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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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TNF-α treatment amplifies CD4U-induced apoptosis. 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) was then added to the samples as indicated and cultures were incubated for an additional 16 h before analysis. (A) The expression of CD4U and CD4U2/6 (top) and α-tubulin (bottom) was determined by immunoblot analysis using a rabbit anti-Vpu polyclonal antibody (U2–3) and a mouse anti–α-tubulin mAb, respectively. (B and C) The cells were evaluated for induction of apoptosis by annexin V assay (B) or TUNEL assay (C), followed by flow cytometry. (D) Cultures were examined for apoptosis-related morphological changes of the nuclei by staining with PI, followed by confocal microscopic analysis. Panel a: Dox+/TNF-α2; panel b: Dox−/TNF-α2; panels c and d: Dox−/TNF-α1 by low and high power magnification, respectively. Arrowheads mark cells containing pyknotic apoptotic bodies.
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fig4: TNF-α treatment amplifies CD4U-induced apoptosis. 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) was then added to the samples as indicated and cultures were incubated for an additional 16 h before analysis. (A) The expression of CD4U and CD4U2/6 (top) and α-tubulin (bottom) was determined by immunoblot analysis using a rabbit anti-Vpu polyclonal antibody (U2–3) and a mouse anti–α-tubulin mAb, respectively. (B and C) The cells were evaluated for induction of apoptosis by annexin V assay (B) or TUNEL assay (C), followed by flow cytometry. (D) Cultures were examined for apoptosis-related morphological changes of the nuclei by staining with PI, followed by confocal microscopic analysis. Panel a: Dox+/TNF-α2; panel b: Dox−/TNF-α2; panels c and d: Dox−/TNF-α1 by low and high power magnification, respectively. Arrowheads mark cells containing pyknotic apoptotic bodies.

Mentions: HeLa-CD4U and as a control HeLa-CD4U2/6 cells were grown in the presence or absence of Dox for 24 h to inhibit or to induce Vpu expression, respectively. Cells were then treated with or without TNF-α for 16 h in the presence or absence of Dox. Induction of CD4U or CD4U2/6 was confirmed by immunoblotting using a Vpu-specific antibody (Fig. 4 A, top). The same blot was subsequently reblotted with an antibody to α-tubulin as a loading control (Fig. 4 A, bottom). The expression levels of CD4U and CD4U2/6 were comparable and were not affected by treatment of the cells with TNF-α. Induction of apoptosis was measured either by annexin V staining (Fig. 4 B), TUNEL assay (Fig. 4 C) or confocal microscopic analysis of nuclear staining with PI (Fig. 4 D). Expression of CD4U (white bars in Fig. 4 B and C) but not CD4U2/6 (black bars in Fig. 4 B and C) in the absence of TNF-α led to a small but detectable increase in the number of apoptotic cells noticeable in all three assay systems (compare TNF-α2 and Dox+/− in Fig. 4 B and C, also compare Fig. 4 D panels a and b). However, the effect of CD4U on apoptosis was significantly more pronounced in cultures treated with TNF-α where 12–15% of the cells were found to be apoptotic (TNF-α1 and Dox+/− in Fig. 4 B and C, and Fig. 4 D panels c and d). PI staining revealed pyknotic nuclear apoptotic bodies, which are typical morphological characteristics of apoptosis, in Dox-depleted CD4U cell lines (indicated by arrowheads in Fig. 4 D) but not in CD4U2/6 cells (data not shown). These results suggest that TNF-α promotes CD4U-induced apoptosis in HeLa cells. The fact that TNF-α alone, i.e., in the absence of CD4U expression, did not cause apoptosis under these experimental conditions suggests that TNF-α–mediated induction of apoptosis is facilitated by the Vpu-dependent suppression of NF-κB–dependent expression of antiapoptotic genes.


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)

TNF-α treatment amplifies CD4U-induced apoptosis. 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) was then added to the samples as indicated and cultures were incubated for an additional 16 h before analysis. (A) The expression of CD4U and CD4U2/6 (top) and α-tubulin (bottom) was determined by immunoblot analysis using a rabbit anti-Vpu polyclonal antibody (U2–3) and a mouse anti–α-tubulin mAb, respectively. (B and C) The cells were evaluated for induction of apoptosis by annexin V assay (B) or TUNEL assay (C), followed by flow cytometry. (D) Cultures were examined for apoptosis-related morphological changes of the nuclei by staining with PI, followed by confocal microscopic analysis. Panel a: Dox+/TNF-α2; panel b: Dox−/TNF-α2; panels c and d: Dox−/TNF-α1 by low and high power magnification, respectively. Arrowheads mark cells containing pyknotic apoptotic bodies.
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fig4: TNF-α treatment amplifies CD4U-induced apoptosis. 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) was then added to the samples as indicated and cultures were incubated for an additional 16 h before analysis. (A) The expression of CD4U and CD4U2/6 (top) and α-tubulin (bottom) was determined by immunoblot analysis using a rabbit anti-Vpu polyclonal antibody (U2–3) and a mouse anti–α-tubulin mAb, respectively. (B and C) The cells were evaluated for induction of apoptosis by annexin V assay (B) or TUNEL assay (C), followed by flow cytometry. (D) Cultures were examined for apoptosis-related morphological changes of the nuclei by staining with PI, followed by confocal microscopic analysis. Panel a: Dox+/TNF-α2; panel b: Dox−/TNF-α2; panels c and d: Dox−/TNF-α1 by low and high power magnification, respectively. Arrowheads mark cells containing pyknotic apoptotic bodies.
Mentions: HeLa-CD4U and as a control HeLa-CD4U2/6 cells were grown in the presence or absence of Dox for 24 h to inhibit or to induce Vpu expression, respectively. Cells were then treated with or without TNF-α for 16 h in the presence or absence of Dox. Induction of CD4U or CD4U2/6 was confirmed by immunoblotting using a Vpu-specific antibody (Fig. 4 A, top). The same blot was subsequently reblotted with an antibody to α-tubulin as a loading control (Fig. 4 A, bottom). The expression levels of CD4U and CD4U2/6 were comparable and were not affected by treatment of the cells with TNF-α. Induction of apoptosis was measured either by annexin V staining (Fig. 4 B), TUNEL assay (Fig. 4 C) or confocal microscopic analysis of nuclear staining with PI (Fig. 4 D). Expression of CD4U (white bars in Fig. 4 B and C) but not CD4U2/6 (black bars in Fig. 4 B and C) in the absence of TNF-α led to a small but detectable increase in the number of apoptotic cells noticeable in all three assay systems (compare TNF-α2 and Dox+/− in Fig. 4 B and C, also compare Fig. 4 D panels a and b). However, the effect of CD4U on apoptosis was significantly more pronounced in cultures treated with TNF-α where 12–15% of the cells were found to be apoptotic (TNF-α1 and Dox+/− in Fig. 4 B and C, and Fig. 4 D panels c and d). PI staining revealed pyknotic nuclear apoptotic bodies, which are typical morphological characteristics of apoptosis, in Dox-depleted CD4U cell lines (indicated by arrowheads in Fig. 4 D) but not in CD4U2/6 cells (data not shown). These results suggest that TNF-α promotes CD4U-induced apoptosis in HeLa cells. The fact that TNF-α alone, i.e., in the absence of CD4U expression, did not cause apoptosis under these experimental conditions suggests that TNF-α–mediated induction of apoptosis is facilitated by the Vpu-dependent suppression of NF-κB–dependent expression of antiapoptotic genes.

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