Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis

Abstract

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.

Bovine Leukemia Virus-Induced Persistent Lymphocytosis in Cattle Does Not Correlate with Increased Ex Vivo Survival of B Lymphocytes

“…This result thus supports the data obtained with the quantitative TUNEL assay and demonstrates that BLV-infected PBMCs from cows with PL are more susceptible to ex vivo apoptotic cell death than those from AL or NI cattle. These observations are unexpected, since we have shown in a previous report that BLV-infected sheep PBMCs are less prone to undergo ex vivo apoptosis than are NI cells (9).…”

“…Two recent studies have demonstrated that BLV is capable of increasing the life span of sheep PB-MCs in ex vivo cultures (9,26). Based on the observation that BLV-expressing cells were completely protected from spontaneous programmed cell death, we have assigned a direct role to the virus in the inhibition of the cellular apoptotic programs in sheep (9). To correlate viral expression with apoptosis in cattle, we analyzed the apoptosis levels within the BLV-infected cells.…”

“…PBMC isolation and culture conditions. Sheep PBMCs were isolated by Percoll gradient centrifugation as previously described (9,10). For isolation of bovine PBMCs, blood samples were first collected by jugular venipuncture and mixed with heparin.…”

“…Semiquantitative PCR analysis. The PCRs were performed directly on blood samples essentially as previously described (9). Briefly, aliquots of blood were mixed with an equal volume (500 l) of lysis buffer (0.32 M sucrose, 10 mM Tris-HCl [pH 7.5], 5 mM MgCl 2 , 1% Triton X-100).…”

“…We and others have previously reported that BLV is able to protect infected sheep B lymphocytes from spontaneous ex vivo programmed cell death (9,26). In addition, supernatants from BLV-infected ovine peripheral blood mononuclear cells (PBMCs) contain factors that indirectly extend survival of naive cells in culture (9).…”