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The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G - J. Exp. Med. (2000)

Bottom Line: The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr.Again, this effect is prevented by addition of recombinant Bcl-2.Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, F-94801 Villejuif, France.

ABSTRACT
Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

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Subcellular localization of Vpr in different cell types. (A) Subcellular localization of transfected Vpr. COS cells were transfected with pcDNA3.1 vector only or with pcDNA-FLAG-Vpr expression vector, followed by immunofluorescence detection (mouse mAb M2 anti-FLAG; Sigma Chemical Co.) of the FLAG (red fluorescence) as well as of the mitochondrial protein Hsp60 (green fluorescence) by confocal microscopy. Fine analysis of the fluorescence distribution (right panel) reveals partial colocalization of Vpr and Hsp60. The micrograph of the FLAG-Vpr–expressing cells represents ∼30% of the transfected cells with a predominantly cytoplasmic staining. The remaining cells showed either a predominantly nuclear/perinuclear Vpr (∼40%) distribution or a mixed (∼20%) phenotype. (B) Subcellular localization of soluble Vpr52-96 added to cells. Primary human PBLs or PHA lymphoblasts derived from them were incubated in the absence (Co.) or presence of 1 μM biotin–Vpr52-96 for 30 min, counterstained with Mitotracker green (100 nM during the last 30 min of culture), fixed, and stained with a streptavidin–PE conjugate (red fluorescence) as well as the DNA intercalating dye Hoechst 33324 (blue fluorescence). Note the clear overlap between green and red fluorescence (yellow), indicating a mitochondrial localization of Vpr52-96 30 min after the addition of the peptide. Aliquots from control cells and biotin–Vpr52-96–treated cells were stained with the potential-sensitive dye JC-1 (right panel). Control cells exhibited an elevated ΔΨm (red fluorescence), whereas Vpr52-96–treated cells had a low ΔΨm (green fluorescence). Micrographs are representative for >90% of the cells, and the experiment was performed four times.
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Figure 7: Subcellular localization of Vpr in different cell types. (A) Subcellular localization of transfected Vpr. COS cells were transfected with pcDNA3.1 vector only or with pcDNA-FLAG-Vpr expression vector, followed by immunofluorescence detection (mouse mAb M2 anti-FLAG; Sigma Chemical Co.) of the FLAG (red fluorescence) as well as of the mitochondrial protein Hsp60 (green fluorescence) by confocal microscopy. Fine analysis of the fluorescence distribution (right panel) reveals partial colocalization of Vpr and Hsp60. The micrograph of the FLAG-Vpr–expressing cells represents ∼30% of the transfected cells with a predominantly cytoplasmic staining. The remaining cells showed either a predominantly nuclear/perinuclear Vpr (∼40%) distribution or a mixed (∼20%) phenotype. (B) Subcellular localization of soluble Vpr52-96 added to cells. Primary human PBLs or PHA lymphoblasts derived from them were incubated in the absence (Co.) or presence of 1 μM biotin–Vpr52-96 for 30 min, counterstained with Mitotracker green (100 nM during the last 30 min of culture), fixed, and stained with a streptavidin–PE conjugate (red fluorescence) as well as the DNA intercalating dye Hoechst 33324 (blue fluorescence). Note the clear overlap between green and red fluorescence (yellow), indicating a mitochondrial localization of Vpr52-96 30 min after the addition of the peptide. Aliquots from control cells and biotin–Vpr52-96–treated cells were stained with the potential-sensitive dye JC-1 (right panel). Control cells exhibited an elevated ΔΨm (red fluorescence), whereas Vpr52-96–treated cells had a low ΔΨm (green fluorescence). Micrographs are representative for >90% of the cells, and the experiment was performed four times.

Mentions: If Vpr acted on mitochondria to induce apoptosis, then at least some Vpr protein should be found in mitochondria from intact cells. To determine the subcellular localization of Vpr, epitope-tagged (FLAG)Vpr was transfected into COS cells and was revealed by a PE-labeled anti-FLAG antibody (red fluorescence). Simultaneously, mitochondria were stained with an FITC-conjugated anti-Hsp60 antibody (green fluorescence). In accord with previous observations of a punctuate cytoplasmic localization of Vpr 57 58, we found that ∼30% of Vpr-expressing cells exhibited an exclusively cytoplasmic Vpr staining pattern ( Fig. 7 A). These cells appear to be programmed to die (not shown), which may explain why they represent only a fraction of the entire population. In such cells, most of the Vpr-dependent red fluorescence colocalizes with the Hsp60 protein, giving rise to a yellow (red plus green) staining pattern. Very little Vpr is localized in the nonmitochondrial compartment (red fluorescence; Fig. 7 A). To confirm this observation in another experimental system, we added biotinylated Vpr52-96 to human primary PBLs or to PHA lymphoblasts. Vpr52-96 was then detected by means of a streptavidin–PE conjugate. Cells were counterstained with Mitotracker green (which labels mitochondria independently from their ΔΨm) and Hoechst 33342 (which labels nuclei) to determine the subcellular distribution of Vpr. After an initial enrichment in the plasma membrane (not shown), biotinylated Vpr52-96 was specifically recruited to mitochondria ( Fig. 7 B).


The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G - J. Exp. Med. (2000)

Subcellular localization of Vpr in different cell types. (A) Subcellular localization of transfected Vpr. COS cells were transfected with pcDNA3.1 vector only or with pcDNA-FLAG-Vpr expression vector, followed by immunofluorescence detection (mouse mAb M2 anti-FLAG; Sigma Chemical Co.) of the FLAG (red fluorescence) as well as of the mitochondrial protein Hsp60 (green fluorescence) by confocal microscopy. Fine analysis of the fluorescence distribution (right panel) reveals partial colocalization of Vpr and Hsp60. The micrograph of the FLAG-Vpr–expressing cells represents ∼30% of the transfected cells with a predominantly cytoplasmic staining. The remaining cells showed either a predominantly nuclear/perinuclear Vpr (∼40%) distribution or a mixed (∼20%) phenotype. (B) Subcellular localization of soluble Vpr52-96 added to cells. Primary human PBLs or PHA lymphoblasts derived from them were incubated in the absence (Co.) or presence of 1 μM biotin–Vpr52-96 for 30 min, counterstained with Mitotracker green (100 nM during the last 30 min of culture), fixed, and stained with a streptavidin–PE conjugate (red fluorescence) as well as the DNA intercalating dye Hoechst 33324 (blue fluorescence). Note the clear overlap between green and red fluorescence (yellow), indicating a mitochondrial localization of Vpr52-96 30 min after the addition of the peptide. Aliquots from control cells and biotin–Vpr52-96–treated cells were stained with the potential-sensitive dye JC-1 (right panel). Control cells exhibited an elevated ΔΨm (red fluorescence), whereas Vpr52-96–treated cells had a low ΔΨm (green fluorescence). Micrographs are representative for >90% of the cells, and the experiment was performed four times.
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Figure 7: Subcellular localization of Vpr in different cell types. (A) Subcellular localization of transfected Vpr. COS cells were transfected with pcDNA3.1 vector only or with pcDNA-FLAG-Vpr expression vector, followed by immunofluorescence detection (mouse mAb M2 anti-FLAG; Sigma Chemical Co.) of the FLAG (red fluorescence) as well as of the mitochondrial protein Hsp60 (green fluorescence) by confocal microscopy. Fine analysis of the fluorescence distribution (right panel) reveals partial colocalization of Vpr and Hsp60. The micrograph of the FLAG-Vpr–expressing cells represents ∼30% of the transfected cells with a predominantly cytoplasmic staining. The remaining cells showed either a predominantly nuclear/perinuclear Vpr (∼40%) distribution or a mixed (∼20%) phenotype. (B) Subcellular localization of soluble Vpr52-96 added to cells. Primary human PBLs or PHA lymphoblasts derived from them were incubated in the absence (Co.) or presence of 1 μM biotin–Vpr52-96 for 30 min, counterstained with Mitotracker green (100 nM during the last 30 min of culture), fixed, and stained with a streptavidin–PE conjugate (red fluorescence) as well as the DNA intercalating dye Hoechst 33324 (blue fluorescence). Note the clear overlap between green and red fluorescence (yellow), indicating a mitochondrial localization of Vpr52-96 30 min after the addition of the peptide. Aliquots from control cells and biotin–Vpr52-96–treated cells were stained with the potential-sensitive dye JC-1 (right panel). Control cells exhibited an elevated ΔΨm (red fluorescence), whereas Vpr52-96–treated cells had a low ΔΨm (green fluorescence). Micrographs are representative for >90% of the cells, and the experiment was performed four times.
Mentions: If Vpr acted on mitochondria to induce apoptosis, then at least some Vpr protein should be found in mitochondria from intact cells. To determine the subcellular localization of Vpr, epitope-tagged (FLAG)Vpr was transfected into COS cells and was revealed by a PE-labeled anti-FLAG antibody (red fluorescence). Simultaneously, mitochondria were stained with an FITC-conjugated anti-Hsp60 antibody (green fluorescence). In accord with previous observations of a punctuate cytoplasmic localization of Vpr 57 58, we found that ∼30% of Vpr-expressing cells exhibited an exclusively cytoplasmic Vpr staining pattern ( Fig. 7 A). These cells appear to be programmed to die (not shown), which may explain why they represent only a fraction of the entire population. In such cells, most of the Vpr-dependent red fluorescence colocalizes with the Hsp60 protein, giving rise to a yellow (red plus green) staining pattern. Very little Vpr is localized in the nonmitochondrial compartment (red fluorescence; Fig. 7 A). To confirm this observation in another experimental system, we added biotinylated Vpr52-96 to human primary PBLs or to PHA lymphoblasts. Vpr52-96 was then detected by means of a streptavidin–PE conjugate. Cells were counterstained with Mitotracker green (which labels mitochondria independently from their ΔΨm) and Hoechst 33342 (which labels nuclei) to determine the subcellular distribution of Vpr. After an initial enrichment in the plasma membrane (not shown), biotinylated Vpr52-96 was specifically recruited to mitochondria ( Fig. 7 B).

Bottom Line: The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr.Again, this effect is prevented by addition of recombinant Bcl-2.Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, F-94801 Villejuif, France.

ABSTRACT
Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Show MeSH
Related in: MedlinePlus