gene in the PV genome.
To elucidate the nature of PV infection in regard to virus ability to regulate cell death, we examined apoptosis and an antiapoptotic function in the PV-infected cells by our highly definitive system described above (Koyama & Miwa, 1997 ; Koyama et al., 2000a
). In addition, we also examined the effect of the virus infection on non-apoptotic necrosis-like death. Necrosis has been defined as another one of two types of eukaryotic cell death (i.e. apoptosis and necrosis), but the characteristics of necrosis are still much less clear than those of apoptosis. Previously, we found that the treatment of HEp-2 with sodium chloride induces death of the treated cells without the characteristics of apoptosis (Koyama et al., 2000a
). Considering that, regardless of the types of cell death, death of the infected cells can bring interruption of virus multiplication, we characterized cell death induced by the sodium chloride treatment and examined the ability of PV to block non-apoptotic death.
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Methods |
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Plaque assay of PV-1 was carried out as follows. Virus samples were diluted with PBS containing 0·1% BSA and added to the confluent monolayers of Vero cells. The infected monolayers were incubated at 35·5 °C in MEM containing 0·5% FBS and 0·6% methylcellulose. After 2 or 3 days of incubation, the infected cells were fixed and stained simultaneously with 0·5% crystal violet solution containing 10% formalin.
Induction of apoptosis and necrosis.
To induce apoptosis, confluent monolayers of HEp-2 cells were incubated at 37 °C in MEM containing 1·0 M sorbitol for 60 min. The treated cells were washed once with prewarmed MEM and further incubated at 37 °C in MEM for 60 min (Koyama et al., 2000a ). For the induction of necrosis, 0·50 M sodium chloride was used instead of sorbitol (Koyama et al., 2000a
).
Detection of chromosomal DNA fragmentation.
Fragmentation of chromosomal DNA was determined by the method described previously (Koyama et al., 1995 ; Koyama & Miwa, 1997
). Briefly, monolayered cells were harvested with trypsin, and fragmented DNA was extracted from the cells by the Hirt method (Hirt, 1967
) with minor modifications and analysed for oligonucleosomal DNA ladders by electrophoresis on a 1·5% agarose gel.
For quantification of the chromosomal DNA extracted in the fragmented DNA fraction, chromosomal DNA was metabolically labelled with [3H]thymidine prior to infection and the radioactivity in fractions of the total and extracted DNA was determined separately. Relative amounts of the fragmented DNA were calculated by a ratio of radioactivity of the extracted DNA to that of the total cellular DNA.
Observation of nuclear morphology.
Morphology of the infected cell nuclei was examined after the cells were fixed and stained with DNA-binding dye Hoechst 33258 (0·05 µg/ml) according to the method used by McGarrity (1979) .
Electron microscopy.
The examination was performed by the pop-off method (Yasuda & Toida, 1986 ). Briefly the cells, cultured on glass slides, were fixed by immersing the slides in 2% glutaraldehyde solution for 30 min and post-fixation was done with 1% OsO4 solution for 1 h in a glass container. Then, cells on the slides were washed with buffer solution and dehydrated with graded ethanol. Epon monomer, mixed with hardener and accelerator, was prepared in O capsules and the capsules were placed upside down over the specimens on the glass slides. Polymerization was carried out at 60 °C for 4 days. After the polymerization was completed, careful trimming and ultrathin sectioning of the block were done, followed by staining with uranyl acetate and lead citrate. The specimens were observed under the electron microscope (JEM, 1200EX).
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Results |
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Fig. 2 also shows the effect of PV-1 infection on the DNA degradation induced by sodium chloride. As reported previously (Koyama et al., 2000a
), the temporal incubation of HEp-2 cells in the medium containing 0·5 M sodium chloride, with the osmotic pressure equivalent to 1 M sorbitol, induces a massive cell death without the apoptotic characteristics. Extensive degradation of chromosomal DNA occurs in the treated cells within 60 min of incubation in the reagent-free medium, but only a smear of DNA, products of a non-specific degradation of chromosomal DNA, is observed by agarose gel electrophoresis (Koyama et al., 2000a
). Not only do they lack a DNA ladder, the sodium chloride-treated cells show a nuclear morphology quite different from that of the apoptotic cells as shown in the latter section. These characteristics of the dead cells after sodium chloride treatment indicate that this death is not apoptosis. The nature of sodium chloride-induced cell death will be described in the latter section of this report.
As shown in the last five lanes from the left in Fig. 2, the infection with PV-1 also suppressed the sodium chloride-induced degradation of chromosomal DNA. When the infected cells were treated with sodium chloride at various times after the infection, a massive DNA smear, instead of the apoptotic ladder of the sorbitol-treated cells, was clearly observed in the cells treated with the reagent at 0 or 2 h p.i. However, this degradation was also noticeably affected by the treatment at 4 h p.i. and almost completely inhibited at 6 or 8 h p.i. These results indicate that the infection with PV-1 can suppress not only apoptotic DNA fragmentation induced by sorbitol but also non-apoptotic DNA degradation induced by sodium chloride.
Quantification of the fragmented DNA revealed the kinetics of virus ability to suppress the degradation of chromosomal DNA in the cells treated with sorbitol or sodium chloride (Fig. 3). Suppression of the sorbitol-induced fragmentation of chromosomal DNA could be detected in the PV-1-infected cells at 4 h p.i. and enhanced with time, reaching a plateau level at 6 h p.i. Almost the same kinetics were observed in the expression of activity to suppress the sodium chloride-induced DNA degradation in the infected cells, although the suppression was more notable than that induced by sorbitol. The similarity of kinetics suggests that a single virus-coded function might be responsible for the blockage of both apoptotic and non-apoptotic responses in the reagent-treated cells.
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Electron microscopic examination of the cells treated with sodium chloride
To further examine the nature of sodium chloride-induced cell death, we examined the reagent-treated cells with an electron microscope. Fig. 6(a
, d
) shows morphology of the mock-treated control HEp-2 cells. The cells had a clear nuclear structure, filled cytoplasm and a smooth cell surface membrane. When the cells were incubated in the medium containing sodium chloride for 60 min, most cells showed a severe disorganization of the cell structure even immediately after the incubation (Fig. 6b
). After 60 min of an additional incubation in the reagent-free medium, many of the cells showed marked generalized swelling (swollen mitochondria, unrecognizable Golgi apparatus etc.), condensation of chromatin and disruption of the plasma membrane (Fig. 6c
). These morphological characteristics of the cell injuries are consistent with those of liquefactive necrosis (Goudie, 1985
). In contrast, the sorbitol-treated cells displayed a mild disorganization of the cell structure immediately after incubation with the reagent (Fig. 6e
), but, after the incubation in the reagent-free medium, the cells showed an extensive surface blebbing, nuclear fragmentation and the formation of many membrane-bounded apoptotic bodies which are composed of cytoplasm and packed organelles with or without nuclear fragments (Fig. 6f
). The observed characteristics show the typical morphology of apoptotic cells (Kerr & Harmon, 1991
). These results indicate that death by sodium chloride is necrosis while death by sorbitol is apoptosis.
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Discussion |
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Antiapoptotic function of PV
Previously, Tolskaya et al. (1995) reported that PV induces apoptosis under conditions non-permissive for virus growth although virus does not induce apoptosis under permissive conditions. Based on the observation that PV infection inhibits apoptosis of HeLa cells induced by cycloheximide or actinomycin D, they concluded that PV carries an apoptosis-preventing function. On the other hand, even under permissive conditions, PV infection has been found to induce apoptosis in a variety of cells (Ammendolia et al., 1999
; Girard et al., 1999
; Lopez-Guerrero et al., 2000
). This apoptosis-inducing ability of the virus was confirmed by the finding that the expression of the PV protease genes induces apoptosis of the transfected cells in vitro (Barco et al., 2000
; Goldstaub et al., 2000
). Considering that (1) the use of these inhibitors to induce apoptosis does not allow normal virus replication and takes a relatively long time to induce apoptosis and (2) the experiments by Tolskaya et al. (1995)
are based mostly on morphological observations, which makes it difficult to discuss the results quantitatively with the kinetics of virus multiplication, we characterized the ability of PV to prevent cell death in relation to virus multiplication to resolve the apparent discrepancy.
PV-1 grows rapidly in HEp-2 cells (Fig. 1) and does not induce apoptosis in the infected cells (Figs 2
and 3
), confirming the observation of Tolskaya et al. (1995)
. Previously we demonstrated that the temporal incubation of HEp-2 cells in the medium containing 1 M sorbitol induces rapid and massive apoptosis of the treated cells, as judged by the classical definition of apoptosis (Kerr & Harmon, 1991
) (i.e. morphology of the cells and cell nuclei as well as oligonucleosomal fragmentation of chromosomal DNA), although biochemical characterizations of intracellular signalling pathway in the treated cells are required to understand precisely the nature of sorbitol-induced apoptosis. By examining the effect of PV-1 infection on sorbitol-induced apoptosis, antiapoptotic activity of the virus was conclusively demonstrated. Comparison of the kinetics of virus multiplication (Fig. 1
) and of the expression of antiapoptotic activity (Fig. 3
) reveals that the expression of antiapoptotic activity is observed at the late stage of the virus multiplication cycle, simultaneously with the formation of progeny viruses. Agol et al. (2000)
obtained a similar conclusion by the morphological studies on the effect of cycloheximide on the viral apoptosis-preventing function, at a drug concentration not inducing apoptosis in the infected HeLa cells but preventing the expression of virus genes. They used the Mahoney strain of PV-1 in their studies while we used the Sabin strain, indicating that the antiapoptotic function of PV-1 is not strain-specific. The apparent discrepancy between the presence of viral antiapoptotic activity and the induction of apoptosis in the infected cells of certain cell lines can be explained possibly by cell type-dependency of the viral antiapoptotic function; the function might be active in HeLa or HEp-2 cells, but not active in other types of cells reported (Ammendolia et al., 1999
; Girard et al., 1999
; Lopez-Guerrero et al., 2000
). As to the existence of a specific antiapoptosis gene, it should be mentioned that we cannot exclude the possibility, although it is not very likely, that the observed suppression of apoptosis in the PV-infected cells might be the result of a bystander effect of the infection or the result of a constellation of multiple cellular and viral activities in the infected cells, because the antiapoptotic gene of the virus has not yet been identified.
In addition, it is noteworthy that the infection of PV in the presence of cycloheximide did not induce apoptosis in HEp-2 cells (data not shown), although HSV-1 and HSV-2 induce apoptosis under the same conditions (Koyama & Adachi, 1997 ; Koyama et al., 1998b
). This result suggests that virion components of PV do not have any ability to induce apoptosis in the infected cells, while those of HSV-1 and HSV-2 can trigger an apoptotic response in the cells.
Characterization of sodium chloride-induced cell death and the effect of PV infection
Eukaryotic cell death has been classified into two categories, apoptosis and necrosis. Apoptosis is considered to be an active physiological process in which cells die in a tightly controlled manner under a cellular death programme, while necrosis is a passive degenerating process in which cells are killed accidentally by a toxic environment. Although death by sodium chloride is not definitely passive degeneration of the cells, the sodium chloride-treated cells can be considered to be killed by a necrosis-like mechanism, because cell death by sodium chloride is not apoptosis by the classical criteria of apoptosis. Morphology of both the dead cells (Fig. 4) and the cell nuclei (Fig. 5
) are quite different from those of apoptotic cells and oligonucleosomal fragmentation (laddering) of chromosomal DNA was not observed (Fig. 2
). Although the characteristics of the cells killed by necrosis are not yet as clear as those of apoptotic cells, electron microscopic observations (Fig. 6c
) revealed that death by this agent shows the characteristics of liquefactive necrosis (generalized swelling, disruption of the cell membrane and condensation of chromatin without fragmentation of the nucleus; Goudie, 1985
), indicating that the temporal treatment of HEp-2 cells with sodium chloride can induce one type of necrosis in HEp-2 cells. A biochemical study is now in progress to reveal a definite mechanism of sodium chloride-induced cell death.
Interestingly, PV infection also suppressed cell death induced by sodium chloride (Figs 2, 3
, 4
and 5
). We conclude that the virus can prevent both apoptotic and necrotic death of the infected cells. It should be noted that, although the virus has the ability to prevent death of the infected cells, these cells died at the end of infection (after the completion of progeny virus formation), probably by a non-specific loss of the regulation of the infected cell metabolism.
Multiplication of PV-1 in the apoptotic cells
In addition to the observations discussed above, PV-1 can grow in the apoptotic cells. When sorbitol-treated HEp-2 cells were infected with PV-1, the virus grew significantly, although massive fragmentation of chromosomal DNA was observed in these infected cells (data not shown). The virus yield decreased to one-quarter of that in the untreated normal cells, but the formation of progeny virus started and reached a plateau with similar kinetics to those in the untreated cells. This result confirms our previous conclusion that virus-induced apoptosis by animal virus infection does not bring about the abortion of virus multiplication by premature death of the infected cells, but renders the infected cells to be phagocytosed by macrophages by presenting recognition signal molecules on the infected cell surface (Koyama et al., 2000b ).
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Acknowledgments |
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References |
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Received 16 May 2001;
accepted 17 August 2001.