JOURNAL OF VIROLOGY, Jan. 1997, p. 630–639 Vol. 71, No. 1 0022-538X/97/$04.00ϩ0 Copyright ᭧ 1997, American Society for Microbiology

Both Wild-Type and Strongly Attenuated Bovine Viruses Protect Peripheral Blood Mononuclear Cells from Apoptosis FRANCK DEQUIEDT,1 EMMANUEL HANON,2 PIERRE KERKHOFS,3 PAUL-PIERRE PASTORET,2 4 1,5 1 1 DANIEL PORTETELLE, ARSE`NE BURNY, RICHARD KETTMANN, AND LUCAS WILLEMS * Department of Molecular Biology and Animal Physiology1 Faculty of Agronomy, B5030 Gembloux, and Department of Microbiology,4 Department of Immunology/Vaccinology, University of Lie´ge, B43 bis, B4000 Lie`ge,2 National Institute of Veterinary Research, B1120 Uccle,3 and Department of Molecular Biology, University of Brussels, B1640 Rhode-St-Gene`se,5 Belgium

Received 17 June 1996/Accepted 7 October 1996

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.

The survival of multicellular organisms is regulated by a tight cellular genes involved in the control of cell proliferation (14, control of the critical balance between cell proliferation and 23, 36, 42). The Tax proteins have an immortalization potential death (reviewed in reference 44). Apoptosis is an encoded in cell culture and collaborate with the Ha-ras oncogene to suicide program allowing for the elimination of cells that have transform primary cells (16, 30, 43, 50, 51, 55). At least two been produced in excess, developed improperly, or sustained other mRNAs encoding putative proteins are transcribed from genetic damage. Apoptotic cell death involves a characteristic alternative open reading frames located in the region between panel of morphological and biochemical changes including cy- the env and the tax/rex genes (R3 and G4 for BLV; p30, p13, toskeletal disruption, cell shrinkage, and membrane blebbing. and p12 for HTLV) (1, 2, 5, 20). HTLV-1 p12 has been shown Furthermore, the biochemical hallmark of apoptosis is the to cooperate with bovine papillomavirus E5 in cell transforma- endonuclease-mediated degradation of the genomic DNA into tion and to bind to the ␤ and ␥ chains of the interleukin-2 internucleosomal fragments. receptor (13, 26). In the HTLV-2 system, deletion of the se- A number of studies reported that deregulation of apoptosis quences located between the env and tax genes does not affect is an important feature of virus-induced pathogenesis (for re- the expression of viral proteins and the immortalization of views, see references 33 and 44). The viruses have developed a blood lymphocytes in culture (17). However, the precise role of series of strategies to either enhance or inhibit apoptosis. Vi- these proteins in the pathogenesis of the viruses remains to be ruses that cause persistent infections may prevent or delay identified. The use of recombinant BLVs has allowed the iden- normal cellular suicide programs. Among these viruses, mem- tification of genes required for the viral infectivity. The dele- bers of the family Oncovirinae are nonacutely lymphotropic tion of the R3 and G4 genes does not alter the infectious that induce leukemia/lymphoma after long latency potential of the virus but drastically decreases the proviral periods. These viruses include (BLV) loads in vivo (52, 53). This observation suggests a biological and human T-lymphotropic virus types 1 and 2 (HTLV-1 and relevance of these genes in vivo. Similar conclusions were -2), which are similar in genetic organization (34, 37). In ad- obtained after infection of recombinant HTLV-2 proviruses in dition to the classical gag, pol, and env retroviral genes, they rabbits (6). The pathologic effects induced by these recombi- contain an X region coding for a number of regulatory genes nants remain unknown. (37, 39). This region contains two genes, tax and rex, that are Because of the lack of an appropriate animal system, it has involved in transcriptional and posttranscriptional regulation been difficult to understand the role of HTLV-1 in the apo- of viral expression (4, 9, 10, 41, 48). BLV Tax and HTLV-1 Tax ptotic process. Depending on the experimental conditions, are nuclear proteins that transactivate long terminal repeat- contradictory conclusions have been obtained in in vitro cell directed gene expression (9, 12, 40, 48). In addition, the Tax culture. The HTLV-1 Tax protein was initially described as an protein from HTLV-1 activates of a number of inducer of apoptosis in murine fibroblasts (54). Indeed, Tax- transformed Rat-1 cells undergo apoptotic cell death after serum deprivation. This Tax-mediated apoptosis is blocked by * Corresponding author. Mailing address: Avenue Mare´chal Juin, the coexpression of the bcl-2 proto-oncogene. In contrast, no. 13, B5030, Gembloux, Belgium. Phone: 32-81-62-21-57. Fax: 32- transfection of the tax gene into Jurkat T cells reduced their 81-61-38-88. susceptibility to anti-APO-1-induced apoptosis (7). Further-

630 VOL. 71, 1997 PROTECTION BY BLV FROM APOPTOSIS 631 more, when Jurkat cells were treated with soluble Tax protein, amplified by 22 cycles (30 s at 94ЊC,30sat60ЊC, and 1 min at 72ЊC). Ten the proportion of cells undergoing apoptosis decreased more microliters of the amplification products were migrated on a 1% agarose gel. After denaturation, the DNA was transferred to a Hybond Nϩ membrane than twofold. This reduction in the apoptotic levels was less (Amersham) and hybridized with a BLV probe (EcoRI insert from plasmid pronounced when primary human lymphocytes were used. The pSGX3A). This plasmid contains the viral sequences (positions 6755 to 7202 specificity of these assays was supported by the seroneutraliza- according to reference 36) derived from a cDNA isolated from the BLV-infected tion of Tax. Both experimental protocols lack an animal model FLK cell line. BLV p24 titration. After 40 h of culture, 2 ϫ 106 PBMCs were harvested by for HTLV to support the biological relevance of their conclu- centrifugation (800 ϫ g, 10 min) and resuspended in 100 ␮l of PBS. The p24 sions. To date, the closest model system where a leukemia major Gag antigen was then titrated from the cells by using an ELISA procedure occurs in vivo is provided by the experimental infection of as previously described (28, 29). Briefly, 96-well microtiter plates were precoated sheep by BLV. We therefore propose to analyze the apoptotic with the anti-p24 monoclonal antibody (MAb) 4ЈG9. The antigen mixtures to be tested were then added to the wells. After washing of the plates, the p24 antigen process in this animal model. was revealed by colorimetric assay using two antibodies (2ЈC1 and 4ЈF5) conju- gated with peroxidase. MAbs and immunophenotyping. As a B-cell marker, we used the anti-bovine MATERIALS AND METHODS immunoglobulin M (IgM) MAb 1H4 (kindly provided by J. J. Letesson, Univer- Peripheral blood mononuclear cell (PBMC) isolations and cultures. Sheep site´ Notre Dame de la Paix, Namur, Belgium), which cross-reacts with ovine infected with the recombinant and wild-type (WT) viruses were previously de- surface (sIgM). The IgM ST8 and IgG1 ST4 MAbs (provided by I. Schwartz, scribed (52, 53). Sheep 234 and 235, sheep 247 and 261, and sheep 265 were URA-INRA, Maisons-Alfort, France) define the sheep CD8 and CD4 homologs, infected with proviruses pBLV344, pBLVIX, and pBLVX3C, respectively. These respectively. Fluorescein isothiocyanate (FITC)-conjugated F(abЈ)2 goat anti- three BLVs are similar in terms of infectivity, replication, and pathogenesis in mouse Ig and phycoerythrin (PE)-conjugated F(abЈ)2 goat anti-mouse Ig were vivo. Sheep 237, 240, and 272 were initially transfected with the pBLVIG4 purchased from DAKO (Gentbrugge, Belgium). For immunophenotyping, MAbs 1H4, ST4, and ST8 were diluted in PBS with provirus, which contains a translational stop codon in the G4 gene (genotype 6 R3ϩ G4Ϫ Taxϩ Rexϩ). The pBLVDX provirus, which harbors deletions of both 10% FCS (PBS-FCS). After their isolation, 2 ϫ 10 PBMCs were incubated for the R3 and G4 genes, was used to infect sheep 245, 246, and 263 (genotype R3Ϫ 30 min at 4ЊCin100␮l of each primary MAb, washed twice in PBS-FCS, and G4Ϫ Taxϩ Rexϩ). In addition, uninfected sheep (sheep 291, 294, 295, 296, 430, incubated in 100 ␮l of FITC-conjugated antiserum (dilution 1:100 in PBS-FCS). and 441) were used as controls. All infected sheep were 4 or 5 years old. The After two washes in PBS-FCS, the cells were resuspended in PBS and analyzed uninfected sheep 294, 295, and 296 were 1 year old, while sheep 430 and 441 were by flow cytometry (fluorescence-activated cell sorting [FACS]) for green fluo- 3 years old. Except for sheep 430 and 441, which were kept in Gembloux, all rescence (FITC, relative antigen expression). sheep were maintained under controlled conditions at the National Institute for In situ detection of apoptosis. An In situ Cell Death Detection Kit, Fluores- Veterinary Research (Uccle, Belgium). At regular intervals of time, the total cein (Boehringer Mannheim) was used to measure apoptosis in individual cells. lymphocyte counts were determined with a model ZN Coulter Counter. Sera The test uses TdT to label free 3Ј-OH ends in genomic DNA with fluorescein- were collected and analyzed for BLV seropositivity by immunodiffusion and dUTP (15). This assay was performed directly on cells after culture or after the indirect gp51 enzyme-linked immunosorbent assay (ELISA) (28, 29). cells had been first labelled with MAb 1H4 and a PE-coupled conjugate. Prior to PBMCs were isolated essentially as described previously (11). In brief, blood the labelling reaction, the cells were washed, fixed in 1% paraformaldehyde for samples were collected by jugular venipuncture, mixed with EDTA, and centri- 15 min at 4ЊC, washed twice, and finally fixed in 70% ethanol at Ϫ20ЊC for 30 fuged at 1,750 ϫ g for 15 min at room temperature. The PBMCs were then min. The labelling was performed essentially according to the manufacturer’s isolated by Percoll gradient centrifugation (60% solution; specific gravity, 1.075; protocol. The fluorescence of individual cells was measured by flow cytometry Pharmacia) and washed twice in phosphate-buffered saline (PBS) containing (FACStar Plus; Becton Dickinson). 0.075% EDTA, with centrifugation steps at 450 ϫ g for 7 min at 10ЊC. The cells For simultaneous detection of apoptosis in infected and uninfected cells, the were washed with PBS (centrifugations at 200 ϫ g for 7 min at 10ЊC) until the protocol was slightly modified as described by Gorczyca et al. (15). After 22 h of supernatant became clear. The PBMCs were then tested for viability by trypan culture, the cells were fixed in paraformaldehyde and ethanol as previously blue exclusion and cultivated at 2 ϫ 106 cells/ml/tube in RPMI 1640 medium described. Incorporation of biotin-dUTP was performed at 37ЊCfor1hin50␮l (Gibco) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine of appropriate buffer (Boehringer Mannheim) containing 10 U of TdT, 1 mM (Gibco), 100 UI of penicillin (Gibco) per ml, and 100 ␮g of streptomycin (Gibco) CoCl2, 0.5 nmol of biotin-dUTP, and 20 ␮M dATP. The cells were then washed per ml. once in PBS-FCS and once in PBS alone. The cells were next incubated for 30 DNA ladder assays. After 22 h of culture, the DNA was extracted essentially min at 4ЊC in the presence of PE-conjugated avidin. Finally, the cells were rinsed as described by Jeurissen et al. (19), with slight modifications. Briefly, the PBMCs in PBS-FCS, labelled with anti-p24 MAb 4ЈG9 and a fluorescein-coupled con- were lysed in DNA extraction buffer (20 mM Tris-HCl [pH 7.5], 2 mM EDTA, jugate, and analyzed by flow cytometry. 0.2% sodium dodecyl sulfate, 0.6 mg of proteinase K per ml) and incubated for DNA fragmentation was also revealed by performing DNA content analysis 1hat37ЊC. The samples were extracted with phenol followed by phenol- (see reference 8 for a review). Cultured PBMCs were collected, washed twice in chloroform-isoamyl alcohol (25:24:1). After ethanol precipitation, the DNA was PBS, and fixed in 70% ethanol at Ϫ20ЊC for 30 min. The cells were then washed resuspended in TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) containing in PBS, treated with RNase (50 ␮g/ml; Sigma) for 20 min at 37ЊC, and incubated RNase (0.6 mg/ml) and incubated for 15 min at room temperature. The DNA for 5 min at room temperature in 50 ␮g of propidium iodide (PI) per ml. The was then directly resolved on a 1.5% agarose gel and visualized by ethidium cells were analyzed for red fluorescence (PI, relative DNA content) by flow bromide. cytometry. After cell sorting, the DNA corresponding to 20,000 cells was isolated as Flow cytometry. Flow cytometric analysis was performed with a Becton Dick- described above except that the DNA was extracted with phenol-chloroform and inson fluorescence-activated cell sorter (FACStar Plus) equipped with an argon precipitated with ethanol after the digestion with RNase. DNA fragmentation laser (ILT air cooled with 100-mW excitation lines at 488 nm). Debris were was detected by using terminal deoxynucleotidyltransferase (TdT) essentially as excluded from the analysis by the conventional scatter gating method. The cells described by Gorczyca et al. (15). The DNA strand breaks were end labelled with or the nuclei doublets were excluded from the analysis by using the pulse 7.5 U of TdT (Boehringer Mannheim) in 20 ␮l of appropriate buffer (0.2 M processor boards (Becton Dickinson). The FITC, PE, and PI emission signals potassium cacodylate, 25 mM Tris-HCl, 1 mM cobalt chloride, 0.25 mg of bovine were collected by using appropriate filters at 530 nm (band pass 30), 575 nm serum albumin per ml) containing 0.4 mmol of [␣-32P]ddATP (5,000 Ci/mmol; (band pass 26), and 630 nm (band pass 22), respectively. Ten thousand events per Amersham). Samples were labelled for1hat37ЊC, extracted with phenol- sample were collected in a list mode, stored, and analyzed by the Consort 32 chloroform, and precipitated with ethanol and ammonium acetate. DNA was system (Becton Dickinson). resuspended in TE buffer and loaded on a 1.5% agarose gel. After electrophore- sis, the gel was dried under vacuum at 80ЊC and autoradiographed. PCR analysis. Blood samples (500-␮l aliquots) were mixed with an equal RESULTS volume of lysis buffer (0.32 M sucrose, 10 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1% Triton X-100). After 20 s of centrifugation, the pellets were resuspended in WT BLV protects sheep PBMCs from spontaneous apopto- 1 ml of lysis buffer. This step was repeated twice. The samples were then sis. Many types of animal cells require signals from other cells resuspended in 500 ␮l of PCR buffer (50 mM KCl, 1.5 mM MgCl2,10mM to survive (3, 31). Therefore, lymphocytes would quickly acti- Tris-HCl [pH 8.3]) and incubated with 6 ␮l of proteinase K (5 mg/ml) for1hat 50ЊC. The digestions were stopped by boiling the samples for 5 min. Twenty- vate their intrinsic suicide program if isolated from the blood- microliter aliquots were amplified by PCR in the presence of 0.2 mM each stream and cultured. Our experimental protocol is based on deoxynucleotide, 200 ng of primers, and1UofTaq DNA polymerase (Boehr- the analysis of this programmed cell death in PBMCs from inger Mannheim). Two oligonucleotides were used: A (position 6450 [34]; 5Ј-T animals chronically infected by BLV. Therefore, blood was GGAAAGAACTAACGCTGACGG-3Ј) and B (position 7200 [34]; 5Ј-ACGGT GGGTCTCGCAGGTGAGCGTGGTGTCGATCC-3Ј). The 50-␮l reactions collected by jugular venipuncture of BLV-infected sheep 234, mixtures were overlaid with mineral oil, denatured for 5 min at 95ЊC, and 261, and 265. PBMCs were isolated by Percoll gradient cen- 632 DEQUIEDT ET AL. J. VIROL.

FIG. 1. Detection of apoptosis in PBMCs culture from WT BLV-infected sheep 234, 261, and 265 and uninfected (NI) control sheep 294, 295, and 296. (A) After 40 h of culture, PBMCs were successively fixed in paraformaldehyde and ethanol, and the DNA strand breaks were labelled by the TUNEL procedure. The incorporation of fluorescein-dUTP was detected by flow cytometric analysis. The percentages of apoptotic cells are indicated on each FACS contour plot. (B) After 40 h of culture, PBMCs were fixed in ethanol and stained with PI. The relative DNA content of the cells was then analyzed by flow cytometry. The percentages on each histogram correspond to the proportion of apoptotic cells (Ap) or to cells in the SϩG2/M phase of the cycle. (C) Internucleosomal fragmentation of DNA by agarose gel electrophoresis. After 22 h of cultivation, DNA was extracted, electrophoresed through a 1.5% agarose gel, and visualized with ethidium bromide. The samples correspond to uninfected (NI) sheep 294 (lane 1) and 295 (lane 2) and to WT BLV-infected sheep 234 (lane 3) and 261 (lane 4). trifugation and cultured for 40 h. As a control, uninfected respectively). These values were extremely stable among two PBMCs were also collected from three animals that were se- independent experiments: the standard variations ranged be- ronegative for BLV (sheep 294, 295, and 296). After culture, tween 1.4% and 4.2%. These experiments thus indicate that the cells were harvested, fixed in paraformaldehyde and etha- the proportion of apoptotic cells in PBMCs from infected nol, and analyzed for programmed cell death by the TdT- sheep is about half that in PBMCs from BLV-free animals. mediated fluorescein-dUTP nick end labeling (TUNEL) pro- These analyses could, however, be altered by the spontane- cedure. This protocol is based on the incorporation of ous multiplication of the cells during the culture. Indeed, the fluorescein-dUTP by the TdT on the free 3Ј ends generated infected PBMCs appeared to enlarge after culture. Therefore, after the degradation of DNA in the apoptotic cells. This the cells were counted at the beginning and at 40 h of culture. procedure thus reflects the level of internucleosomal DNA Under our culture conditions, the total number of cells (2 fragmentation that is a major characteristic of apoptosis. The million per ml) did not change with time (for culture times up cell samples were next analyzed by flow cytometry in order to to 40 h). In contrast, the proportion of dead cells, as revealed estimate the proportion of cells undergoing apoptosis (Fig. by blue trypan exclusion, increased during the culture. These 1A). After 40 h of culture, the majority of the uninfected results paralleled the percentages obtained by the TUNEL PBMCs had incorporated the fluorescein marker (68, 71, and procedure (data not shown). 61% for sheep 294, 295, and 296, respectively). This observa- To confirm the occurrence of apoptosis, we performed an tion is not surprising since the cells from most organs will analysis of the relative DNA content at 40 h of cultivation. undergo apoptosis if cultured individually in the absence of Ethanol-fixed PBMCs were stained with PI and analyzed by specific survival factors (reviewed in reference 31). In contrast, flow cytometry to evaluate the proportion of the cells within only about half of these apoptotic rates were found in PBMCs the different phases of the (Fig. 1B). Very few cells infected by BLV (36, 34, and 29% for sheep 234, 261, and 265, (less than 13%) were present in the S or G2/M phase of the VOL. 71, 1997 PROTECTION BY BLV FROM APOPTOSIS 633 cycle, supporting our previous observations concerning the lack of cellular division during the cell culture (Fig. 1B, SϩG2/ M). A major peak was observed at a relative DNA content of

400, corresponding to the G0/G1 phase of the cell cycle. A second main fraction of the cells contained a smaller relative

DNA content and appeared as a sub-G0/G1 peak. The appear- ance of such cells with lower DNA stainability is a marker of cell death by apoptosis (Fig. 1B, Ap). The proportion of cells in the sub-G0/G1 peak was much lower in BLV-infected than in uninfected PBMCs (41, 41, and 28% versus 53, 60, and 54%). The analysis of the relative DNA content thus correlated with the data obtained by the TUNEL protocol. A third parameter characteristic of cells undergoing apopto- sis is the regular DNA fragmentation between the nucleo- somes. This internucleosomal cleavage of the genomic DNA is a characteristic biochemical marker of apoptosis. These breaks generate a typical DNA ladder after gel electrophoresis. Therefore, DNA was isolated from short-term-cultured PB- MCs and migrated on an agarose gel. A typical DNA ladder indeed appeared, demonstrating the apoptotic process in both infected and uninfected PBMCs (Fig. 1C). The level of DNA fragmentation was, however, reduced in BLV-infected cells (compare lanes 3 and 4 with lanes 1 and 2). This result thus supported the data obtained in situ by the quantitative TUNEL protocol and by the DNA content analysis (see above). Altogether, these data demonstrate that under the experi- mental conditions used, PBMCs from BLV-infected sheep are less prone to undergo apoptosis than their counterparts from BLV-free animals. BLV-infected cells in an infected animal are not prone to apoptosis. In vivo, the main target cell for BLV is the B lym- phocyte (25, 27, 38). To analyze the apoptotic process within B lymphocytes, the cells were first labelled with antibody 1H4, which recognizes sIgMs, and with a PE-coupled conjugate. The cells were then fixed and analyzed for programmed cell death by the TUNEL procedure (Fig. 2A). In control uninfected FIG. 2. Detection of apoptosis in B lymphocytes from uninfected (NI) sheep sheep 291, 294, and 295, the proportion of B cells undergoing 291, 294, and 295 and WT BLV-infected sheep 234, 261, 265. PBMCs were apoptosis after culture ranged from 70 to 79% (Fig. 2B). In- cultivated for 22 h and labelled with antibody 1H4 (directed against sIgM) and deed, no more than 30% of the B lymphocytes survive even at a PE-coupled conjugate. The cells were then fixed, and the DNA strand breaks 22 h of culture (21, 30, and 25% for sheep 291, 294, and 295, were labelled with fluorescein-dUTP by the TUNEL procedure. (A) Bivariate flow cytometric analyses of the labelled cells presented as dot plots. On the basis respectively). In contrast, the majority of the B lymphocytes of control staining, each distribution was separated into four quadrants. The isolated from BLV-infected sheep did not undergo apoptosis numbers indicate the percentages of positively stained PBMCs in each quad- (74, 84, and 83% for sheep 234, 261, and 265, respectively). rants. (B) Percentages of apoptotic (solid bars) and nonapoptotic (hatched bars) These data indicate that most of the B lymphocytes isolated cells within the B-lymphocyte subset. The percentages were calculated from the from uninfected sheep were prone to spontaneous apoptosis. results presented in panel A. On the other hand, most of the B cells from BLV-infected sheep appeared to be protected against programmed cell death. grated on an agarose gel (Fig. 3). In agreement with the data The blood cells undergo constant renewal from hematopoi- mentioned above, DNA fragmentation was detected in the etic progenitors. This also implies a permanent destruction of total population. Furthermore, the presence of a DNA ladder the B lymphocytes. Among this population, a fraction is in- pattern revealed massive cell death in the noninfected cells fected by the virus. We hypothesized that BLV could have (lane VϪ). By contrast, the cells expressing the p24 major developed a strategy to interfere with this process of cell de- capsid protein were almost completely protected from apopto- struction. To gain insight into a possible direct role of the virus sis (lane Vϩ). Only a faint ladder was observed in the infected in the protection against apoptosis, the infected cells were cells isolated from one of the three sheep (sheep 234). sorted by flow cytometry as follows. PBMCs were isolated from These data demonstrate that most (if not all) of the cells that three infected sheep (234, 261, and 265) and cultivated for undergo apoptosis are not infected by BLV. In contrast, the 22 h. The cells were then fixed with paraformaldehyde and presence of the virus within a lymphocyte drastically decreases, ethanol and incubated in the presence of MAb 4ЈG9, directed if not abolishes, the programmed cell death. This observation toward the major viral capsid protein p24. After coupling to an assigns a direct role to the virus that enables its host cell to FITC-conjugated secondary antiserum, the infected, uninfect- survive the stress conditions after the isolation and cultivation ed, and total cells were sorted by flow cytometry. The quality of of PBMCs. the sorting procedure was checked by a subsequent flow cyto- Even strongly attenuated BLVs protect PBMCs from apo- metric analysis of the three populations (the purities of the ptosis. We have previously described the construction of re- sorted cells ranged from 95 to 99%). The DNA was extracted combinant BLVs with deletions of the R3 and G4 genes (53). from 20,000 sorted cells, labelled with [␣-32P]ddATP, and mi- These two genes, whose functions are still unknown, corre- 634 DEQUIEDT ET AL. J. VIROL.

ptosis, we analyzed the programmed cell death in sheep PB- MCs infected with mutant proviruses as follows. Blood was collected by jugular venipuncture of sheep chronically infected by recombinant viruses (DX mutants) with deletions of both the R3 and G4 genes (sheep 245, 246, and 263) or only the G4 gene (G4 mutants) (sheep 237, 240, and 272). PBMCs were isolated by Percoll gradient centrifugation and cultured for 1, 12, 22, or 40 h. As controls, uninfected PBMCs were also collected from three animals that were seronegative for BLV infection (sheep 294, 295, and 296) and from three sheep infected with WT provirus (sheep 234, 261, and 265). After culture, the cells were harvested, fixed in paraformaldehyde and ethanol, and labelled for programmed cell death by the TUNEL procedure. The cells were next analyzed by flow cy- tometry in order to estimate the proportion of cells undergoing apoptosis (Table 1). After1hofcultivation, the cell mortalities were low (around 10%) and did not significantly differ among the different samples. Within 12 h, the percentages of dead cells greatly increased both in the PBMCs from uninfected FIG. 3. Detection of DNA fragmentation in BLV-infected cells. PBMCs from BLV-infected sheep 234, 261, and 265 were cultivated for 22 h and fixed in animals (294, 295, and 296) and in cells isolated from BLV- paraformaldehyde and ethanol. The cells were then stained with MAb 4ЈG9 infected sheep. After 12 h, a marked difference in the apopto- (directed against the viral p24 protein) and coupled to an FITC-conjugated tic rates between the infected and noninfected PBMCs cultures antiserum. Twenty thousand cells from BLV-infected (Vϩ), uninfected (VϪ), (24 to 38% and 42 to 54% of apoptotic cells, respectively) was and total (T) PBMCs were then sorted by flow cytometry. The DNA was ex- tracted from the three cell populations, end labelled with [␣-32P]ddATP, elec- observed. The apoptosis analysis at 22 and 40 h extended these trophoresed through a 1.5% agarose gel, and autoradiographed. observations and confirmed our previous results (compare re- sults in Table 1 with those in Fig. 1). We next calculated the mean apoptotic rates accordingly to the kind of virus (Fig. 4A). spond to the HTLV p12 and p30 sequences. In cell culture, the Surprisingly, after 40 h of cultivation, the proportions of dead deletion of these open reading frames does not affect the cells were similar in the PBMCs isolated from animals infected expression of BLV or HTLV (35, 52). Furthermore, primary with WT and mutant (G4 and DX) recombinant proviruses cells can be transformed by a recombinant HTLV-2 provirus (Fig. 4). Since we have previously reported that viral loads are that has been deleted in these frames (17). However, these drastically decreased in animals infected by the recombinant genes appear to have a biological function since as in the BLV virus (53), one could have expected to obtain values similar to system, their deletion drastically decreases the proviral loads in those for the uninfected PBMCs. Moreover, there was no vivo (53). The propagation of the recombinant virus within its significant difference in the kinetics of apoptosis between the host thus appears affected in vivo. WT and mutant BLV-infected PBMCs (Fig. 4). In fact, most of To investigate the effect of viral load in the process of apo- the apoptotic cells from WT-, G4-, or DX-infected sheep were

TABLE 1. Kinetic analysis of the apoptotic levels in PBMCs from mutant BLV-infected sheepa

Positive PBMC (%) before d c % Apoptotic cells (mean Ϯ SD) Date of Lymphocytes cultivation Sheep 3 b seroconversion (mm ) 1H4 ST4/ST8 1 h 12 h 22 h 40 h (mean Ϯ SD) WT infected 234 11/91 6,333 85 Ϯ 7.1 12 19 38 Ϯ 4.2 41.5 Ϯ 6.4 36 261 10/92 7,006 82.5 Ϯ 3.5 14 4 32.5 Ϯ 2.1 35.5 Ϯ 3.5 35 Ϯ 1.4 265 10/92 3,305 49.5 Ϯ 7.8 39 7 24.5 Ϯ 2.1 26 Ϯ 4.2 32 Ϯ 4.2 G4 infected 237 4/93 2,614 31.5 Ϯ 4.9 52 8 36.5 Ϯ 3.5 41 Ϯ 4.2 41.5 Ϯ 3.5 240 6/93 1,820 28.5 Ϯ 6.4 60 7 26 Ϯ 1.4 32 Ϯ 1.4 38.5 Ϯ 0.7 272 11/93 2,714 24.5 Ϯ 4.9 22 6 30 Ϯ 1.4 32 Ϯ 2.8 31.5 Ϯ 2.1 DX infected 245 11/93 3,593 39.5 Ϯ 9.2 25 11 37 Ϯ 5.6 40 Ϯ 7.1 42 Ϯ 4.2 246 5/92 5,637 26 Ϯ 1.4 28 4 34 Ϯ 2.8 35.5 Ϯ 4.9 33 Ϯ 2.8 263 10/92 2,617 36.5 Ϯ 3.5 56 8 33 Ϯ 0 36.5 Ϯ 3.5 40 Ϯ 2.8 Not infected 294 3,882 41 Ϯ 7.1 34 11 54.5 Ϯ 2.1 61.5 Ϯ 4.2 69 Ϯ 1.4 295 4,912 45.5 Ϯ 4.9 31 9 52.5 Ϯ 2.1 60 Ϯ 069Ϯ2.8 296 3,679 NDe ND ND 42 52 61

a PBMCs from sheep infected with attenuated G4 and DX proviruses were cultivated for up to 40 h. PBMCs from uninfected sheep or WT BLV-infected sheep were used as controls. b Determined with a Coulter Counter. c Determined from flow cytometric analysis of 10,000 cells. d Measured by the TUNEL procedure and flow cytometry at 1, 12, 22, and 40 h of cultivation. e ND, not determined. VOL. 71, 1997 PROTECTION BY BLV FROM APOPTOSIS 635

lymphocytosis stage of the disease (with lymphocyte counts above 10,000 up to 300,000). We next evaluated the relative number of cells within the lymphocyte populations. The per- centages of sIgMϩ and CD4ϩ CD8ϩ cells were determined by flow cytometry using antibodies 1H4 and ST4/ST8, respec- tively. Animals 234 and 261 exhibited a marked increase in the proportion of sIgMϩ B cells and a reduction of the CD4ϩ CD8ϩ T-cell population. The other BLV-infected sheep in- fected with WT or mutant BLV provirus exhibited no obvious hematological perturbations when compared to uninfected an- imals. It thus appears that the lower ex vivo apoptotic rate ob- served in the PBMC cultures from BLV-infected sheep does not correlate with changes in the relative proportions of the sIgMϩ B lymphocytes and the CD4ϩ CD8ϩ T cells. Moreover, despite significant differences in the absolute and the relative cell numbers, PBMCs infected with WT or recombinant virus yielded comparable apoptotic rates. To correlate the apoptotic rates to the viral expression, we next measured the synthesis of the major capsid antigen in the cultured PBMCs (Fig. 5). The levels of p24 antigen were mea- sured at 40 h of cultivation by an ELISA procedure (28, 29). It appeared that the amounts of p24 protein were much lower in the samples corresponding to the R3-G4 recombinants (G4 and DX) than in those corresponding to WT virus. As controls for background levels, no p24 antigen was detected in cells from uninfected sheep. These observations confirm our previ- ous report (53) and further demonstrate that the differences in the levels of apoptosis are not correlated to the amounts of p24 protein titrated from the cultivated cells. Finally, the proviral loads were measured by semiquantita- tive PCR. Aliquots of blood were collected in parallel to those used for the analysis of apoptosis. The genomic DNA was extracted, and the sequences corresponding to the viral X FIG. 4. Kinetic analysis of the apoptotic levels in PBMCs from mutant BLV- region were amplified by PCR. These samples were then ana- infected sheep. PBMCs from sheep infected with attenuated G4 (sheep 237, 240, lyzed by Southern blot hybridization using a BLV X probe and 272) and DX (sheep 245, 246, and 263) proviruses were cultivated for up to 40 h. PBMCs from uninfected (NI) sheep 294, 295, and 296 or WT BLV-infected (Fig. 6). The PCR conditions were determined in order to sheep 234, 261, and 265 were used as controls. The percentages of apoptotic cells compare the relative amounts of proviral sequences in the measured by the TUNEL procedure and flow cytometry analysis are from Table blood samples. Therefore, the number of cycles was restricted 1. (A) Mean values according to the type of virus were calculated from the to 22. Under these conditions, the PCR-hybridization analysis apoptotic rates presented in Table 1. These mean values (uninfected [ଙ] and WT [F], G4 [E], or R3 [✳]-infected cells) are schematically represented in a kinetic yielded semiquantitative results, as demonstrated by the am- analysis after 1, 12, 22, and 40 h of cultivation. (B) The increase of the mean plification of serial dilutions from WT proviral DNA (1/1 to apoptotic rates from uninfected (ଙ), G4-infected (E), and R3-infected (✳) cells 1/1,000 from sheep 261 DNA). As controls for PCR contami- was calculated in comparison with the data obtained with the WT virus and nations, no signal was observed in the uninfected sheep DNAs schematically represented at 1, 12, 22, and 40 h of cultivation. dead at 12 h of cultivation. On the other hand, the apoptotic rates corresponding to uninfected animal still increased be- tween 12 and 40 h (Fig. 4B). These data were confirmed by relative DNA content analysis at 40 h (data not shown). To confirm our observations, we performed a series of con- trol experiments. First, the lower apoptotic rates observed in the PBMC cultures from BLV-infected sheep (either WT or mutant) could change proportionally to the number of the sIgMϩ B cells. Indeed, after extended latency periods (up to several years), the virus induces hematological disorders in the infected animals. A major perturbation is a higher number of sIgMϩ B cells and a depletion of the T-cell population. It is thus important to correlate the differences in the apoptotic rates between BLV-infected and noninfected sheep to the rel- ative numbers within the lymphocyte subsets. Therefore, blood was collected by jugular venipuncture of the sheep used in the FIG. 5. Ex vivo expression of BLV p24 in PBMC cultures. The p24 major experiment described above. Cell populations were then de- Gag antigen was titrated from the cells by an ELISA procedure after 40 h of termined by examination under a light microscope (Table 1). cultivation. OD ϫ dil. values are equivalent ELISA optical densities (OD) according to the antigen dilutions (dil.). PBMCs from uninfected (NI) sheep and The highest numbers were obtained for BLV-infected sheep from animals infected with WT virus and G4 or R3 mutant virus were analyzed 234 and 261, but none of the BLV-infected sheep was in the for viral expression. 636 DEQUIEDT ET AL. J. VIROL.

onstrating that infected cells within the PBMC population do not undergo apoptosis. Indeed, only a minority (if any) of the infected cells were prone to apoptotic death (2 to 4% versus 27 to 40% [Fig. 7, WT]). The analysis of PBMCs infected with G4 (sheep 272) and DX (sheep 245) mutant proviruses yielded similar values, since only 2% of the PBMCs were plotted as double positives. These results confirm that BLV protects the infected cell from apoptotic death. Furthermore, the deletion of G4 and/or R3 genes does not impede the mechanism of direct protection conferred by the virus to its host cell. Culture supernatants from infected cells protect uninfected FIG. 6. Semiquantitative PCR-hybridization analysis of viral sequences in blood samples from infected sheep. DNA was extracted from 500-␮l aliquots of PBMCs from apoptosis. Our data demonstrate that about 30% blood and amplified by 22 cycles of PCR. The PCR products were then analyzed of the total number of cells were protected from programmed by Southern blot hybridization using a BLV probe. Blood samples from unin- cell death in the presence of either WT or mutant virus (Fig. fected (NI) sheep were used as controls for PCR contaminations. The semiquan- 4B). Since very few cells are infected by the mutant viruses in titative analysis was supported by the amplification of serial dilutions from WT proviral DNA (1/1 to 1/1,000 from sheep 261). The size of the amplification vivo (Fig. 6 and 7), we hypothesized that the viruses also product obtained with the DX virus-infected sheep is 360 bp, compared with 750 indirectly protect uninfected cells in the animals. To test this bp for that obtained with the WT- and G4-infected animals. hypothesis, PBMCs were isolated from sheep infected with the WT or DX mutant virus (respectively from sheep 265 and 245). After 22 h of culture to allow viral expression, the supernatants (294, 295, and 296). We should mention here that the oligo- were recovered by centrifugation, diluted 10- or 100-fold, and nucleotides used for the PCR amplifications surround the re- added to PBMCs isolated from two uninfected animals (sheep gion that has been deleted in the R3- and G4-minus DX 430 and 441). The apoptotic rates were then determined by the mutant. This explains why the size of the amplification product TUNEL procedure (Fig. 8). As a control, we used culture is reduced in the samples corresponding to sheep 245, 246, and supernatants from uninfected PBMCs (isolated from sheep 263 (360 bp versus 750 bp). The PCR amplification using the 291). Under these conditions, about half (49%) of the unin- genomic DNA from sheep infected with the WT virus revealed fected cells from sheep 430 underwent apoptosis at 42 h (Fig. similar amounts of viral sequences (lanes 234, 261, and 265). In 8A). When the cells were cultivated in 10-fold-diluted super- contrast, a very weak signal (less than 1,000-fold) was obtained natants from WT-infected cells, the apoptotic rates decreased for genomic DNA from sheep infected with the DX and G4 to 31%. This finding demonstrates that supernatants from WT mutant viruses (sheep 237, 240, 245, 246, and 263). The highest virus-infected cultures can indirectly rescue uninfected cells signal corresponding to the recombinant viruses was obtained from programmed cell death (protection rate of 18% [Fig. 8B, with DNA from sheep 272. However, the intensity of this signal sheep 430]). As one could have expected considering our pre- was still 100-fold weaker than that obtained with animals in- vious results (Fig. 4), a similar protection rate was obtained fected with the WT virus. These results confirm our previous with a supernatant from DX virus-infected cells (18% protec- report on the attenuation of the R3-G4 recombinant viruses tion [Fig. 8B, sheep 430]). An efficient protection (11 and 13% (53) and demonstrate that the deletion of the R3 and G4 genes for the DX and WT viruses, respectively) was observed even does not affect the global protection from apoptosis in the when supernatants were diluted up to 100-fold (Fig. 8B, sheep infected PBMCs. Finally, since the proviral loads differ drasti- 430). All of these experiments were repeated with uninfected cally among different sheep yielding similar apoptotic rates, PBMCs isolated from sheep 441; in this case, the protection one can conclude that the global level of programmed cell rates were even higher, ranging between 20 and 28% (Fig. 8B). death is independent of the amount of provirus in the animals. These results clearly demonstrate that supernatants from The R3- and G4-deleted mutant proviruses directly protect BLV-infected cells can indirectly rescue uninfected cells from the infected cells from apoptosis. The presence of WT BLV programmed cell death. Furthermore, the R3 and G4 genes do within a cell is able to prevent its apoptosis (Fig. 3). To gain not appear to play a major role in this process since their insight into a possible role of the R3 and G4 genes in this direct absence in the mutated viruses does not affect this protection mechanism, we analyzed the apoptotic process in cells infected against apoptosis. with mutant proviruses. Blood samples were collected from sheep 245 and 272, which were infected with a DX mutant DISCUSSION virus and a G4 mutant virus, respectively. As controls, PBMCs were also collected from three sheep infected with WT provi- Most cells are programmed to commit suicide if they do not rus (235, 247, and 265) and from one uninfected animal (sheep receive specific signals from their environment (3, 31). There- 294). PBMCs were isolated by Percoll gradient centrifugation fore, lymphocytes would quickly undergo apoptosis when iso- and cultured for 22 h. After culture, the cells were fixed in lated from the blood and cultured individually without mito- paraformaldehyde and ethanol, and DNA strand breaks were genic activation. The modulation of this programmed cell labelled with biotin-dUTP by using TdT. The incorporation of death is a hallmark of virus-induced pathogenesis. Our data biotin-dUTP was then revealed by PE-coupled avidin. Finally, show that BLV, a member of the Oncovirinae subfamily of the cells were incubated with MAb 4ЈG9, which recognizes the retroviruses, decreases the apoptotic process within the host BLV capsid protein p24, and a fluorescein-coupled conjugate. cell. The presence of the virus within a lymphocyte drastically Flow cytometric analysis of these cells thus allows the detection decreases, if not abolishes, the programmed death of the cell of apoptosis concomitantly in the uninfected and infected cell (Fig. 3). This observation assigns a direct role to the virus that subsets (Fig. 7). As a control for background level, very few enables its host cell to survive the stress conditions after iso- cells expressing the p24 capsid protein (4%) and dually la- lation and cultivation. The reduction in the susceptibility to belled cells (2%) were detected in uninfected PBMCs (sheep apoptosis could unravel a general mechanism developed by the 294). As a positive control, dot plot patterns corresponding to virus to disturb the cell homeostasis in vivo. This process could sheep infected with the WT provirus (sheep 235, 247, and 265) allow infected cells to escape immune surveillance and favor confirmed our previous results (compare Fig. 3 and 7), dem- the expansion of a B-cell population with the potential to VOL. 71, 1997 PROTECTION BY BLV FROM APOPTOSIS 637

FIG. 7. In situ detection of apoptosis in BLV-infected cells. PBMCs from WT-, G4-, and DX-infected sheep were cultivated for 42 h. Cells from an uninfected (NI) sheep were used as a control. After culture, the cells were fixed in paraformaldehyde and ethanol. The DNA strand breaks were then labelled with biotin-dUTP by using TdT. The cells were then incubated with anti-p24 antibody 4ЈG9, which recognizes the viral capsid protein, and an FITC-conjugated secondary antiserum. The dual-immunofluorescence FACS analyses are presented as dot plots. Numbers represent the percentages of positively stained cells in each quadrant. develop into a leukemia/lymphoma in vivo. During the late provides a selective advantage to the infected cells. As men- phase of the disease, the protection from cell death might give tioned in the introduction, the HTLV Tax protein could di- a survival advantage to the infected cells and thus play a major rectly protect T lymphocytes from apoptosis. However, the role role in oncogenesis and the development of tumors. of BLV Tax in this process could not be analyzed in vivo In contrast, lentiviruses have developed strategies based on because this gene is essential to the infectious potential of the apoptosis to destroy the host immune system. For example, virus. This problem could be circumvented by obtaining a mu- human immunodeficiency virus (HIV) induces apoptosis of tant tax gene that still transactivates viral transcription but that both CD4ϩ and CD8ϩ cells (24). Kinetic analysis showed that lacks a putative potential to decrease the susceptibility to ap- the DNA fragmentation was observed at 12 h after infection optosis. We were unable to obtain this mutant since most (22). The viral Tat transactivator has been found to be directly within the tax gene destroy its transactivation activ- involved in the induction of apoptosis (21). Furthermore, the ity (49, 51). picomolar concentrations of Tat that were found to be effective BLV thus appears to have developed a strategy to resist in cell culture were also detected in sera from HIV-infected apoptosis under stress conditions after culture of the infected patients (45, 56). It thus appears that lentiviruses and oncovi- cells. Aside from this direct mechanism, the virus is also able to ruses have developed opposite strategies either to enhance or interfere with the apoptotic process of other cells. The most to inhibit apoptosis of target immune cells. straightforward interpretation for this observation is that an The mechanism by which BLV interferes with apoptosis is immunological response occurs in vitro during the isolation or unknown. As described for other systems, the virus might en- cultivation of the cells. We think that this hypothesis is unlikely code a factor involved in the regulation pathways of the apo- because of kinetic data. The development of an immunological ptotic process. For instance, the adenovirus E1B 19K protein, response in vitro is thought to require a succession of multiple which has similarities with Bcl-2, has been shown to inhibit events including the expression of viral antigens, their recog- apoptosis directly (32, 46, 47). Another strategy appears to be nition by responsive cells, and the synthesis of secondary mol- used by Epstein-Barr virus, whose LMP-1 gene activates ex- ecules that trigger the activation and multiplication of specific pression of the bcl-2 proto-oncogene (18). This mechanism cells. Our data show that very few cells enter the cell cycle even 638 DEQUIEDT ET AL. J. VIROL.

most straightforward explanation for this observation is that the virus synthesizes an antiapoptotic protein interfering with the cell death pathways. However, we cannot exclude the pos- sibility that BLV preferentially infects a specific B-cell subset that would be less susceptible to spontaneous apoptosis. In both cases, it appears that infected cells can survive severe environmental conditions after their isolation from the blood- stream. This observation could be related to an extended life of the infected cell in the peripheral blood. The reduced suscep- tibility to apoptosis could thus prevent the destruction of the infected cell and allow the clonal expansion of the virus. Perhaps the most unexpected observation from this study is that recombinant BLV proviruses harboring deletions in the R3 and G4 genes also decrease the susceptibility of the PBMCs to global apoptotic death. A direct conclusion from this obser- vation is that the BLV R3 and G4 genes are not involved in the resistance to apoptotic cell death in vivo. These genes are required neither for expression nor for the infectious potential of BLV (52). However, they are involved in the propagation of the virus and in the maintenance of high proviral loads (53). Furthermore, R3 and G4 appear to play a key role in the pathogenesis of the virus (19a). Unfortunately, the function of these genes remains unknown. The use of recombinant provi- ruses has allowed us to draw a second conclusion. These mu- tant proviruses were indeed attenuated when the experiments on apoptosis were performed. Since the levels of apoptosis corresponding to recombinant mutant or WT virus-infected FIG. 8. Protection of uninfected PBMCs by culture supernatants from BLV- PBMCs are similar, one can conclude that the global level of infected cells. (A) PBMCs from uninfected (NI), DX-infected, or WT-infected programmed cell death is independent of the viral load inside sheep were isolated and cultivated for 2 days. The corresponding cell culture supernatants were added to PBMCs from uninfected (NI) sheep 430 and 441. the animals. In other words, a single provirus yields the same After 42 h of cultivation in 10- or 100-fold-diluted NI, DX, or WT culture rates of apoptosis as more than a thousand viruses. supernatants, the PBMCs were fixed in paraformaldehyde and ethanol, and the percentages of apoptotic cells were measured by the TUNEL procedure and FACS analysis. (B) The decrease of the apoptotic rates corresponds to the ACKNOWLEDGMENTS difference between the percentages measured with the NI- and the DX (hatched bars) or the WT (open bars) supernatants. This work was supported by the Caisse Ge´ne´rale d’Epargne et de Retraite, the Belgian Service de Programmation de la Recherche Sci- entifique (PAI15), the Belgian Cancer Association, the Bekales Foun- dation, and the Fond National de la Recherche Scientifique. F.D., at 40 h of culture. Because the majority of the cells had un- E.H., R.K., and L.W. are, respectively, Te´le´vie Fellow, Research As- dergone apoptosis at 12 h, the protective effect should be sistant, Directeur de Recherches, and Maıˆtre de Recherches of the evidenced within the first hours of cultivation (Fig. 4). This Fonds National de la Recherche Scientifique. short delay does not allow the elaboration of an indirect im- We warmly thank P. Griebel for helpful suggestions about PBMC munological stimulation in culture. More convincingly, the isolation and culture experiments. The antisera were kindly provided data in Fig. 8 demonstrate that uninfected PBMCs can directly by J. J. Letesson (Universite´ Notre Dame de la Paix, Namur, Belgium) be protected from apoptosis with culture supernatants from and I. Schwartz (URA-INRA, Ecole Nationale Ve´te´rinaire, Maisons- BLV-infected cells. Therefore, we favor another hypothesis Alfort, France). Skillful technical help was provided by R. Martin, P. that is based on the secretion of a viral or cellular factor by the Ridremont, and G. Vandendaele. infected cells. This factor could diffuse into the culture medium REFERENCES and provide an effective protection to the uninfected cells. It is important to mention here that the decreased susceptibility to 1. Alexandersen, S., S. Carpenter, J. Christensen, T. Stordgaard, B. Viuff, Y. Wannemuehler, J. Belousov, and J. Roth. 1993. 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