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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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Expression of CD4U but not CD4U2/6 causes spontaneous apoptosis in HeLa cells. Inducible HeLa-CD4U and CD4U2/6 cell lines were cultured after the removal of Dox for the times indicated. The cells were then evaluated for induction of apoptosis by staining with PE-conjugated annexin V, followed by flow cytometry. Error bars reflect SDs from three independent experiments.
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fig3: Expression of CD4U but not CD4U2/6 causes spontaneous apoptosis in HeLa cells. Inducible HeLa-CD4U and CD4U2/6 cell lines were cultured after the removal of Dox for the times indicated. The cells were then evaluated for induction of apoptosis by staining with PE-conjugated annexin V, followed by flow cytometry. Error bars reflect SDs from three independent experiments.

Mentions: The experiments presented in the previous sections demonstrate that Vpu significantly contributes to the induction of apoptosis in HIV-1–infected T cells independent of Vpr. However, it is nevertheless possible that Vpu alone is insufficient for induction of apoptosis and requires other viral protein(s). We have previously reported on the inducible expression of CD4-Vpu or CD4-Vpu2/6 chimeras using a tetracycline/Dox-inducible vector system in stable HeLa cell lines (16). The CD4U and CD4U2/6 chimeric molecules were found to have biological activities indistinguishable to those of wild-type Vpu and Vpu2/6 (16, 27, 28). To assess the effect of Vpu on apoptosis in the absence of other HIV-1–specific proteins, we made use of these inducible cell lines. In a first set of experiments we compared the induction of apoptosis over time in the CD4U and CD4U2/6 lines after removal of Dox. Cells were analyzed at various times after induction by annexin V staining (Fig. 3) . The results of this experiment demonstrate that induction of CD4U but not CD4U2/6 caused a dramatic increase in the number of annexin V-positive cells. Therefore, Vpu alone is sufficient for the induction of apoptosis. Furthermore, the fact that induction of CD4U2/6 did not increase the number of apoptotic cells with time indicates that the observed effect of CD4U is specific and not the result of a nonspecific toxicity caused by the overexpression of a heterologous protein in these cells.


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)

Expression of CD4U but not CD4U2/6 causes spontaneous apoptosis in HeLa cells. Inducible HeLa-CD4U and CD4U2/6 cell lines were cultured after the removal of Dox for the times indicated. The cells were then evaluated for induction of apoptosis by staining with PE-conjugated annexin V, followed by flow cytometry. Error bars reflect SDs from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195969&req=5

fig3: Expression of CD4U but not CD4U2/6 causes spontaneous apoptosis in HeLa cells. Inducible HeLa-CD4U and CD4U2/6 cell lines were cultured after the removal of Dox for the times indicated. The cells were then evaluated for induction of apoptosis by staining with PE-conjugated annexin V, followed by flow cytometry. Error bars reflect SDs from three independent experiments.
Mentions: The experiments presented in the previous sections demonstrate that Vpu significantly contributes to the induction of apoptosis in HIV-1–infected T cells independent of Vpr. However, it is nevertheless possible that Vpu alone is insufficient for induction of apoptosis and requires other viral protein(s). We have previously reported on the inducible expression of CD4-Vpu or CD4-Vpu2/6 chimeras using a tetracycline/Dox-inducible vector system in stable HeLa cell lines (16). The CD4U and CD4U2/6 chimeric molecules were found to have biological activities indistinguishable to those of wild-type Vpu and Vpu2/6 (16, 27, 28). To assess the effect of Vpu on apoptosis in the absence of other HIV-1–specific proteins, we made use of these inducible cell lines. In a first set of experiments we compared the induction of apoptosis over time in the CD4U and CD4U2/6 lines after removal of Dox. Cells were analyzed at various times after induction by annexin V staining (Fig. 3) . The results of this experiment demonstrate that induction of CD4U but not CD4U2/6 caused a dramatic increase in the number of annexin V-positive cells. Therefore, Vpu alone is sufficient for the induction of apoptosis. Furthermore, the fact that induction of CD4U2/6 did not increase the number of apoptotic cells with time indicates that the observed effect of CD4U is specific and not the result of a nonspecific toxicity caused by the overexpression of a heterologous protein in these cells.

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