The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation - PubMed (original) (raw)

The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and inhibits Bak activation

Shawn T Wasilenko et al. J Virol. 2005 Nov.

Abstract

Many viruses have evolved strategies to counteract cellular immune responses, including apoptosis. Vaccinia virus, a member of the poxvirus family, encodes an antiapoptotic protein, F1L. F1L localizes to mitochondria and inhibits apoptosis by preventing the release of cytochrome c by an undetermined mechanism (S. T. Wasilenko, T. L. Stewart, A. F. Meyers, and M. Barry, Proc. Natl. Acad. Sci. USA 100:14345-14350, 2003; T. L. Stewart, S. T. Wasilenko, and M. Barry, J. Virol. 79:1084-1098, 2005). Here, we show that in the absence of an apoptotic stimulus, F1L associates with Bak, a proapoptotic member of the Bcl-2 family that plays a pivotal role in the release of cytochrome c. Cells infected with vaccinia virus were resistant to Bak oligomerization and the initial N-terminal exposure of Bak following the induction of apoptosis with staurosporine. A mutant vaccinia virus missing F1L was no longer able to inhibit apoptosis or Bak activation. In addition, the expression of F1L was essential to inhibit tBid-induced cytochrome c release in both wild-type murine embryonic fibroblasts (MEFs) and Bax-deficient MEFs, indicating that F1L could inhibit apoptosis in the presence and absence of Bax. tBid-induced Bak oligomerization and N-terminal exposure of Bak in Bax-deficient MEFs were inhibited during virus infection, as assessed by cross-linking and limited trypsin proteolysis. Infection with the F1L deletion virus no longer provided protection from tBid-induced Bak activation and apoptosis. Additionally, infection of Jurkat cells with the F1L deletion virus resulted in cellular apoptosis, as measured by loss of the inner mitochondrial membrane potential, caspase 3 activation, and cytochrome c release, indicating that the presence of F1L was pivotal for inhibiting vaccinia virus-induced apoptosis. Our data indicate that F1L expression during infection inhibits apoptosis and interferes with the activation of Bak.

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Figures

FIG. 1.

FIG. 1.

Characterization of a recombinant VV devoid of F1L. (A) Agarose gel analysis of PCR products amplified from VV(Cop)- and VV(Cop)ΔF1L-infected cells compared to plasmid containing wild-type (WT) F1L and the plasmid used to generate VV(Cop)ΔF1L by homologous recombination. (B) Microscopic analysis of plaques generated from CV-1 cells infected with VV(Cop) and VV(Cop)ΔF1L. (C) Single-step growth analysis of VV(Cop) and VV(Cop)ΔF1L in CV-1 cells.

FIG. 2.

FIG. 2.

VV(Cop)ΔF1L is unable to protect cells from staurosporine-induced apoptosis. (A) Jurkat cells and Jurkat cells overexpressing Bcl-2 were mock infected or infected with VV(Cop) or VV(Cop)ΔF1L at an MOI of 10 and treated with 500 nM staurosporine for 90 min to induce apoptosis. Apoptosis was assessed by TMRE fluorescence, which measures loss of the inner mitochondrial membrane potential. (B) Jurkat cells and Jurkat cells overexpressing Bcl-2 were either mock infected, infected with VV(Cop), or infected with VV(Cop)ΔF1L and treated with 500 nM staurosporine. Cytochrome c release was monitored by Western blot analysis. (C) Jurkat cells and Jurkat cells overexpressing Bcl-2 were either mock infected, infected with VV(Cop), or infected with VV(Cop)ΔF1L at an MOI of 10 and treated with 500 nM staurosporine. Caspase 3 activation was monitored by Western blot analysis.

FIG. 3.

FIG. 3.

VV(Cop)ΔF1L induces apoptosis in Jurkat cells. (A) Jurkat cells were infected with VV(Cop) or VV(Cop)ΔF1L at an MOI of 10 for 15 h, and apoptosis was assessed by TMRE fluorescence. (B) Jurkat cells were infected with VV(Cop) or VV(Cop)ΔF1L at an MOI of 10 for 5, 10, and 15 h. Cytochrome c release and caspase 3 activation were monitored by Western blot analysis. (C) Bcl-2 overexpression is sufficient to inhibit VV(Cop)ΔF1L-induced apoptosis. Jurkat cells were infected with VV(Cop) or VV(Cop)ΔF1L at an MOI of 10 for 5, 10, and 15 h in the presence and absence of zVAD.fmk. Cytochrome c release and caspase 3 activation were monitored by Western blot analysis.

FIG. 4.

FIG. 4.

F1L interacts with endogenous Bak but not other Bcl-2 family proteins. HeLa cell lysates from mock-infected cells or cells infected with VV(WR)Flag-F1L at an MOI of 10 were applied to an anti-F1L immunoaffinity column. Bound proteins were eluted by linear addition of elution buffer containing 100 mM glycine and 0.5 M NaCl, pH 2.7. The eluted fractions were monitored by Western blotting for Flag-F1L, Bax, Bak, Bcl-2, Bcl-xL, and Mcl-1.

FIG. 5.

FIG. 5.

F1L interacts with Bak in the presence and absence of VV(Cop) infection. (A) Ectopic expression of F1L and Bak demonstrates interaction between F1L and Bak. HEK 293T cells were cotransfected with either pEGFP or pEGFP-F1L in the presence of pcDNA-HA-Bak. EGFP-F1L interacts with HA-Bak. IP, immunoprecipitate. (B) F1L interacts with endogenous Bak. HeLa cells were transfected with pEGFP or pEGFP-F1L. EGFP-F1L, but not EGFP, interacts with endogenous Bak. (C) F1L associates with endogenous Bak during virus infection. HeLa cells were infected with VV(Cop) at an MOI of 10 and transfected with pSC66 or pSC66-Flag-F1L to express Flag-F1L during infection. F1L associates with endogenous Bak.

FIG. 6.

FIG. 6.

F1L and endogenous Bak localize at the mitochondria during vaccinia virus infection. HeLa cells were infected with VV(WR)Flag-F1L at an MOI of 5 for 8 h. The localization of Flag-F1L was visualized with a fluorescein isothiocyanate-conjugated mouse anti-Flag antibody (a and c). Endogenous Bak was detected using an anti-Bak (G23) antibody, followed by detection with the Alexa-Fluor 546-conjugated goat anti-rabbit antibody (b and c).

FIG. 7.

FIG. 7.

F1L expression inhibits staurosporine-induced Bak activation. (A) Bak oligomerization is inhibited by VV(Cop)-EGFP infection but not infection with VV(Cop)ΔF1L. Jurkat cells were infected with VV(Cop)-EGFP or VV(Cop)ΔF1L at an MOI of 10, and 5 h postinfection, they were treated with 1 μM staurosporine for 2 h to induce apoptosis. Bak oligomerization was assessed by gel filtration analysis and detected by Western blotting. (B) F1L expression is necessary for VV(Cop)-EGFP to inhibit the N-terminal exposure of Bak. Jurkat cells, Jurkat cells overexpressing Bcl-2, or Jurkat cells devoid of Bak and Bax were infected with VV(Cop)-EGFP or VV(Cop)ΔF1L at an MOI of 10 and treated with 250 nM staurosporine for 2 h to induce apoptosis. Exposure of the N terminus of Bak was monitored by flow cytometry using a conformation-specific N-terminal anti-Bak antibody or an isotype control antibody. Untreated cells, open histogram; staurosporine-treated cells, shaded histogram.

FIG. 8.

FIG. 8.

F1L inhibits tBid-induced Bak activation. (A) F1L expression inhibits release of cytochrome c initiated by tBid. Mitochondria were isolated from wild-type (WT) MEFs, Bax-deficient (Bax−/−) MEFs, and Bak- and Bax-deficient (Bak−/−Bax−/−) MEFs that had previously been infected with VV(Cop) or VV(Cop)ΔF1L at an MOI of 20. The mitochondria were treated with increasing amounts of tBid and assessed for cytochrome c release by Western blotting. (B) F1L expression is necessary to inhibit Bak oligomerization initiated by tBid. Mitochondria from Bax-deficient MEFs were treated with tBid and cross-linked with BMH. VV(Cop)EGFP infection, but not VV(Cop)ΔF1L infection, inhibits Bak oligomerization. *, Bak homo-oligomers; **, monomeric intramolecularly cross-linked Bak species. (C) F1L expression is necessary to inhibit the N-terminal exposure of Bak induced by increasing amounts of tBid. After exposure to tBid, isolated mitochondria were treated with trypsin and Bak conformation was monitored by Western blotting. As a control, the presence of Mn SOD was also monitored by Western blotting.

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