The Gamma-2-Herpesvirus Bovine Herpesvirus 4 Causes Apoptotic Infection in Permissive Cell Lines

Virology 277, 27–39 (2000) doi:10.1006/viro.2000.0575, available online at http://www.idealibrary.com on

The Gamma-2-Herpesvirus Bovine Herpesvirus 4 Causes Apoptotic Infection in Permissive Cell Lines Maria Teresa Sciortino,* Donata Perri,† Maria Antonietta Medici,* Mariella Foti,‡ Bianca Maria Orlandella,‡ and Antonio Mastino* ,1 *Department of Microbiological, Genetic, and Molecular Sciences, and ‡Department of Pathology, Infectious and Parasitic Diseases, and Food Inspection, University of Messina, Messina, Italy; and †Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata,” Rome, Italy Received December 6, 1999; returned to author for revision January 5, 2000; accepted August 11, 2000 Increasing evidence suggests that regulation of apoptosis in infected cells is associated with several viral infections. The gammaherpesvirus bovine herpesvirus 4 (BHV-4) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by BHV-4 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell targets. Apoptosis induced by BHV-4 was inhibited by (1) treatment with doses of heparin, which completely inhibited virus attachment and infectivity; (2) UV treatment, which completely abrogated infectivity; and (3) treatment with a dose of phosphonoacetic acid, which blocked virus replication. Virus-induced apoptosis was associated with a down-regulation of Bcl-2 expression and was reduced by Z-VAD-FMK, but not by Z-DEVD-FMK (caspase-3-specific) caspase inhibitors. Inhibition of apoptosis by Z-VAD-FMK treatment during infection did not modify virus yield. Therefore, despite the presence of antiapoptotic genes in its genoma, BHV-4 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of BHV-4 and other gammaherpesviruses in vivo. © 2000 Academic Press

INTRODUCTION

chicken as well as in various cell lines from these and other species, including humans, as recently demonstrated (Egyed, 1998). Viruses have been found to have selected, during evolution, specific mechanisms to escape host defense, using different strategies (reviewed by Ploegh, 1998). Apoptosis, the form of cell death initially described by Kerr et al. (1972), could have been selected during evolution as a highly conservative nonspecific host response to limit virus spread, thus contributing to the control of the infectious focus (reviewed in O’Brien, 1998; Razvi and Welsh, 1995; Shen and Shenk, 1995). This seems to be the reason why a number of viruses have genes able to sustain inhibitory effects on apoptosis upon infection (reviewed in Teodoro and Branton, 1997). It was demonstrated that BHV-4 carries a gene which encodes a protein that inhibits Fas- and TNFR1-induced apoptosis by interacting with caspase-8 (Wang et al., 1997). In addition, a bcl-2 homolog, whose antiapoptotic activity was recently demonstrated (Bellows et al., 2000), was found in the BHV-4 genome. The products of these genes are strictly related to proteins encoded by other ␥-2-herpesviruses, such as HHV-8. However, no information is available on the real ability of these viruses to regulate apoptosis following productive infection. Obviously, it is difficult to obtain this information studying viruses,

Bovine herpesvirus 4 (BHV-4) is a member of the Gammaherpesvirinae subfamily of herpesviruses. It belongs to the ␥-2-herpesviruses on the basis of its thymidine kinase activity and its genomic similarity to other viruses of this subgroup, such as herpesvirus Saimiri (HVS) and human herpesvirus 8 (HHV-8), the ethiological agent of Kaposi’s sarcoma in humans (Bublot et al., 1991, 1992; Goltz et al., 1994; Lomonte et al., 1992, 1996; Thiry et al., 1992). Unlike other herpesviruses, BHV-4, originally isolated by Bartha et al. (1966), is characterized by an unusually wide host range both in vivo and in vitro. In fact, it has been demonstrated that BHV-4 strains can infect American bison, African buffalo, and goats as well as nonruminant species such as the lion and, under particular conditions, the cat (Kruger et al., 1990; Moreno-Lopez et al., 1989; Rossiter et al., 1989; Todd and Storz, 1983). In vitro, BHV-4 replicates in a variety of cell cultures, including established primary kidney cell cultures from cattle, sheep, goats, dogs, cats, rabbits, pigs, and

1 To whom correspondence and reprint requests should be addressed at University of Messina, Department of Microbiological, Genetic, and Molecular Sciences, Salita Sperone 31, Messina 98166, Italy. Fax: ⫹39 090 392733. E-mail: [email protected].

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0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Fluorescence microscopy analysis of BHV-4-infected cells. BS/BEK and AU-BEK cells, mock-infected or infected with the LVR 140 reference strain of BHV-4, at a m.o.i. of 10 PFU/cell, were analyzed 48 h p.i. after staining with the fluorescent DNA-binding dye acridine orange. In infected samples, many cells show the morphological characteristics of apoptosis, with nuclei resembling one or more groups of featureless, bright, spherical beads. Magnification ⫻400.

such as HHV-8, that cannot yet be grown efficiently in cell culture. Since increasing evidence would indicate that several viruses could simultaneously carry genes able to both block and induce apoptosis (Teodoro and Branton, 1997), we have used the availability of an efficient experimental model of infection in vitro for BHV-4 to investigate whether the presence of antiapoptotic genes in the genoma of a ␥-2-herpesvirus effectively and invariably leads to the inhibition of apoptosis during productive infection. RESULTS Bovine herpesvirus 4, strain LVR 140, induced apoptosis in permissive cell lines To define whether infection by BHV-4 could be associated with the ability of the virus to interact with apoptotic signals, we investigated the characteristics of cell death caused by infection with the LVR 140 strain of the virus in the BS/BEK cell line, derived from bovine embryo kidney, as a model of a permissive cell line. In this cell line, infected at a multiplicity of infection (m.o.i.) of 10 PFU/cell, the cytopathic effect appeared on Day 2 postinfection (p.i.) and reached a massive effect on Days 3–4 p.i., when most of the cells were seen to be detached from the bottom of the flask and were floating in the supernatant. Results showed a marked increase in the percentage of apoptotic cells following LVR 140 infection, when compared with control cells, which were uninfected or mock-infected,

using both acridine orange and Hoechst staining. In the representative experiment shown in Fig. 1, the level of apoptosis, detected by acridine orange staining, reached about 90% 48 h p.i. Similar, but lesspronounced, effects using the same dose of virus inoculum and at the same time p.i., were observed in the AU-BEK (Fig. 1) and in the MDBK (data not shown) BHV-4-permissive cell lines. Uninfected controls showed low levels of spontaneous apoptosis (⬍5%), as did mock-infected cells (data not shown). Similar effects were obtained when Hoechst was used as a DNA-staining dye (data not shown). To confirm the above-reported results, we utilized flow cytometry analysis of isolated nuclei, according to a technique to detect apoptosis, which allows us to distinguish apoptosis from necrosis and viability, as we recently demonstrated (Matteucci et al., 1999). Results unconfutably confirmed morphological analysis. In the experiment shown in Fig. 2, the percentage of hypodiploid nuclei obtained from cultures infected with a dose of 10 PFU/cell of LVR 140 was still rather low 24 h after infection (17%, Fig. 2C), while it rose to 81% 48 h after infection (Fig. 2D). Moreover, to verify whether necrosis could be distinguished from apoptosis and viability in BS/BEK using our technique, combined flow cytometry analysis for red fluorescence and forward-scatter (FSC) versus side-scatter (SSC) was applied in other experiments, in parallel to control untreated cells, to cells that were heat-shocked at 60°C for 1 h to induce necrosis and to cells infected by

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FIG. 2. Induction of apoptosis by BHV-4-infection in BS/BEK cells, detected by flow cytometry. After 24 h (A, C) and 48 h of culture (B, D), nuclei from control, mock-infected cultures (A, B) or from cells infected with BHV-4, strain LVR 140, at a m.o.i. of 10 PFU/cell (C, D) were isolated and stained by treatment with a hypotonic solution containing detergent and propidium iodide and analyzed by flow cytometry. The dashed lines indicate the boundaries among the peaks of diploid and hypodiploid nuclei and presumably of debris, which were arbitrarily set on untreated samples and maintained for all other samples. The numbers in the cytograms represent the percentages of hypodiploid nuclei, based on a total number of nuclei from which debris were excluded.

BHV-4 to induce apoptosis. As shown in Fig. 3, nuclei from viable cells (Fig. 3A) and from heat-shocked necrotic cells (Fig. 3C) fell into the same fluorescence emission peaks, while many of those from infected cells fell into the typical peak of hypodiploid apoptotic nuclei (Fig. 3E). However, using the FSC/SSC analysis, nuclei derived from BS/BEK cells in which necrosis was induced (Fig. 3D) were remarkably different from nuclei derived from viable or BHV-4-infected cells (Figs. 3B and 3F, respectively). In fact, they showed higher FSC and SSC values, as we previously described for lymphoid cells (Matteucci et al., 1999). Nuclei with similar characteristics, typical of necrotic cells, were not detected in BHV-4-infected samples. Apoptosis in BS/BEK cells infected with LVR 140 was also evaluated using the TUNEL technique, an assay able to directly detect DNA fragmentation at the single-cell level. In the experiment illustrated in Fig. 4, 24 h after infection at a m.o.i. of 5 PFU/cell, we ob-

tained 13% positive apoptotic cells (Fig. 4C) and 63% positive apoptotic cells at 48 h p.i. (Fig. 4D), in comparison with 1 and 2% positive cells from mock-infected cultures at 24 or 48 h p.i., respectively (Figs. 4A and 4B) and 98% in the positive control consisting of DNase I-treated cells (not shown). Figure 5 summarizes the results of four independent experiments in which apoptosis induced by LVR 140 at various PFU/ cell ratios was evaluated by morphological analysis of cells after staining with acridine orange, as described above. Low levels of apoptosis were detected using this technique at 24 h following infection at all m.o.i. tested as well as at a m.o.i. of 1 PFU/cell at 48 h p.i., while at 72 h p.i. cultures infected at all m.o.i. tested showed a high percentage of characteristic apoptotic cells in a dose-dependent fashion. Variability from experiment to experiment was rather high, as indicated by standard deviation values. Nevertheless, induction of apoptosis was invariably detected following

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FIG. 3. Combined flow cytometry analysis by red fluorescence (A, C, E) and by two-parameter FSC/SSC (B, D, F) analysis of nuclei from viable, necrotic, or apoptotic BS/BEK cells. BS/BEK cells were left either untreated (A, B), heat shocked at 60°C for 1 h in a water bath to induce necrosis (C, D), or infected with LVR 140 at a m.o.i. of 10 PFU/cell to induce apoptosis 48 h after infection (E, F) and stained as described in Fig. 2. The dashed lines in (A), (C), and (E) indicate the boundaries among the peaks of diploid and hypodiploid nuclei and presumably of debris, which were arbitrarily set on untreated samples and maintained for all other samples. The numbers in the cytograms represent the percentages of hypodiploid nuclei, based on a total number of nuclei from which debris were excluded.

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FIG. 4. Apoptosis of BHV-4-infected BS/BEK cells, detected using the TUNEL technique. Apoptosis of BS/BEK cells infected with LVR 140 at a m.o.i. of 5 PFU/cell, was analyzed by flow cytometry using the TUNEL technique for detecting DNA breaks. Mock-infected cultures at 24 h p.i. (A) and at 48 h p.i. (B), respectively. Infected cultures at 24 h p.i. (C) and at 48 h p.i. (D), respectively. The dashed lines indicate the boundaries between negative and positive cells, which were arbitrarily set in control samples incubated with FITC-dUTP solution alone, without the addition of Tdt. Control FITC-dUTP samples showed less than 1% positive cells (not shown) and boundaries were maintained in the corresponding FITC-dUTP-Tdt-treated samples. The numbers in the cytograms represent the percentages of apoptotic-positive cells.

infection and was dependent on the dose of virus inoculum and on the length of time of culture following infection. Induction of apoptosis by BHV-4 in BS/BEK cells was associated with significant levels of viral production and was not restricted to the LVR-140 strain We were then interested to confirm whether cell death by apoptosis was compatible with a significant level of viral production. To this purpose, the infecting activity of supernatants from LVR 140-infected BS/BEK cells was evaluated by detecting plaque formation at Day 7 in the same cell line. Figure 6A shows a time course of virus yield in supernatants following exposure to LVR 140 for 1 h at a m.o.i. of 10 PFU/ml. Productive infection was clearly demonstrated by a progressive increase in released virus with the ongoing of the culture. Active replication of LVR 140 in BS/BEK cells was also confirmed by using a BHV-4-specific monoclonal antibody and fluorescence microscopy. Using this technique to detect virus replication, the first appearance of positivity for BHV-4 proteins was evident at 12 h p.i., with a high percentage

of positive cells for viral proteins at 24 h p.i. and almost all positive cells at 48 h p.i. (Fig. 6B). Simultaneous detection of viral protein expression and apoptosis still showed a very low level of apoptotic cells at 24 h p.i. (Fig. 6B). Conversely, at 48 h p.i. cells showed the characteristics of both apoptotic and infected cells (Fig. 6B). Double-fluorescence observation demonstrated that part of the cells positive for virus-specific proteins was still apparently intact, whereas apoptosis was never observed in virus-protein-negative cells (data not shown). Moreover, to verify whether induction of apoptosis in permissive cells was dependent on the particular strain of BHV-4 utilized, the effects of LVR 140 were compared with those obtained using the BHV-4/F1 strain. Both strains were able to induce apoptosis following infection of BS/BEK cells (data not shown). Treatment with heparin or with an inhibitor of virus replication and exposure of BS/BEK cells to UVinactivated LVR 140, inhibited virus-induced apoptosis To obtain information on the mechanisms involved in cell death by apoptosis caused by BHV-4, we then tested

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FIG. 5. Kinetic- and dose-dependency of BHV-4-induced apoptosis in permissive cells. Apoptosis induced by BHV-4, strain LVR 140, following infection at indicated PFU/cell ratios, was evaluated by calculating the percentage of cells showing nuclear morphology of apoptosis using fluorescence microscopy analysis of cells after staining with the DNAbinding dye acridine orange. Results are expressed as means ⫾ SD from four independent experiments. All comparisons among the mockinfected group and groups infected with a different m.o.i. at the same time were significant except for: all groups at time 24 h, control versus m.o.i. 1, and m.o.i. 10 versus m.o.i. 100 at time 48 h. All comparisons among groups infected with the same m.o.i. at different times were significant except for: control mock-infected groups at any time, time 24 h versus time 48 h at a m.o.i. of 1, time 48 h versus time 72 h at a m.o.i. of 10 and 100.

whether treatment with heparin, which prevents virion attachment, or exposure of BS/BEK cells to UV-inactivated LVR 140, which allows attachment but prevents infectivity, or addition of phosphonoacetic acid (PAA), a well-known inhibitor of herpesvirus DNA polymerase, could interfere with the induction of apoptosis. Heparin addition prevented apoptosis at 48 h p.i. In particular, at doses of 100 and 500 U/ml, apoptosis (as well as virus infection) was completely inhibited in comparison with

control cultures (data not shown). Moreover, as evaluated by fluorescence microscopy following acridine orange staining, PAA prevented LVR 140-induced apoptosis at 48 h p.i. in a dose-dependent manner (Fig. 7A). The same experiments showed that PAA by itself induced a low level of apoptosis, but only at the highest dose of 300 ␮g/ml. In this case, too, simultaneous detection of viral replication by IFA showed a direct, dose-dependent correlation between prevention of apoptosis and inhibition of viral protein expression (Fig. 7A). We further investigated the relationships between apoptosis and virus replication. After UV irradiation of the virus we compared the inactivation of infectivity with that of apoptosis-inducing activity. As shown in Fig. 7B both infectivity and apoptosis-inducing activity were similarly suppressed by increasing exposure to UV light. LVR 140-induced apoptosis in BS/BEK cells was associated with a down-modulation of Bcl-2 and was reduced by the caspase inhibitor Z-VAD-FMK Expression of the bcl-2 gene is one of the mechanisms associated with the modulation of cell apoptosis during viral infections (Levine et al., 1996; Olsen et al., 1996). Using flow cytometry, we thus analyzed Bcl-2 protein cellular levels in BS/BEK cells following LVR 140 infection. Results showed an evident down-modulation of Bcl-2 protein in most of the infected cells in experimental conditions able to induce apoptosis (Fig. 8). No downregulation of Bcl-2 was observed at 24 h p.i. (data not shown). Moreover, to obtain further information on the mechanisms regulating BHV-4-induced apoptosis, the possible involvement of the caspase cascade was investigated by adding synthetic, cell-permeable, noncleavable peptide analogs of caspase substrates to infected

FIG. 6. Time course of virus production, viral protein expression, and apoptosis in BS/BEK cells infected with BHV-4. (A) Virus released in supernatants by BS/BEK cells was titrated by plaque assay soon after the removal of virus inoculum (time 0) and at 12, 24, 36, and 48 h after infection with LVR 140 at a m.o.i. of 10 PFU/cell. (B) Expression of viral proteins, following staining with a BHV-4-specific commercial antibody (histograms), and apoptosis, calculated by counting the percentage of cells showing nuclear morphology of apoptosis following staining with acridine orange (line), were evaluated by fluorescence microscopy in the same infected cultures utilized for virus titration of (A). Results are expressed as means ⫾ SD obtained from triplicate cultures in one of three experiments performed with similar results. All comparisons among sample groups were significant, except for virus production, time 0 against time 12 h.

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FIG. 7. Effect of phosphonoacetic acid (PAA) and UV treatment on apoptosis and viral protein expression in cells infected with BHV-4. (A) Apoptosis (histograms) and percentage of viral-protein-positive cells (line), evaluated 48 h p.i. using fluorescence microscopy, by calculating the percentage of cells showing nuclear morphology of apoptosis after staining with acridine orange or percentage of positive cells after staining with a BHV-4-specific commercial antibody, respectively, in BS/BEK cells infected with BHV-4, strain LVR 140, at a m.o.i. of 10 PFU/cell and left untreated or treated with PAA at the indicated doses. Apoptosis was also detected in control uninfected cells. Results are expressed as means ⫾ SD obtained from triplicate cultures in one of the four experiments performed with similar results. IFA, all comparisons among sample groups were significant except for: PAA 37.5 ␮g/ml versus PAA 75 ␮g/ml, PAA 150 ␮g/ml versus PAA 300 ␮g/ml. Apoptosis, comparisons among sample groups were not significant except for: PAA 150 ␮g/ml versus PAA 300 ␮g/ml, PAA 18.7 ␮g/ml and PAA 0 ␮g/ml (untreated control) versus all other groups. (B) Percentage of apoptosis (histograms) and percentage of viral-protein-positive cells (line), evaluated at 48 h p.i. as reported in (A), in BS/BEK cells infected at a m.o.i. of 10 PFU/cell with BHV-4, strain LVR 140, inactivated by increasing time of exposure to UV light. Apoptosis was also detected in control uninfected cells. Data from one of the two experiments performed with similar results are shown.

and control cultures, to irreversibly inhibit protease activity. As shown in Fig. 9A, Z-VAD-FMK, but not Z-DEVDFMK, significantly reduced apoptosis of infected cultures in comparison with control-treated cultures at 48 h p.i. Moreover, Fig. 9B shows that daily treatment with a dose of Z-VAD-FMK able to completely block apoptosis, did not substantially alter virus yield, measured as PFU/ml in supernatants from infected cultures, during 6 days of culture. DISCUSSION It has been postulated that apoptosis represents a well-conserved ancestral form of resistance to virus infection. A number of studies, in fact, have demonstrated that viruses belonging to a variety of families, particularly those characterized by persistence, should harbor, in their genomes, genes that are able to counteract apoptotic mechanisms (Aubert and Blaho, 1999; Bellows et al., 2000; Galvan and Roizman, 1998; Galvan et al., 1999; Jerome et al., 1998, 1999; Koyama and Miwa, 1997; Leopardi and Roizman, 1996). BHV-4 as well as other ␥-2herpesviruses have been shown to possess such antiapoptotic machinery (Cheng et al., 1997; Derfuss et al., 1998; Hu et al., 1997; Sarid et al., 1997; Thome et al., 1997; Wang et al., 1997). However, results reported in the present study demonstrate that apoptosis was the only detectable form of cytopathic effect in several permissive cell lines for BHV-4. Recently, another member of the Herpesviridae family, bovine herpesvirus 1 (BHV-1), has been shown to cause apoptosis in vitro (Devireddy and Jones, 1999; Hanon et

al., 1997, 1998). Indeed, some similarities exist between cell death associated with infection by the two bovine herpesviruses. First, in both BHV-1 and BHV-4 infections, cells do not undergo apoptosis early after virus exposure, but in a later phase, i.e., starting 24–36 h following infection. Second, in both BHV-4 and BHV-1 infection, in vitro apoptosis is associated with a true productive cycle, with a high level of virus release, and cannot be efficiently induced by inactivated virus. Finally, induction of apoptosis by both BHV-1 and BHV-4 is caspase-dependent and can be greatly inhibited using the caspase inhibitor Z-VAD-FMK. However, when Z-VAD-FMK was used to reduce apoptosis caused by BHV-1, a significant increase in virus yield was observed. In contrast, inhibition of apoptosis using the same treatment did not modify virus yield in BHV-4-infected cells, unlike that also observed for other viruses (Chinnaiyan et al., 1997; Chiou and White, 1998). This distinction could be related to differences in the virus replication cycle of the two subfamilies of herpesviruses. In contrast with alphaherpesviruses, members of the Gammaherpesvirinae subfamily cause infections that usually occur without production of infectious progeny (Roizman and Sears, 1996). Interestingly, we have shown here for the first time that even when a productive infection of a ␥-2-herpesvirus does occur, as in the case of BHV-4, it seems to be associated with apoptosis and not with a lytic phase, despite the presence of antiapoptotic genes. Three experimental findings reported in studies concerning HHV-8 could be relevant to this point, the first of which concerns the difficulty to establish efficient transmission

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FIG. 8. Bcl-2 levels in BS/BEK cells following infection with BHV-4. BS/BEK cells were infected with BHV-4 at a m.o.i. of 10 PFU/cell (INFECTED) or mock infected (MOCK INFECTED). After 48 h p.i. flow cytometry analysis of intracellular Bcl-2 protein following permeabilization and staining with an FITC-conjugated monoclonal antibody against human Bcl-2 (Bcl-2) or an irrelevant control monoclonal antibody (FITC) was performed. The dashed lines indicate reference boundaries, which were arbitrarily set for the samples stained with irrelevant antibody and maintained in the corresponding anti-Bcl-2-stained samples. Similar results were obtained in four independent experiments.

of HHV-8 infection in vitro (Schultz, 1998). Recently, this difficulty was partly overcome by using experimental stratagems which, at least in one case, allowed researchers to detect apoptosis in infected cells (Friborg et al., 1998). Thus, it is possible to hypothesize that difficulty in finding a good experimental cell model permissive to HHV-8 could be because of the apoptosis-inducing capacity of the virus. The second finding is that recent results suggest that chemical induction of HHV-8 DNA replication and transcription in primary effusion lymphoma cell lines overlaps with cell death by apoptosis (Yu et al., 1999). Investigators have attributed apoptosis to chemical treatment, but we cannot exclude that expression of viral genes contributes to the phenomenon. Finally, it has recently been demonstrated that apoptosis is well detected in early Kaposi’s sarcoma lesions, i.e., in a phase which could precede the latency of HHV-8 infection, while it is practically absent in late-stage lesions, when expression of K13/v-FLIP gene of HHV-8 increases (Sturzl et al., 1999). Regarding the mechanisms involved in BHV-4-induced apoptosis, our results raise several points worthy of consideration. The first is that we observed a strict correlation between the suppression of apoptosis-inducing

activity and that of infectivity, as shown by experiments with heparin, PAA, or UV treatment. This distinguishes apoptosis induced by BHV-4 from that caused in lymphoid or monocytoid cells by the members of Alphaherpesvirinae subfamily bovine herpesvirus 1 and herpes simplex 2, which have been demonstrated to maintain, at least in part, their ability to induce apoptosis following UV inactivation (Hanon et al., 1996; Mastino et al., 1997). As a consequence, BHV-4 should not have apoptosisinducing structural proteins at all or, alternatively, should concomitantly harbor one or more highly active apoptosis-blocking molecules in its virion. Expression of viral genes in the late phase of BHV-4 infection could modify this initial apoptosis-blocking balance. The second consideration concerns the role of caspases, key mediators of apoptosis (reviewed in Cohen, 1997; Porter and Janicke, 1999; Salvesen and Dixit, 1997). We found that the caspase inhibitor Z-VAD-FMK almost completely inhibited apoptosis caused by BHV-4, indicating that activation of the caspase cascade is not dispensable in virusinduced apoptosis. Interestingly, the specific caspase-3 inhibitor Z-DEVD-FMK did not produce the same effect. A simple interpretation of these results is that caspases other than caspase-3, such as effector caspase-6 or

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FIG. 9. Effect of synthetic, cell-permeable caspase inhibitors on apoptosis and on virus yield in permissive cells following BHV-4 infection. (A) Z-VAD-FMK and Z-DEVD-FMK, peptide analogs of caspase substrates, were added at various concentrations to uninfected BS/BEK cells or to BS/BEK cells infected with LVR 140 at a m.o.i. of 10 PFU/cell immediately after removing virus inoculum. At 48 h p.i. apoptosis was detected by calculating the percentage of cells showing nuclear morphology of apoptosis using fluorescence microscopy analysis of cells after staining with acridine orange. Results from one of the three experiments performed with similar results are shown. (B) BS/BEK cells were infected with BHV-4 as described in (A) and left untreated or treated daily with Z-VAD-FMK or DMSO at the concentration present in the diluent. Z-VAD-FMK was added at a full dose of 50 ␮M immediately after removing virus inoculum, and at half dose during the following days. Virus yield released in supernatants from untreated infected control cells (control) or from cells infected and treated with Z-VAD-FMK (Z-VAD-FMK) or DMSO (DMSO), respectively, was titrated by plaque assay. Data obtained from triplicate samples are expressed as means ⫾ SD PFU/ml. Differences among groups were not significant at all times tested.

caspase-7, could be activated in a caspase-3-independent manner. Z-VAD-FMK, acting upstream in caspase cascade activation (Cohen, 1997), could in any case block virus-induced apoptosis, while Z-DEVD-FMK, acting downstream specifically on caspase-3, does not produce the same effect. More complex explanations of our results, however, cannot be excluded. The caspase cascade can be activated in both death-receptor-mediated and death-receptor-independent apoptosis (Sun et al., 1999). Unfortunately, the lack of related reagents in the bovine system prevented us from investigating this aspect. Conversely, the availability of an anti-human cross-reacting antibody allowed us to verify that BHV-4-induced apoptosis was associated with antiapoptotic Bcl-2 protein down-regulation. The kinetics of Bcl-2 down-regulation overlaps that of induction of apoptosis. This evidence suggests that the presence of optimal levels of Bcl-2 could counteract BHV-4-induced apoptosis and that Bcl-2 down-regulation, occurring specifically or not following infection, could be linked to induction of apoptosis. However, our results do not allow us to fully understand the pathways involved in BHV-4-induced apoptosis and further studies are necessary. Nevertheless, whatever the exact mechanism, our results could lead to a revision of the general dogma claiming that apoptosis of infected cells and efficient virus production cannot coexist. But how could this happen? One of the characteristics of herpesviruses seems to be that nucleocapsids bud through the nuclear membrane, where they acquire their envelope and tegument

to become mature virions. Viruses belonging to other families complete these last phases of their replicative cycles at the external membrane level. Modifications in the inner nuclear membrane occur only during late stages of apoptosis, as also recently shown (DubandGoulet et al., 1998), while those involving the external membrane occur very rapidly once apoptosis has been triggered. As a consequence, it can be hypothesized that members of the Herpesviridae family selected the peculiar final phase of their replicative cycle to escape, at least in part, the aspecific host-defense mechanisms represented by the triggering of apoptosis in virus-infected cells. Thus, mature, fully infective virions could be released by herpesviruses even from apoptosis-undergoing cells, or eventually, transmitted to apoptotic bodies capturing phagocytes. BHV-4, as well as other ␥-2-herpesviruses, could have selected a replication strategy, in which apoptosis of infected cells at a late stage of infection, which had its protective effect toward the host, was converted into an efficient mechanism of virus spread. The absence of an inflammatory response, which is associated in vivo with cell death by necrosis but not with apoptosis, could provide the virus with a subtle trick to escape host defense and to facilitate its persistence. Relevant to this is the new and interesting finding that exposure to necrotic cells, in addition to presentation of peptides on the cell surface, induces activation in dendritic cells, while exposure to apoptotic cells does not trigger expression of costimulatory molecules in these cells (Sauter et al., 2000). In the absence of costimulatory molecules on their surface, presentation

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of viral antigens by dendritic cells could lead to a selected inactivation of specific T cells rather than to their activation. In conclusion, our results demonstrate a virus inoculum-dependent and time-dependent induction of apoptosis in cells productively infected by BHV-4 in vitro. As a consequence, we identified as “apoptotic infection” that caused by BHV-4 in permissive cell lines. Finally, the availability of the experimental model of apoptotic BHV-4 infection in vitro could also be useful for future identification of viral genes involved in the control of apoptosis by other ␥-2-herpesviruses for which productive infection in vitro represents a serious problem. MATERIALS AND METHODS Viruses, cells, and treatments The LVR 140 reference strain of BHV-4 (kindly provided by Dr. M. Luini) was used in most of the experiments. In some experiments a field isolate, designated as BHV-4/ FI, deriving from a bovine affected by postpartum metritis (characterized and kindly provided by Dr. C. Buonavoglia, University of Bari, Italy), was utilized. Virus stocks were produced by infecting cultures of BS/BEK cells (Ferrari et al., 1991), originally obtained from the Istituto Zooprofilattico Sperimentale (Brescia, Italy), propagated in complete medium (CM) consisting of Eagle’s MEM (HyClone Europe, Cramlington, UK) containing 10% fetal calf serum (FCS; Life Technologies, Gaithersburg, MD), 5 mM Lglutamin, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, at 37°C in a CO 2 incubator. This cell line, as well as the AU-BEK (Rossi and Kiesel, 1973) and the Madin– Darby bovine kidney (MDBK, originally obtained from ATCC, Rockville, MD) cell lines that were used in some experiments, were all characterized by productive infection by BHV-4, and were found to be free of mycoplasma and bovine viral diarrhea virus. After 72 h of infection, cell extracts, obtained by three cycles of freezing and thawing, were pooled, collected, and stored in aliquots at ⫺80°C. The virus-containing cell-extract stocks were successively tested and gave a titer of 1 ⫻ 10 8 PFU/ml in BS/BEK cells 7 days after infection. For experimental infections, 24 h after cultures had been split, cells grown to approximately 80% confluency were inoculated in CM with BHV-4 at different PFU/cell ratios and incubated at 37°C in 5% CO 2. Control cultures were inoculated with equivalent quantities of medium only. Alternatively, for mock infection, target cells were exposed to control cellular extracts collected from uninfected BS/BEK cells, manipulated, and then stored in the same way as those containing the virus. At 1 h p.i., medium containing virus inoculum was removed from the cultures to which fresh CM was then added for the remaining culture time. Uninfected, BHV-4-infected, and mock-infected cells were grown at 37°C in CM. In experiments requiring inactivated virus, virus suspensions were placed in

35-mm petri dishes and, in constant agitation, a volume of 1 ml was exposed for varying times to UV light at an intensity of 30 W from a germicidal lamp situated 19 cm above the sample. Phosphonoacetic acid (PAA; Sigma, St. Louis, MO) was added after removing virus inoculum, together with CM, at doses from 18.7 to 300 ␮g/ml. The higher concentration utilized completely inhibited virus replication, while only slightly affecting cell viability in preliminary experiments. In caspase-inhibition experiments, Z-VAD-FMK and ZDEVD-FMK (Calbiochem, San Diego, CA) cell-permeable peptide analogs of caspase substrates were added to infected and control cultures at concentrations ranging from 12.5 to 100 ␮M. Productive infection was detected by titrating extracellular or total virus from experimental cultures. Virus titers were determined by plaque assay on BS/BEK cells, as previously described for determining titers of virus stocks. In some experiments, necrosis was induced in BS/BEK cells by heat-shock treatment at 60°C for 1 h in a water bath. Evaluation of apoptosis At different times following infection, adherent cells, removed from the culture substrate by treatment with trypsin-EDTA solution, were again mixed with cells previously collected by centrifugation in supernatant from the same flask and resuspended at an adequate concentration in PBS. Thus, the entire cell population of the culture was reconstituted and processed for further analysis. The percentage of apoptotic cells from infected or control cultures was then evaluated using different techniques. Morphological analysis, following staining with acridine orange or Hoechst chromatin dyes, was performed as previously described (Mastino et al., 1997). Briefly, a minimum of 600 cells, including those showing apoptotic characteristics, were counted using a fluorescence microscope (Biomed, Leitz, Wetzlar, Germany). The identification of apoptotic cells was based on the presence of uniformly stained nuclei showing chromatin condensation and nuclear fragmentation. The percentage of apoptotic cells was calculated as follows: % apoptosis ⫽

Total no. of cells with apoptotic nuclei ⫻ 100. Total no. of cells counted

Flow cytometry analysis of isolated nuclei was performed using a method that we recently described for lymphoid cells, which distinguishes apoptosis from necrosis and viability (Matteucci et al., 1999). Briefly, cells collected as previously described were washed twice in PBS in 15-ml polypropylene tubes (Falcon/Becton Dick-

APOPTOSIS IN PERMISSIVE CELLS BY BHV-4

inson Labware, Lincoln Park, NJ). The cell pellet was immediately resuspended gently at room temperature in 1 ml of hypotonic solution consisting of 50 ␮g/ml propidium iodide (Becton Dickinson, Mountain View, CA), 0.1% sodium citrate (Merck, Darmstadt, Germany), and 1% Triton X-100 (Sigma) in distilled water. The tubes were placed at 4°C in the dark overnight. Flow cytometry analysis was subsequently performed using a FACscan flow cytometer (Becton Dickinson). A correct threshold value was experimentally selected to exclude the majority of cell debris. Data collection was gated using adequate values of forward- and side-angle scatter to exclude remaining cell debris and large nuclei aggregates, and to include nuclei from apoptotic, necrotic, and living cells. For each sample 5000 events were acquired. Direct analysis of DNA fragmentation was carried out at the single-cell level using the TUNEL technique. Labeling of DNA strand breaks with fluorescein-dUTP by terminal deoxynucleotidyl transferase was performed using a commercial kit (In situ cell death detection kit, fluorescein; Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. Positive control consisted of cells treated with DNase I (1 mg/ml) for 10 min at room temperature. Analysis of labeled cells was performed by either fluorescence microscopy or flow cytometry. For flow cytometry, labeled cells were acquired and analyzed on a FACScan (Becton Dickinson) using the FACScan program (Becton Dickinson), following adequate gating by scatter parameters to exclude cell debris and large cell aggregates, and to include presumably apoptotic, necrotic, and living cells. For each sample, 5000 events were acquired. Immunofluorescence To analyze Bcl-2 expression by flow cytometry, cells collected as previously described were transferred at 2 ⫻ 10 6 cells/sample to 12 ⫻ 75-mm polystrene tubes (Falcon/Becton Dickinson Labware). Cell pellets were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature in a total volume of 0.5 ml. A volume of 1 ml of 0.1% Triton X-100 in PBS with 0.1% FCS was then added and left for 5 min. After washing twice in PBS with 3% FCS, permeabilized cells were incubated with a fluorescein-conjugated monoclonal antibody against human Bcl-2 (clone 124; Dako, Copenhagen, Denmark) or with an isotype control fluorescein-conjugated antibody of unknown, irrelevant specificity (clone MOPC-21, mouse IgG 1,k; BD, Pharmingen, San Diego, CA) for 30 min on ice and washed twice in PBS with 0.1% sodium azide. Preliminary experiments proved this anti-human Bcl-2 antibody to react with bovine cells. Stained cells were acquired and analyzed on a FACScan (Becton Dickinson) as previously described for the TUNEL technique. For detection of viral proteins by fluorescence microscopy, without simultaneous analysis of apoptosis, after

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washing in PBS, cells were transferred to a multiwell slide for immunofluorescence (about 1 ⫻ 10 4 cells/well; BioMerieux, Marcy l’Etoile, France) and allowed to dry before fixing and permeabilizing for 5 min in acetone at ⫺20°C. Samples were blocked with PBS containing 3% FCS. Staining of cells was then performed in a wet chamber for 1 h at 37°C with a fluorescein-conjugated commercial monoclonal antibody, specific for BHV-4 proteins (dilution, 1:20 in PBS, plus Evans blue solution; Bio-x, Brussels, Belgium). For double-fluorescence microscopy analysis, to detect both virus protein and apoptosis, cells fixed and permeabilized as previously described for the analysis of Bcl-2 expression were stained by the simultaneous addition of the fluorescein-conjugated monoclonal antibody specific for BHV-4 and the DNA-binding dye Hoechst (50 ␮g/ml), without the addition of Evans blue solution. Statistical analysis Data analysis was performed using the SPSS statistical software system (version 6.0 for Windows; SPSS, Chicago, IL). Comparisons of means were carried out using the Duncan test, as a multiple range test with 0.05 significance level. ACKNOWLEDGMENTS We are grateful to Prof. Enrico Garaci for his support, insights, and helpful discussion throughout this work. We thank Alison Inglis, B.A., for her helpful linguistic assistance. These studies were supported by M.U.R.S.T., National Project “Apoptosis-Regulation and Viral Infections,” coordinated by Prof. E. Garaci; by the C.N.R., Special Project “Biology of Aging”; and by the Italian Ministry of Health, AIDS Project.

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