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The human immunodeficiency virus type 1 accessory protein Vpu induces apoptosis by suppressing the nuclear factor kappaB-dependent expression of antiapoptotic factors.

Akari H, Bour S, Kao S, Adachi A, Strebel K - J. Exp. Med. (2001)

Bottom Line: Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity.The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated.Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for betaTrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including IkappaB. Remarkably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of IkappaB, resulting in the suppression of nuclear factor (NF)-kappaB activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity. The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.

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Vpu-induced apoptosis involves activation of the caspase pathway. CD4U and CD4U2/6 cell lines were cultured in complete DMEM medium in the presence or absence of Dox for 24 h. TNF-α (20 ng/ml) and z-VAD-fmk (50 μM) were then added where indicated and the cultures were incubated for an additional 16 h before analysis. (A) Cultures were evaluated for induction of apoptosis by annexin V staining. Error bars reflect SDs from three independent experiments. (B) The same cultures were analyzed by flow cytometry for the expression of the active form of caspase-3 using a FITC-conjugated rabbit antiactive caspase-3 polyclonal antibody. The numbers indicate percentages of FITC-positive cells.
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fig5: Vpu-induced apoptosis involves activation of the caspase pathway. CD4U and CD4U2/6 cell lines were cultured in complete DMEM medium in the presence or absence of Dox for 24 h. TNF-α (20 ng/ml) and z-VAD-fmk (50 μM) were then added where indicated and the cultures were incubated for an additional 16 h before analysis. (A) Cultures were evaluated for induction of apoptosis by annexin V staining. Error bars reflect SDs from three independent experiments. (B) The same cultures were analyzed by flow cytometry for the expression of the active form of caspase-3 using a FITC-conjugated rabbit antiactive caspase-3 polyclonal antibody. The numbers indicate percentages of FITC-positive cells.

Mentions: In view of our observation that Vpu has the ability to suppress spontaneous and TNF-α-induced NF-κB activation (16), it seems likely that Vpu-induced apoptosis is the result of an indirect activation of the caspase pathways by downmodulating the expression levels of antiapoptotic factor(s). To address this issue, we initially tested whether Vpu-induced apoptosis is dependent on the caspase pathway. For that purpose, we determined the effect of a broad-range inhibitor of caspases, Z-VAD-fmk, on CD4U-induced and TNF-α–enhanced apoptosis. HeLa-CD4U cells were cultured in the absence of Dox for 24 h and then treated for 16 h with TNF-α (10 ng/ml) either in the presence or absence of Z-VAD-fmk as indicated in Fig. 5 . Cells were then reacted with PE-conjugated annexin V and analyzed by flow cytometry (Fig. 5 A). The results of this experiment show that treatment of cells with the caspase inhibitor reduced the level of annexin V-positive cells to background levels despite the presence of CD4U and TNF-α. Of note, treatment with Z-VAD-fmk did not affect the level of CD4U expression by Dox deprivation (data not shown).


The human immunodeficiency virus type 1 accessory protein Vpu induces apoptosis by suppressing the nuclear factor kappaB-dependent expression of antiapoptotic factors.

Akari H, Bour S, Kao S, Adachi A, Strebel K - J. Exp. Med. (2001)

Vpu-induced apoptosis involves activation of the caspase pathway. CD4U and CD4U2/6 cell lines were cultured in complete DMEM medium in the presence or absence of Dox for 24 h. TNF-α (20 ng/ml) and z-VAD-fmk (50 μM) were then added where indicated and the cultures were incubated for an additional 16 h before analysis. (A) Cultures were evaluated for induction of apoptosis by annexin V staining. Error bars reflect SDs from three independent experiments. (B) The same cultures were analyzed by flow cytometry for the expression of the active form of caspase-3 using a FITC-conjugated rabbit antiactive caspase-3 polyclonal antibody. The numbers indicate percentages of FITC-positive cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195969&req=5

fig5: Vpu-induced apoptosis involves activation of the caspase pathway. CD4U and CD4U2/6 cell lines were cultured in complete DMEM medium in the presence or absence of Dox for 24 h. TNF-α (20 ng/ml) and z-VAD-fmk (50 μM) were then added where indicated and the cultures were incubated for an additional 16 h before analysis. (A) Cultures were evaluated for induction of apoptosis by annexin V staining. Error bars reflect SDs from three independent experiments. (B) The same cultures were analyzed by flow cytometry for the expression of the active form of caspase-3 using a FITC-conjugated rabbit antiactive caspase-3 polyclonal antibody. The numbers indicate percentages of FITC-positive cells.
Mentions: In view of our observation that Vpu has the ability to suppress spontaneous and TNF-α-induced NF-κB activation (16), it seems likely that Vpu-induced apoptosis is the result of an indirect activation of the caspase pathways by downmodulating the expression levels of antiapoptotic factor(s). To address this issue, we initially tested whether Vpu-induced apoptosis is dependent on the caspase pathway. For that purpose, we determined the effect of a broad-range inhibitor of caspases, Z-VAD-fmk, on CD4U-induced and TNF-α–enhanced apoptosis. HeLa-CD4U cells were cultured in the absence of Dox for 24 h and then treated for 16 h with TNF-α (10 ng/ml) either in the presence or absence of Z-VAD-fmk as indicated in Fig. 5 . Cells were then reacted with PE-conjugated annexin V and analyzed by flow cytometry (Fig. 5 A). The results of this experiment show that treatment of cells with the caspase inhibitor reduced the level of annexin V-positive cells to background levels despite the presence of CD4U and TNF-α. Of note, treatment with Z-VAD-fmk did not affect the level of CD4U expression by Dox deprivation (data not shown).

Bottom Line: Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity.The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated.Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for betaTrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including IkappaB. Remarkably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of IkappaB, resulting in the suppression of nuclear factor (NF)-kappaB activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity. The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.

Show MeSH
Related in: MedlinePlus