Feline calicivirus replication induces apoptosis in cultured cells

Virus Research 94 (2003) 1
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Feline calicivirus replication induces apoptosis in cultured cells Stanislav V. Sosnovtsev a,*, Elena A. Prikhod’ko b, Gae¨l Belliot a, Jeffrey I. Cohen b, Kim Y. Green a a

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive MSC8007, Building 50, Room 6316, Bethesda, MD 20892-8007, USA b Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Received 31 December 2002; received in revised form 24 March 2003; accepted 24 March 2003

Abstract Infection of Crandell
/Rees feline kidney (CRFK) cells by feline calicivirus (FCV) causes rapid cytopathic effects followed by cell death. In this study, we observed that FCV replication in cells results in the induction of changes characteristic of apoptosis, including translocation of phosphatidyl serine to the cell outer membrane, chromatin condensation, and oligonucleosomal DNA fragmentation. FCV infection was associated with increases in the activities of caspase-3, -8, and -9, with the level of activation of caspase-3 higher than those of caspases-8 and -9. Caspase activation in CRFK cells was not observed when cells were inoculated with UV-inactivated FCV or when cycloheximide was present during virus infection, indicating that FCV replication and de novo synthesis of virus proteins are critical for induction of apoptosis. Published by Elsevier Science B.V. Keywords: Feline calicivirus; Apoptosis; Caspases

1. Introduction The family Caliciviridae is comprised of small (27
/40 nm), positive strand RNA viruses that infect a broad range of mammalian hosts including humans (Green et al., 2000). Caliciviruses are linked to a variety of animal diseases including a fatal liver disease in rabbits caused by rabbit hemorrhagic disease virus (RHDV) in the genus Lagovirus (Ohlinger et al., 1990), upper respiratory tract disease in cats caused by feline calicivirus (FCV) in the genus Vesivirus (Gaskell, 1985), and diarrhea associated with porcine enteric calicivirus (PEC), a member of the genus Sapovirus (Saif et al., 1980). In humans, caliciviruses in the genus Norovirus are major etiologic agents of epidemic nonbacterial gastroenteritis (Green et al., 2001; Kapikian et al., 1972). Tissue tropism and pathogenesis studies are restricted to caliciviruses that replicate in cell culture and to those

* Corresponding author. Tel.: /1-301-594-1666; fax: /1-301-4805031. E-mail address: [email protected] (S.V. Sosnovtsev). 0168-1702/03/$ - see front matter. Published by Elsevier Science B.V. doi:10.1016/S0168-1702(03)00115-1

for which suitable animal models are available such as RHDV, FCV, and PEC. The mechanisms that caliciviruses use to enhance spread of viral progeny in the host are not clear. Recent studies of RHDV-infected rabbits showed that apoptosis of hepatocytes might account for the lethal fulminant hepatitis associated with the disease (Alonso et al., 1998; Jung et al., 2000). Apoptotic cells were observed in a number of cell types from different organs including macrophages in the lung, liver, spleen, and lymph nodes and tubular cells in the kidney (Alonso et al., 1998). Signs of apoptotic changes in cells from infected tissues were also reported for wild hares infected with the European Brown Hare Syndrome calicivirus (Alonso et al., 1998). Apoptosis is a highly regulated biochemical process in multicellular organisms directed at cellular self-destruction in response to a wide range of stimuli. One of the main functions of apoptosis is the removal of cells during developmental events in embryogenesis or cells that are damaged due to external factors such as oxygen deprivation, UV-irradiation or virus infection. Cells undergoing apoptosis exhibit a number of characteristic changes including alterations in cellular morphology, changes in outer cell membrane composition, nuclear

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chromatin compaction and fragmentation. One of the main components of apoptosis is a cascade activation of cellular cysteine proteases, termed caspases. Caspases are responsible for the selective cleavage of a number of key cellular proteins involved in maintenance of the structural and functional integrity of the cell (reviewed in Cryns and Yuan, 1998). A number of cellular proteins are involved in the complex regulatory control of apoptosis (reviewed in Green, 2000; Shi, 2002; Vander Heiden and Thompson, 1999). Induction of apoptosis in virus-infected cells can be triggered by a number of mechanisms including dysregulation of the cell cycle, virus modulation of cellular anti-apoptotic effectors, and activation of caspases. A number of viruses are known to express proteins that have an inhibitory effect on apoptosis (Hay and Kannourakis, 2002; Roulston et al., 1999). It has been proposed that delay or inhibition of the cell’s selfdestructive process by these viruses may allow an increased time for viral amplification. In contrast, certain viruses may depend on apoptosis to destroy the integrity of cells near the end of the replication cycle, thus facilitating the release and spread of virus progeny. Consistent with this idea is the identification of certain individual viral proteins that induce programmed cell death pathways when expressed alone (Hay and Kannourakis, 2002; Roulston et al., 1999; Teodoro and Branton, 1997). An understanding of the mechanisms responsible for the induction or inhibition of apoptosis by viruses may yield new strategies for control of viral infections (Barber, 2001). The role of apoptosis in calicivirus replication and pathogenesis has not been well established because of the inability to grow many of these viruses in cell culture. The efficient replication of FCV in cultured feline cells and the availability of a reverse genetic system for this virus have allowed its use as a model for the study of calicivirus replication (Sosnovtsev and Green, 1995; Sosnovtsev et al., 2002). In the present study, we investigated whether apoptosis contributes to the death of cultured cells infected by FCV. We report several features characteristic of apoptosis in FCVinfected Crandell
/Rees feline kidney (CRFK) cells and demonstrate that active viral replication is required for induction of apoptosis.

2. Materials and methods 2.1. Cells and virus infection CRFK cells were maintained in Eagle’s minimum essential medium containing amphotericin B (2.5 mg/ml), chlortetracycline (25 mg/ml), penicillin (250 U/ml), and streptomycin (250 mg/ml) and supplemented with 10% heat-inactivated fetal bovine serum. The FCV Urbana

strain isolated from a kitten with acute respiratory illness was provided by Dr G. Sherba, University of Illinois. The virus was plaque-purified three times in CRFK cells, amplified to a titer of 5/107 pfu/ml, aliquoted, and frozen at /70 8C. All virus infections were conducted using aliquots from the same virus stock. CRFK cell monolayers were inoculated with the virus at a multiplicity of infection (MOI) of 1 and incubated for 1 h at 37 8C. A mockinfected CRFK cell control was prepared concurrently, in which the same procedure was followed in the absence of virus. After 1 h, the inoculum was replaced with fresh growth medium and cells were allowed to incubate further at 37 8C. Thereafter, cells were collected at different time points for further analysis. 2.2. DAPI staining To visualize nuclear morphology, mock- and FCVinfected CRFK cells fixed with methanol were stained with 4?,6-diamidino-2-phenylindole (DAPI) (Roche Molecular Biochemicals) (1 mg/ml) in PBS buffer for 20 min. After several washes with PBS buffer, cells were visualized by fluorescence microscopy using filters for UV excitation. 2.3. Annexin V binding assay To detect translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane of mock- or FCV-infected CRFK cells, approximately 0.5 /106 of the cells (including floating cells) were collected and incubated with fluorescein isothiocyanate (FITC)-labeled annexin V (Pharmingen). Binding and incubation conditions were performed according to the manufacturer’s protocol. After incubation with annexin V-FITC, cells were washed with annexin V-binding buffer (Pharmingen) and analyzed by flow cytometry. The percentage of annexin V-positive cells was calculated as the ratio of the number of annexin V-positive cells to the total number of forward scatter/side scatter (FSC/SSC) gated cells. 2.4. DNA fragmentation assay Isolation of total DNA and gel electrophoresis of DNA fragments were carried out as described previously (Ikeda et al., 1998). 2.5. Detection of activated caspases using FITC-VADFMK Activated caspases were quantitated by incubating 0.5 /106 CRFK cells with a 10 mM FITC-Val-Ala-Aspfluoromethylketone (FITC-VAD-FMK) in situ marker (Promega) for 20 min at 37 8C. After incubation, cells

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were washed with PBS and analyzed by flow cytometry. Selection of the cell population for counting was carried out as described above. The percent of caspase-positive cells was calculated as the ratio of the number of FITCVAD-FMK-positive cells to the total number of FSC/ SSC gated cells. To visualize cells with activated caspases, FCV- and mock-infected cells were incubated with FITC-VADFMK as described above, fixed with 2% paraformaldehyde, washed with PBS and analyzed by immunofluorescence microscopy. To detect expression of the virus capsid protein, the FITC-VAD-FMK-labeled cells were stained with hyperimmune serum prepared against purified FCV virions followed by staining with secondary Alexa Fluor568-conjugated antibodies (Molecular Probes, Inc.) as described previously (Sosnovtsev and Green, 1995). 2.6. Colorimetric analysis of caspase activity A colorimetric analysis of caspase activity was conducted using Caspase-3, -8 and -9 Colorimetric Protease Assays (BioSource International, Inc.) according to the manufacturer’s protocol. Briefly, 8 /106 mock- or FCV-infected CRFK cells were collected at different times post infection, resuspended in 300 ml of Cell Lysis Buffer (BioSource International, Inc.) and incubated on ice for 10 min. Cell lysates were clarified by centrifugation for 1 min at 10 000 /g , and the supernatants were transferred to fresh tubes. The concentration of the protein was adjusted with Cell Lysis Buffer to 1 mg/ml. Fifty ml-aliquots of the protein solutions were incubated with p- nitroanilide (p NA) peptide substrates for 4 h at 37 8C. Ile
/Glu
/Thr
/Asp (IETD)
/pNA was used for assaying caspase-8 activity, Leu
/Glu
/His
/Asp (LEHD)
/p NA for caspase-9, and Asp
/Glu
/Val
/Asp (DEVD)
/p NA for caspase-3. The optical density of the samples at 405 nm was measured using the MRX microplate reader (Dynex Technologies, Inc.). 2.7. UV-inactivation of virus UV-treatment of FCV was conducted as previously described (Kang et al., 1999; Parquet et al., 2002). Virus stocks (6 ml at 5 /107 pfu/ml) were placed on ice and irradiated with UV (254 nm) light for 15 min. The source of UV light was a Spectroline UV lamp (Spectronics Inc.) positioned at a distance of 5 cm above the dish. UV-treated virus was analyzed for infectivity on CRFK monolayers and used for further analysis. 2.8. Western blot analysis Immunoblot analysis of virus proteins in lysates of FCV-infected cells was performed as previously described (Sosnovtsev et al., 1998). Virus proteins were

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detected using serum from an FCV-infected cat (a gift from Dr. William Mengeling, USDA).

3. Results 3.1. FCV infection induces apoptotic changes in CRFK cells FCV growth in cell culture is accompanied by rapid cytopathic effects (CPE). When cells are inoculated with virus at MOI /1, CPE can be observed as early as 5 h post infection and the monolayer is destroyed by 14 h (Fig. 1, panel A). Virus replication occurs in the cytoplasm of cells and infected cells undergo significant membrane rearrangements associated with the production of numerous intracellular vesicles (Green et al., 2002; Studdert and O’Shea, 1975). In the later stages of virus replication, from 5 to 8 h post infection, infected cells become rounded (Fig. 1, panel A). In addition, beginning at 5 h post infection, changes are observed in the nuclei as the nuclear chromatin condenses into one or two dense round bodies (Fig. 1, panel B) (Crandell and Madin, 1960; Love and Sabine, 1975; Studdert and O’Shea, 1975). Finally, round-shaped cells detach (8
/14 h post infection) and undergo lysis. Because FCV infection is accompanied by dramatic morphological changes of infected cells, we examined whether CPE in FCV infection is due to apoptosis. One of the early markers of apoptosis is the translocation of PS from the inner to the outer leaflet of the plasma membrane. This early apoptotic event can be detected by the analysis of binding of annexin V to the surface of cells. During the late stages of apoptosis, when the integrity of cellular membranes becomes compromised, annexin V can also interact with the inner layer of the cell membrane. To distinguish dead cells that might stain with annexin V due to loss of membrane integrity from intact cells in the early stages of apoptosis, we used propidium iodide (PI), a nucleic acid stain that does not penetrate the membranes of viable cells. Staining of FCV-infected CRFK cells with both annexin V-FITC and PI confirmed that during infection, most of the cells undergo membrane modifications (Fig. 2). Starting at 5 h post infection, we observed an increased percentage of FCV-infected cells stained with annexin V-FITC, but not with PI. The highest percentage of annexin V-FITC-positive, PI-negative, cells (34.9%) was observed at 8 h post infection and began to decrease over time */10.7% at 14 h and 2.7% at 18 h. This finding was consistent with the presence of a population of viable infected cells undergoing apoptotic changes in their cellular membranes. The percentage of double-positive cells (that were presumably dead) increased as infection progressed and represented 95.1% of the cells by 18 h post infection (Fig. 2).

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Fig. 1. Effect of FCV infection on CRFK cell monolayer. CRFK cell monolayers were infected with FCV at MOI/1, then fixed with methanol at different times post infection and stained with DAPI. (A) Phase-contrast appearance of the mock- and FCV-infected CRFK cells, 100/ magnification. (B) Fluorescent microscopic visualization of DAPI-stained nuclei of the same cells, 400/ magnification.

As shown in Fig. 1, panel B, condensed chromatin bodies could be easily identified in cell nuclei using DAPI staining at 5 h post infection and later (Fig. 1, panel B). To investigate whether DNA of infected cells undergoes the fragmentation characteristic of apoptosis, we isolated cellular DNA at different time-points post infection (data not shown). Characteristic oligonucleosomal DNA fragments became detectable starting at 8
/ 10 h post infection (Fig. 3). DNA isolated from noninfected CRFK cells remained predominantly intact, showing no signs of specific fragmentation. 3.2. Apoptosis in FCV-infected CRFK cells is associated with activation of caspases The nuclear DNA oligonucleosomal fragmentation in apoptotic cells occurs at the final phase of cell death and is coupled to the activity of the caspases. Caspases are activated through the proteolytic processing of their zymogen forms constitutively expressed in living cells. To examine whether caspase activation takes place in FCV-infected cells, we incubated infected and mock-

infected cells at 8 h post infection with a cell-permeable fluorogenic peptide marker, FITC-VAD-FMK that binds to most activated caspases. Fluorescence microscopy analysis of FCV-infected cells showed numerous FITC-positive cells (Fig. 4A). Counterstaining of these cells with antibody to FCV virions confirmed efficient virus replication in the same cells (Fig. 4A, inset). When the FITC-VAD-FMK peptide was used for flow cytometry analysis of FCV-infected cells, the percentage of FITC-positive cells gradually increased starting at 5 h post infection and reaching a plateau at 14
/18 h. At 18 h post infection 96% of the total cell population was FITC-positive (Fig. 4B). This finding demonstrates caspase activation in FCV-infected cells and shows that it occurs concomitantly with PS translocation and initiation of DNA fragmentation. 3.3. Colorimetric assay of caspase activity in infected cells shows preferential activation of caspase-3 Analysis of the activation of caspase-8 (associated with death receptor-mediated binding of external li-

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Fig. 2. Effect of FCV replication on plasma membrane integrity of CRFK cells. Mock- and FCV-infected CRFK cells were incubated with annexin V-FITC and PI at different times post infection. Annexin V and PI binding was analyzed by flow cytometry and the results of a representative experiment are shown as dot plots of fluorescence (annexin V-FITC vs. PI) staining of mock- and FCV-infected CRFK cells. Quadrant borders were set empirically using mock-infected CRFK cells collected at 0 h post infection. The lower left quadrant (low-fluorescence PI and FITC signals) contains normal viable cells and the lower right quadrant (low-fluorescence PI and high-fluorescence FITC signals) defines cells in the early stages of apoptosis. The two upper quadrants (high-fluorescence PI signal) define dead cells. The numbers indicate the percentages of the different populations of cells.

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were significantly lower at 14 h postinfection (data not shown) consistent with the loss of cell membrane integrity observed in flow cytometry experiments with annexin V and PI. 3.4. Caspase activation is dependent on virus protein synthesis

Fig. 3. Internucleosomal DNA degradation in FCV-infected CRFK cells. Total cellular DNA isolated from FCV-infected CRFK cells was analyzed by electrophoresis in a 1.7% agarose gel. The 100 bp DNA ladder (Invitrogen, Inc.) was used as a molecular mass marker and the arrow indicates the 600-bp DNA. Lane 1, total DNA extracted from mock-infected CRFK cells 8 h post infection. Lanes 2 and 3, total DNA extracted from FCV-infected CRFK cells at 8 and 10 h post infection, respectively.

gands) and caspase-9 (associated with cytotoxic stimuli and the formation of an apoptosome) has been used to examine potential mechanisms involved in the initiation of the proteolytic pathways that lead to apoptosis. Both caspases -8 and -9 are involved in activation of effector caspases-3, -6, and -7, which are primarily responsible for the cleavage of specific cellular proteins. Using differences in substrate specificity of caspases-8 and -9, we attempted to identify a specific induction pathway of apoptosis in FCV-infected cells. Activation of each of the caspases was assayed by preparing infected cell lysates and incubating these lysates with specific substrates coupled to p-NA as a chromophore molecule. The caspase-8 substrate was IETD-p NA and the caspase-9 substrate was LEHD-p NA. Activity was detected by measuring the absorbance of released p NA at 405 nm. We observed a 7
/9-fold increase in the activity of caspases-8 and -9 at 8 h post infection (Fig. 5), indicating that both caspases were active at low levels. In contrast, assay of caspase-3 activity in parallel experiments (with substrate DEVD-pNA) showed an 18-fold increase (Fig. 5). All detected caspase activities

To establish whether apoptotic changes in FCVinfected cells could be initiated by binding of the virus particles to the cell surface, we inoculated cells with virus subjected to UV-treatment. Fifteen-minute treatment of FCV at 254 nm was found to reduce plaqueforming activity of virus by eight log(10) units (data not shown). However, UV-treated virus particles maintained their ability to bind to the cell surface and bound particles could be detected by staining of live cells with virion-specific antibodies (data not shown). Flow cytometry analysis of cells stained with FITC-VAD-FMK showed that the percentage of FITC-positive cells inoculated with UV-treated FCV did not increase over time (Fig. 4B). In contrast, the percentage of FITCpositive cells increased over time for cells infected with the nontreated virus. We then infected CRFK cells with FCV and incubated them in the presence of cycloheximide (CHX), an inhibitor of protein synthesis in eukaryotic cells, in order to examine whether caspase induction was linked to de novo viral protein synthesis and subsequent viral replication. Two concentrations (0.5 and 1 mg/ml) of CHX did not induce apoptotic changes in mock-infected CRFK cells after an 8 h-incubation (Fig. 6A). As expected, caspase activation was observed in the FCVinfected cell control (Fig. 6A). However, no evidence for caspase activity was found when FCV-infected cells were incubated with CHX for 8 h. Western-blot analysis of the CHX-treated FCV-infected cell lysates showed no expression of the virus capsid protein or nonstructural proteins (Fig. 6B) consistent with virus replication being blocked by the presence of CHX in the growth medium. These data further support the observation that viral replication is required for the induction of apoptosis.

4. Discussion In this study we demonstrate that infection of cultured monolayers of CRFK cells with a calicivirus, FCV, leads to the onset of apoptosis. We observed increased annexin V binding, changes in nuclear chromatin, DNA fragmentation, and caspase activation, all characteristic of apoptosis. Our findings are consistent with the identification of apoptotic cells in rabbits infected with RHDV (Alonso et al., 1998; Jung et al., 2000), and suggests that caliciviruses in the genus

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Fig. 4. Analysis of caspase activation in FCV-infected cells using FITC-VAD-FMK. CRFK cells were mock-infected or infected with UV-treated or nontreated FCV. At different times post infection the cells were treated with FITC-VAD-FMK. (A) Fluorescent microscopy observation of mock- or FCV-infected cells labeled with FITC-VAD-FMK at 8 h post infection, 100/ magnification. The left inset panel shows FCV-infected cells labeled with FITC-VAD-FMK at 8 h post infection. The right inset panel shows the same cells incubated with anti-FCV virion serum followed by staining with secondary Alexa Fluor568-conjugated antibodies. (B) Time-course analysis of caspase activation in CRFK cells. FITC-VAD-FMK-labeled cells were analyzed by flow cytometry and the percentage of fluorescent cells was calculated for each time point. The data were normalized to the percentage of fluorescent cells in mock-infected cell populations and mean values 9/S.E. for two replica experiments were plotted.

Fig. 5. Colorimetric analysis of caspase-3, -8, and -9 activities in FCVinfected CRFK cells. Caspase-3, -8, and -9 activities were determined by incubation of cell lysates collected over time with chromogenic caspase-specific peptide substrates, DEVD-p NA, IETD-p NA, and LEHD-p NA, respectively. Proteolytic activities were quantified by measuring release of p NA (absorbance at 405 nm). The data was normalized by subtracting background absorbance from mock-infected CRFK cell lysates. Results are mean values 9/S.E. of two replica experiments.

Vesivirus may also induce apoptosis in the cells of infected animals. Virus infections known to induce apoptosis trigger the cascade of corresponding events at varying stages of virus replication. Several viruses (vesicular stomatitis virus, Sindbis virus, avian leukosis virus, reovirus, and vaccinia virus) induce apoptosis in infected cells at an early stage of infection when virus particles interact with receptors on the cell surface or at the time of fusion with cell membrane and disassembly (Barton et al., 2001; Brojatsch et al., 1996; Connolly and Dermody, 2002; Gadaleta et al., 2002; Jan et al., 2000; Ramsey-Ewing and Moss, 1998). For these viruses, induction of apoptosis requires receptor binding or internalization, but does not require protein translation or genome replication. In this study, we found that UV-inactivated FCV that retained its ability to bind to CRFK cells did not trigger apoptosis. Furthermore, induction of apoptosis in FCV-infected CRFK cells was dependent on de novo synthesis of virus proteins, because apoptosis was blocked by CHX. The triggering of apoptosis in FCVinfected CRFK cells was apparently concurrent with the progression of FCV replication. FCV replication occurs

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Fig. 6. Effect of CHX on caspase activation in FCV-infected CRFK cells. Mock- and FCV-infected CRFK cells were incubated in the presence of 0, 0.5 or 1.0 mg/ml of CHX for 8 h. (A) The cells were harvested, labeled with FITC-VAD-FMK and their fluorescence was measured by flow cytometry. The bar graph depicts the percentage of fluorescence positive cells. (B) In parallel, cells were collected, lysed and analyzed by Western blot using serum from an FCV-infected cat. Lane M, SeeBlue Plus molecular mass marker (Invitrogen Inc.)

in the cytoplasm and involves synthesis of two major types of polyadenylated RNA templates: a plus-sense genome-sized RNA and an approximately 2.5 kb subgenomic RNA whose 3?-end is co-terminal with the genomic RNA (Herbert et al., 1996). Of interest, a large increase in RNA replicative activity was observed in the membranous complexes isolated after the 4 h post infection-time-point that correlated with a marked increase in viral protein synthesis (Green et al., 2002). This is also the time at which cells begin to show initial evidence of CPE with rounding of cells. In our present study, the peak in the number of round-shaped cells observed in a monolayer of FCV-infected cells correlated with the peak in number of apoptotic cells identified by flow cytometry analysis. Flow cytometry analysis of infected cells using annexin V-FITC staining

showed that at this time (5
/8 h post infection) a significant proportion of the population of the cells was undergoing changes in the plasma membrane characteristic of the early stage of apoptosis with PS translocation to the outer layer. Certain viruses induce apoptosis at the late stages of replication, providing a mechanism for dissemination of progeny virus (Teodoro and Branton, 1997), and it is possible that FCV may exploit apoptosis for this purpose since apoptosis in FCV-infected cells correlates temporally with replication. It should be mentioned that in parallel with the appearance of a population of annexin V-positive cells and later annexin V- and PIpositive cells that were consistent with the accumulation of cells undergoing death by apoptosis, we detected a number of annexin V-negative cells that lost their ability

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to exclude the PI. This observation was consistent with the accumulation of cells in the population with defects in their membrane transport function that were not undergoing apoptosis. The final decrease in the number of these cells might be explained by further alterations in membrane structure due to virus replication followed by cell death. Taken together, these observations suggest that calicivirus-infected cells may undergo both apoptosis and a virus-related lytic death that enables release and dissemination of progeny. We observed increasing numbers of cells with chromatin condensation between 5 and 8 h post infection. Analysis of DNA degradation showed the presence of oligonucleosomal-size DNA fragments by 8 h post infection. DNA fragmentation is one of the hallmark features of the apoptotic pathway and represents an irreversible event leading to cell death (Compton, 1992). Mediated by a caspase-activated DNAse, it starts with the appearance of 50
/300 kb fragments and proceeds to the accumulation of oligonucleosomal-size fragments (Enari et al., 1998; Sakahira et al., 1998). Activation of this DNAse is linked to the caspase-3 cleavage of a DNAse inhibitor complexed with the DNAse molecule (Sakahira et al., 1998). Consistent with the observed DNA degradation, we found that FCV infection resulted in an 18-fold increase in the activity of caspase-3 that occurred at the time of translocation of PS to the outer cytoplasmic membrane. The marked increase in caspase-3 levels in FCVinfected cells was noteworthy because caspase-3 represents a group of effector caspases that are involved directly in the execution of cellular disassembly. They cleave proteins involved in maintaining the filamentous network of the cytoplasm and in retaining the structural organization of nuclear membranes and chromatin, as well as proteins involved in DNA replication and repair (reviewed in Cryns and Yuan, 1998; Earnshaw et al., 1999). Caspase-3 can be proteolytically activated by initiator caspases-8, or -9. The peak activity of caspases8 and -9 in FCV-infected cells occurred at a similar time but at a much lower level than caspase-3. Attempts to verify cleavage of these caspases in FCV-infected feline kidney cells using commercial antibodies were unsuccessful because the human and murine antibodies we tested failed to recognize caspases in feline cells (data not shown). Similar problems were reported in studies of parvovirus-induced apoptosis in CRFK cells (Best et al., 2002). The relatively low level of caspase-8 and -9 activation might reflect the existence of additional mechanisms used by FCV to induce apoptosis. Alternatively, the low level of these caspases might be sufficient to activate effector caspases. In addition, some of the activation of caspases-8 and -9 might also be the result of either direct or indirect cleavage of these caspases by caspase-3 (Srinivasula et al., 1996; Tang et al., 2000).

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Increasing numbers of viral proteins have been implicated in the modulation of apoptotic pathways. Large DNA-containing viruses that undergo latency often encode proteins that inhibit apoptosis (adenoviruses, baculoviruses, herpesviruses, papovaviruses) (Hay and Kannourakis, 2002; Roulston et al., 1999; Teodoro and Branton, 1997). In contrast, smaller RNAcontaining viruses have often been shown to induce apoptosis. For example, the Langat flavivirus major envelope glycoprotein induces apoptosis through the caspase-3 pathway and the viral serine protease NS3 physically interacts with caspase-8 (Prikhod’ko et al., 2001, 2002). It was demonstrated that a cytopathogenic variant of hepatitis A virus directly induces apoptosis while a wild-type, noncytopathogenic, strain does not show any proapoptotic abilities (Brack et al., 2002, 1998). Poliovirus also induces apoptosis in infected cells (Ammendolia et al., 1999; Lopez-Guerrero et al., 2000; Tolskaya et al., 1995) and has been associated with viral proteinases 3C (Barco et al., 2000) and 2A (Tolskaya et al., 1995). Certain calicivirus proteins may also have proapoptotic activities, and current efforts are underway to identify these proteins. Stable cell lines expressing modified forms of these proteins may be useful for the propagation of FCV or other calicivirus replicons.

Acknowledgements We would like to thank Kyeong-OK Chang, LID, NIAID, NIH, for constructive discussions. We appreciate the assistance of Tanaji Mitra. We would like to extend our appreciation to Albert Z. Kapikian, and Robert H. Purcell, LID, NIAID, NIH, for support.

References Alonso, C., Oviedo, J.M., Martin-Alonso, J.M., Diaz, E., Boga, J.A., Parra, F., 1998. Programmed cell death in the pathogenesis of rabbit hemorrhagic disease. Arch. Virol. 143, 321
/332. Ammendolia, M.G., Tinari, A., Calcabrini, A., Superti, F., 1999. Poliovirus infection induces apoptosis in CaCo-2 cells. J. Med. Virol. 59, 122
/129. Barber, G.N., 2001. Host defense, viruses and apoptosis. Cell. Death Differ. 8, 113
/126. Barco, A., Feduchi, E., Carrasco, L., 2000. Poliovirus protease 3C(pro) kills cells by apoptosis. Virology 266, 352
/360. Barton, E.S., Chappell, J.D., Connolly, J.L., Forrest, J.C., Dermody, T.S., 2001. Reovirus receptors and apoptosis. Virology 290, 173
/ 180. Best, S.M., Wolfinbarger, J.B., Bloom, M.E., 2002. Caspase activation is required for permissive replication of Aleutian mink disease parvovirus in vitro. Virology 292, 224
/234. Brack, K., Frings, W., Dotzauer, A., Vallbracht, A., 1998. A cytopathogenic, apoptosis-inducing variant of hepatitis A virus. J. Virol. 72, 3370
/3376. Brack, K., Berk, I., Magulski, T., Lederer, J., Dotzauer, A., Vallbracht, A., 2002. Hepatitis A virus inhibits cellular antiviral

10

S.V. Sosnovtsev et al. / Virus Research 94 (2003) 1
/10

defense mechanisms induced by double-stranded RNA. J. Virol. 76, 11920
/11930. Brojatsch, J., Naughton, J., Rolls, M.M., Zingler, K., Young, J.A., 1996. CAR1, a TNFR-related protein, is a cellular receptor for cytopathic avian leukosis-sarcoma viruses and mediates apoptosis. Cell 87, 845
/855. Compton, M.M., 1992. A biochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metastasis Rev. 11, 105
/119. Connolly, J.L., Dermody, T.S., 2002. Virion disassembly is required for apoptosis induced by reovirus. J. Virol. 76, 1632
/1641. Crandell, R.A., Madin, S.H., 1960. Experimental studies on a new feline virus. Am. J. Vet. Res. 1, 551
/556. Cryns, V., Yuan, J., 1998. Proteases to die for. Genes Dev. 12, 1551
/ 1570. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383
/424. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S., 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43
/50. Gadaleta, P., Vacotto, M., Coulombie, F., 2002. Vesicular stomatitis virus induces apoptosis at early stages in the viral cycle and does not depend on virus replication. Virus Res. 86, 87
/92. Gaskell, R.M., 1985. Viral-induced upper respiratory tract diseases. In: Chandler, E.A., Gaskell, C.J., Hilbery, A.D.R. (Eds.), Feline Medicine and Therapeutics. Blackwell Scientific Publications, Oxford, pp. 257
/270. Green, D.R., 2000. Apoptotic pathways: paper wraps stone blunts scissors. Cell 102, 1
/4. Green, K.Y., Ando, T., Balayan, M.S., Clarke, I.N., Estes, M.K., Matson, D.O., Nakata, S., Neill, J.D., Studdert, M.J., Thiel, H.-J., 2000. Caliciviridae . In: Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy: The Classification and Nomenclature of Viruses (The seventh report of the International Committee on Taxonomy of Viruses). Springer, Vienna, pp. 725
/735. Green, K.Y., Chanock, R.M., Kapikian, A.Z., 2001. Human caliciviruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, vol. 1 (2 vols). Lippincott Williams & Wilkins, Philadelphia, PA, pp. 841
/874. Green, K.Y., Mory, A., Fogg, M.H., Weisberg, A., Belliot, G., Wagner, M., Mitra, T., Ehrenfeld, E., Cameron, C.E., Sosnovtsev, S.V., 2002. Isolation of enzymatically active replication complexes from feline calicivirus-infected cells. J. Virol. 76, 8582
/8595. Hay, S., Kannourakis, G., 2002. A time to kill: viral manipulation of the cell death program. J. Gen. Virol. 83, 1547
/1564. Herbert, T.P., Brierley, I., Brown, T.D., 1996. Detection of the ORF3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic, subgenomic mRNA. J. Gen. Virol. 77, 123
/127. Ikeda, Y., Shinozuka, J., Miyazawa, T., Kurosawa, K., Izumiya, Y., Nishimura, Y., Nakamura, K., Cai, J., Fujita, K., Doi, K., Mikami, T., 1998. Apoptosis in feline panleukopenia virus-infected lymphocytes. J. Virol. 72, 6932
/6936. Jan, J.T., Chatterjee, S., Griffin, D.E., 2000. Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J. Virol. 74, 6425
/6432. Jung, J.Y., Lee, B.J., Tai, J.H., Park, J.H., Lee, Y.S., 2000. Apoptosis in rabbit haemorrhagic disease. J. Comp. Pathol. 123, 135
/140. Kang, J.I., Park, S.H., Lee, P.W., Ahn, B.Y., 1999. Apoptosis is induced by hantaviruses in cultured cells. Virology 264, 99
/105. Kapikian, A.Z., Wyatt, R.G., Dolin, R., Thornhill, T.S., Kalica, A.R., Chanock, R.M., 1972. Visualization by immune electron micro-

scopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J. Virol. 10, 1075
/1081. Lopez-Guerrero, J.A., Alonso, M., Martin-Belmonte, F., Carrasco, L., 2000. Poliovirus induces apoptosis in the human U937 promonocytic cell line. Virology 272, 250
/256. Love, D.N., Sabine, M., 1975. Electron microscopic observation of feline kidney cells infected with a feline calicivirus. Arch. Virol. 48, 213
/228. Ohlinger, V.F., Haas, B., Meyers, G., Weiland, F., Thiel, H.J., 1990. Identification and characterization of the virus causing rabbit hemorrhagic disease. J. Virol. 64, 3331
/3336. Parquet, M.C., Kumatori, A., Hasebe, F., Mathenge, E.G., Morita, K., 2002. St. Louis encephalitis virus induced pathology in cultured cells. Arch. Virol. 147, 1105
/1119. Prikhod’ko, G.G., Prikhod’ko, E.A., Cohen, J.I., Pletnev, A.G., 2001. Infection with Langat flavivirus or expression of the envelope protein induces apoptotic cell death. Virology 286, 328
/335. Prikhod’ko, G.G., Prikhod’ko, E.A., Pletnev, A.G., Cohen, J.I., 2002. Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis. J. Virol. 76, 5701
/5710. Ramsey-Ewing, A., Moss, B., 1998. Apoptosis induced by a postbinding step of vaccinia virus entry into Chinese hamster ovary cells. Virology 242, 138
/149. Roulston, A., Marcellus, R.C., Branton, P.E., 1999. Viruses and apoptosis. Annu. Rev. Microbiol. 53, 577
/628. Saif, L.J., Bohl, E.H., Theil, K.W., Cross, R.F., House, J.A., 1980. Rotavirus-like, calicivirus-like, and 23-nm virus-like particles associated with diarrhea in young pigs. J. Clin. Microbiol. 12, 105
/111. Sakahira, H., Enari, M., Nagata, S., 1998. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96
/99. Shi, Y., 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 9, 459
/470. Sosnovtsev, S., Green, K.Y., 1995. RNA transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require VPg for infectivity. Virology 210, 383
/390. Sosnovtsev, S.V., Sosnovtseva, S.A., Green, K.Y., 1998. Cleavage of the feline calicivirus capsid precursor is mediated by a virusencoded proteinase. J. Virol. 72, 3051
/3059. Sosnovtsev, S.V., Garfield, M., Green, K.Y., 2002. Processing map and essential cleavage sites of the nonstructural polyprotein encoded by ORF1 of the feline calicivirus genome. J. Virol. 76, 7060
/7072. Srinivasula, S.M., Fernandes-Alnemri, T., Zangrilli, J., Robertson, N., Armstrong, R.C., Wang, L., Trapani, J.A., Tomaselli, K.J., Litwack, G., Alnemri, E.S., 1996. The Ced-3/interleukin 1beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for the apoptotic mediator CPP32. J. Biol. Chem. 271, 27099
/27106. Studdert, M.J., O’Shea, J.D., 1975. Ultrastructural studies of the development of feline calicivirus in a feline embryo cell line. Arch. Virol. 48, 317
/325. Tang, D., Lahti, J.M., Kidd, V.J., 2000. Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3dependent manner during staurosporine-mediated apoptosis. J. Biol. Chem. 275, 9303
/9307. Teodoro, J.G., Branton, P.E., 1997. Regulation of apoptosis by viral gene products. J. Virol. 71, 1739
/1746. Tolskaya, E.A., Romanova, L.I., Kolesnikova, M.S., Ivannikova, T.A., Smirnova, E.A., Raikhlin, N.T., Agol, V.I., 1995. Apoptosisinducing and apoptosis-preventing functions of poliovirus. J. Virol. 69, 1181
/1189. Vander Heiden, M.G., Thompson, C.B., 1999. Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? Nat. Cell. Biol. 1, E209
/E216.