Vaccinia virus deletion mutant VV811 is unable to protect cells from staurosporine-induced apoptosis. (A) Schematic representation of the vaccinia virus (Cop) deletion mutants. VV759 is missing ORFs B13R-B29R. VV811 is missing ORFs between B13R-B29R and C23L-F4L. (B) DNA fragmentation is blocked by infection with vaccinia virus (Cop) and VV759 but not VV811. (C) VV759 infection but not VV811 infection protects cells from apoptotic events downstream of the mitochondria. The cleavage of PARP, caspases 3 and 9, and cytochrome c were monitored by Western blot analysis. *, Crossreacting protein in vaccinia virus-infected lysates.

The product of the vaccinia virus F1L ORF localizes to mitochondria. (A) Live HeLa cells were transiently transfected with either pEGFP (a) or an EGFP-tagged version of F1L, pEGFP-F1L (d) and visualized by confocal microscopy. Mitochondria were labeled with Mitotracker Red (b and e). Merged images (c and f) indicate that EGFP-F1L localizes to the mitochondria. (B) HeLa cells were infected with vaccinia virus strain Copenhagen and transfected with pSC66 or pSC66-F1L-FLAG-N. Mitochondria were labeled by using an anti-cytochrome c Ab (c and g). The nucleus and DNA-containing viral factories were labeled with Hoechst (a and e). The localization of FLAG-tagged F1L was visualized with a FITC-conjugated mouse-anti-FLAG Ab (b and f). Merged images from f and g indicate that F1L-FLAG-N localized to the mitochondria during vaccinia virus infection (h). (C) The C-terminal hydrophobic domain of F1L is necessary and sufficient for mitochondria localization. HeLa cells were transfected with a truncated version of F1L, pEGFP-F1L (1-206), or with pEGFP-F1L (199-226) containing the C-terminal region of F1L. Mitochondria were labeled with Mitotracker Red (b and e). Merging of a and b indicated that EGFP-F1L (1-205) did not localize with Mitotracker Red (c) whereas pEGFP-F1L (199-226) (d) localized to the mitochondria as indicated by the merged image (f).

Vaccinia virus (Cop) protects cells from staurosporine-induced apoptosis. (A) The cleavage of PARP caspases 3 and 9 were monitored by Western blot analysis. *, Crossreacting protein in vaccinia virus-infected lysates. (B) To detect cytochrome c release, cells were permeabilized with digitonin and fractionated into mitochondrial-containing membranous fractions and cytosolic fractions.

The vaccinia virus F1L ORF restores the ability of VV811 to inhibit apoptosis. Jurkat cells were infected with VV811 and transfected with 1 μgofthe reporter plasmid pSC66-EGFP in combination with 4μg of pSC66 or pSC66-F1L. (A) At 5 h after infection/transfection, cells were treated with 1μM staurosporine for 2 h, and the inner mitochondrial membrane potential was assessed by TMRE fluorescence. (B) Graphical representation of the experiment is described in A. SDs were calculated from three independent experiments.

F1L inhibits cytochrome c release from mitochondria. (A) HeLa cells were transfected with pEGFP (a-f) or with pEGFP-F1L (g-l). Transfected cells were treated with anti-Fas, and cytochrome c was detected by using anti-cytochrome c. DNA was stained with Hoechst to identify nuclei (c, f, i, and l). Untreated transfected cells showed mitochondrial localization of cytochrome c (b, c, h, and i). After treatment with anti-Fas, EGFP-transfected cells showed release of cytochrome c (e and f) denoted by arrow. Cytochrome c was retained in cells transfected with EGFP-F1L (k and i) denoted by arrows. (B) F1L expression inhibits cytochrome c release. Jurkat cells transfected with the BMGneo empty vector (a and d), overexpressing Bcl-2 (b and e) or expressing F1L (c and f) were treated with staurosporine. (C) F1L inhibits disruption of the mitochondrial membrane potential. Jurkat cells were treated with staurosporine, and the loss of the membrane potential was determined by using TMRE fluorescence.