JOURNAL OF VIROLOGY, Jan. 1997, p. 630–639 Vol. 71, No. 1 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology Both Wild-Type and Strongly Attenuated Bovine Leukemia 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 b and g 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 retroviruses that induce leukemia/lymphoma after long latency potential of the virus but drastically decreases the proviral periods. These viruses include bovine leukemia virus (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 transcription 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 948C,30sat608C, and 1 min at 728C). 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 N1 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 3 106 PBMCs were harvested by for HTLV to support the biological relevance of their conclu- centrifugation (800 3 g, 10 min) and resuspended in 100 ml 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) 49G9. 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 (29C1 and 49F5) 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(ab9)2 goat anti- three BLVs are similar in terms of infectivity, replication, and pathogenesis in mouse Ig and phycoerythrin (PE)-conjugated F(ab9)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 R31 G42 Tax1 Rex1). The pBLVDX provirus, which harbors deletions of both 10% FCS (PBS-FCS). After their isolation, 2 3 10 PBMCs were incubated for the R3 and G4 genes, was used to infect sheep 245, 246, and 263 (genotype R32 30 min at 48Cin100ml of each primary MAb, washed twice in PBS-FCS, and G42 Tax1 Rex1). In addition, uninfected sheep (sheep 291, 294, 295, 296, 430, incubated in 100 ml 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.