Antiapoptotic activity of the herpesvirus saimiri-encoded Bcl-2 homolog: stabilization of mitochondria and inhibition of caspase-3-like activity

Abstract

Viruses have evolved different strategies to interfere with host cell apoptosis. Herpesvirus saimiri (HVS) and other lymphotropic herpesviruses code for proteins that are homologous to the cellular antiapoptotic Bcl-2. In this study HVS-Bcl-2 was stably expressed in the human leukemia cell line Jurkat and in the murine T-cell hybridoma DO to assess its antiapoptotic spectrum and to gain further insight into its mode of action. HVS- Bcl-2 prevented apoptosis that occurs as a result of a disturbance of intracellular homeostasis by, for example, DNA damage or menadione, which gives rise to oxygen radicals. In Jurkat cells, HVS-Bcl-2 also inhibited apoptosis mediated by the death receptor CD95. In DO cells, HVS-Bcl-2 did not interfere with CD95-mediated apoptosis but blocked dexamethasone-induced cell death. Mitochondrial damage is a central coordinating event in apoptosis induced by different stimuli. To assess the integrity of mitochondria, we used rhodamine 123, which is released upon disturbance of the mitochondrial membrane potential, and determined the release of cytochrome c into the cytosol. Both signs of mitochondrial damage were prevented by HVS-Bcl-2. This viral protein also inhibited the generation of caspase-3-like DEVDase activity and blocked the cleavage of poly(ADP-ribose) polymerase, a natural substrate of caspase-3-like proteases. In conclusion, HVS-Bcl-2 protects against a great variety of apoptotic stimuli, stabilizes mitochondria, and acts upstream of the generation of caspase-3-like activity.

FIG. 1

Expression of HVS-Bcl-2 and Bcl-x_L in transfected cell lines. (A) Transcripts of HVS-Bcl-2 were detected by RNase protection. Different HVS- bcl-2-transfected clones derived from Jurkat cells (Ju) or DO cells were analyzed. Control cells contained the expression vector pCEP4 without insert. In cell lines transfected with HVS-bcl-2, the riboprobe of 700 nucleotides (nt) specifically protected a fragment of 321 nucleotides. This protected fragment of 321 nucleotides consists of 250 nucleotides of HVS-bcl-2, 48 nucleotides of pCR2.1, and 23 nucleotides of pCEP4. As a positive control, we used T-cell cultures from Callithrix jacchus that release infectious virus and transcribe HVS-bcl-2 (31). When RNA from these cells is used, the applied riboprobe protects a fragment of about 250 nucleotides, representing the calculated length of the viral transcript that is mirrored in the riboprobe. (B) The expression of Bcl-x_L in transfected Jurkat cells was detected by Western blotting.

FIG. 2

HVS-Bcl-2 and Bcl-x_L protect Jurkat cells (Ju) from apoptosis mediated by CD95 (A), irradiation (B), and menadione (C). Four different clones transfected with HVS-Bcl-2, four clones transfected with Bcl-x_L, and four control clones were treated with anti-CD95 (A) or irradiated (B) at the indicated dosage. The specific cell death (mean ± standard deviation [SD]) was determined after 24 h (A) or 72 h (B) as described in Materials and Methods. The data for the individual cell lines expressing HVS-Bcl-2 or Bcl-x_L and for the control cells were pooled for presentation in panels A and B. To determine the sensitivity to menadione-induced cell death, two different clones of each group were studied and the data were pooled (C). The development of apoptosis was quantified by a histone release enzyme-linked immunosorbent assay and the enrichment factor (mean ± SD) was calculated as described in Materials and Methods.

FIG. 3

HVS-Bcl-2 blocks apoptosis mediated by dexamethasone but not by CD95 in DO cells. (A and C) DO cells transfected with HVS-Bcl-2 or control vector were treated with 10 nM dexamethasone and assessed for DNA fragmentation after 48 h (A). The development of cell death after dexamethasone treatment was quantified by measuring PI uptake after 48 h (C). Four clones transfected with HVS-Bcl-2 and five control clones were analyzed. The specific cell death (mean ± SD) is indicated (C). (B and D) To assess the sensitivity to CD95-mediated apoptosis, the same transfectants were treated with FLAG-CD95 ligand and anti-FLAG MAb. These cells were analyzed for DNA fragmentation 24 h after treatment with 10 ng of CD95 ligand per ml (B). Development of cell death (mean ± SD) was quantified by measuring PI uptake after 24 h, and the specific cell death (mean ± SD) is indicated (D).

FIG. 4

HVS-Bcl-2 and Bcl-x_L block CD95-induced exposure of phosphatidylserine. The indicated Jurkat transfectants were treated with the CD95-specific MAb CH-11 at 100 ng/ml, and binding of annexin-V was determined at the indicated times after addition of the MAb.

FIG. 5

HVS-Bcl-2 and Bcl-x_L prevent mitochondrial damage. The indicated transfectants were cultured with anti-CD95 (10 ng/ml) or left untreated. After 16 h, the cells were stained with rhodamine 123 for 30 min at 37°C, washed, suspended in PBS containing PI, and analyzed by flow cytometry.

FIG. 6

HVS-Bcl-2 and Bcl-x_L block the release of cytochrome c into the cytosol. The indicated transfectants were treated for 4 h with the indicated concentrations of anti-CD95. Subsequently, the cytosolic fraction was prepared and analyzed by Western blotting for the presence of cytochrome c.

FIG. 7

HVS-Bcl-2 and Bcl-x_L block the appearance of caspase-3-like DEVDase activity. The different transfectants were treated with anti-CD95 at the indicated concentrations for 4 h or left untreated. The DEVDase activity in the cell lysates was measured by using DEVD-aminomethylcoumarin as a substrate. The specific DEVDase activity was calculated as the difference of DEVDase activity in the absence and presence of the inhibitor DEVD-CHO. The increase in the DEVDase specific activity after CD95 engagement was determined. The mean values for three individual cell clones expressing HVS-Bcl-2, of three clones expressing Bcl-x_L, and of a control cell line are shown.

FIG. 8

HVS-Bcl-2 and Bcl-x_L block the CD95-induced cleavage of PARP. The indicated transfectants had been treated with the indicated concentrations of anti-CD95 for 4 h. The cleavage of PARP was analyzed by Western blotting.

FIG. 9

HVS-Bcl-2 and Bcl-x_L prevent the CD95-induced cleavage of caspase-3. The indicated transfectants had been treated with different concentrations of anti-CD95 for 4 h. The content of the 32-kDa uncleaved proform of caspase-3 was analyzed by Western blotting.