A novel checkpoint in the Bcl-2-regulated apoptotic pathway revealed by murine cytomegalovirus infection of dendritic cells

Abstract

Infection with murine cytomegalovirus (MCMV) has contributed to understanding many aspects of human infection and, additionally, has provided important insight to understanding complex cellular responses. Dendritic cells (DCs) are a major target for MCMV infection. Here, we analyze the effects of MCMV infection on DC viability, and show that infected DCs become resistant to apoptosis induced by growth factor deprivation. The precise contribution of changes in the expression of Bcl-2 family proteins has been assessed and a new checkpoint in the apoptotic pathway identified. Despite their resistance to apoptosis, MCMV-infected DCs showed Bax to be tightly associated with mitochondria and, together with Bak, forming high molecular weight oligomers, changes normally associated with apoptotic cell death. Exposure of a constitutively occluded Bax NH2-terminal epitope was blocked after infection. These results suggest that MCMV has evolved a novel strategy for inhibiting apoptosis and provide evidence that apoptosis can be regulated after translocation, integration, and oligomerization of Bax at the mitochondrial membrane.

Figure 1.

MCMV inhibits apoptosis in DCs. (a) The viability of D1 DCs infected with MCMV (MOI >3) for 4 d was measured after culture in the presence or absence of growth factor CM for 16 h. Data represent mean ± SD; n = 6. (b) The viability of uninfected, MCMV-infected or LPS-stimulated D1 DCs cultured in the presence or absence of CM for the indicated times was measured. Data represent mean ± SD; n = 6. (c) Caspase activity in uninfected, LPS-stimulated or MCMV-infected D1 DCs grown in the presence or absence of CM for 12 h was determined by cleavage of the caspase-3 substrate Ac-DEVD-pA. Data represent mean ± SD; n = 3. (d) Viability of Flt3L expanded MCMV-infected or control DCs cultured in medium containing 10% FCS and Flt3L (cells grown in CM), or 1% FCS (cells grown in the absence of CM). Data represent mean ± SD; n = 3. Shaded bars represent cells grown in CM. White bars represent cells grown in the absence of CM.

Figure 2.

Inhibition of PI3 kinase does not affect the viability of MCMV-infected DCs. (a) Percentage viability of immature or MCMV-infected D1 DCs cultured in normal growth medium, medium containing 50 μM LY 294002, or medium containing 10 μM UO126 for 16 h. (b) Viability of MCMV-infected, LPS-stimulated or control D1 DCs grown in medium containing 25 μM LY 294002. Data presented in a and b represent mean ± SD; n = 6. Shaded bars represent uninfected immature DCs. White bars represent MCMV-infected DCs.

Figure 3.

Withdrawal of growth factor from DCs leads to increased Bim expression. (a) Total cell lysates were prepared from uninfected immature D1 DCs grown in the presence or absence of growth factor for the indicated times. Cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against the indicated proteins. (b) D1 DCs were grown in the presence or absence of growth factor for 8 h. Cells were subjected to isotonic lysis and mitochondrial (Mito) and cytoplasmic (Cyto) fractions isolated. Proteins were resolved by SDS-PAGE, transferred to PVDF membrane, and expression of the indicated proteins determined by immunoblot. White lines indicate that intervening lanes have been spliced out.

Figure 4.

MCMV infection or LPS stimulation of DCs affects the expression of Bcl-2 family members. Total cell lysates were prepared from uninfected, LPS-stimulated, or MCMV-infected D1 DCs, resolved by SDS-PAGE and transferred to PVDF membrane. Membranes were immunoblotted with the indicated antibodies.

Figure 5.

Bax localizes to the mitochondria in MCMV-infected DCs. Mitochondrial (Mito) and cytoplasmic (Cyto) fractions were isolated from control or MCMV-infected (a) D1 DCs or (b) Flt3L expanded DCs. Lysates were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with the indicated antibodies.

Figure 6.

ΔΨm is not altered despite Bax integration to mitochondrial membranes. (a) Mitochondrial fractions prepared from uninfected or MCMV- infected D1 DCs were incubated in 0.1 M Na₂CO₃, pH 11.5, and centrifuged at 100,000 g for 45 min to yield supernatant (S) and pellet (P) fractions containing alkali-sensitive and -resistant proteins, respectively. Samples were resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with the indicated antibodies. (b) ΔΨm was analyzed in uninfected or MCMV- infected DCs labeled with MitoTracker red CMXRos in the presence or absence of the mitochondrial uncoupler CCCP, as indicated. Dual staining with the nuclear dye Hoechst 33258 allows visualization of all the cells in the field.

Figure 6.

ΔΨm is not altered despite Bax integration to mitochondrial membranes. (a) Mitochondrial fractions prepared from uninfected or MCMV- infected D1 DCs were incubated in 0.1 M Na₂CO₃, pH 11.5, and centrifuged at 100,000 g for 45 min to yield supernatant (S) and pellet (P) fractions containing alkali-sensitive and -resistant proteins, respectively. Samples were resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with the indicated antibodies. (b) ΔΨm was analyzed in uninfected or MCMV- infected DCs labeled with MitoTracker red CMXRos in the presence or absence of the mitochondrial uncoupler CCCP, as indicated. Dual staining with the nuclear dye Hoechst 33258 allows visualization of all the cells in the field.

Figure 7.

Bax and Bak elute as high molecular weight oligomers in MCMV-infected DCs, but exposure of a Bax NH₂-terminal epitope is inhibited. (a) MCMV-infected or uninfected control D1 DCs were grown in the presence or absence of growth factor for 8 h and total cell lysates prepared using 2% CHAPS lysis buffer. Bax protein was immunoprecipitated using the 6A7 conformation-specific antibody. Precipitated proteins were resolved by SDS-PAGE and detected by immunoblot. (b and c) Uninfected or MCMV-infected DCs were grown in the presence or absence of growth factor for 12 h. Mitochondria were isolated by differential centrifugation, treated with 2% CHAPS lysis buffer, and solubilized proteins were separated by gel filtration. The indicated fractions were resolved by SDS-PAGE and immunoblotted with antibodies against (b) Bax and (c) Bak.

Figure 7.

Bax and Bak elute as high molecular weight oligomers in MCMV-infected DCs, but exposure of a Bax NH₂-terminal epitope is inhibited. (a) MCMV-infected or uninfected control D1 DCs were grown in the presence or absence of growth factor for 8 h and total cell lysates prepared using 2% CHAPS lysis buffer. Bax protein was immunoprecipitated using the 6A7 conformation-specific antibody. Precipitated proteins were resolved by SDS-PAGE and detected by immunoblot. (b and c) Uninfected or MCMV-infected DCs were grown in the presence or absence of growth factor for 12 h. Mitochondria were isolated by differential centrifugation, treated with 2% CHAPS lysis buffer, and solubilized proteins were separated by gel filtration. The indicated fractions were resolved by SDS-PAGE and immunoblotted with antibodies against (b) Bax and (c) Bak.

Figure 8.

Candidate MCMV anti-apoptotic proteins do not bind Bax or inhibit apoptosis. Flag-tagged M36, M37, and M45 MCMV viral ORFs were transiently transfected into COS cells. (a) Cells were lysed in the presence of 2% CHAPS and Flag-tagged proteins immunoprecipitated. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-Bax specific antibody. The expression of the Flag-tagged constructs was confirmed by immunoblotting of total cell lysates. White lines indicate that intervening lanes have been spliced out. (b) Cells were grown in the presence (shaded bars) or absence (white bars) of staurosporine (250 nM) for 18 h and the number of viable cells counted. Data represent the mean ± SD, n = 4.