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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 affects the expression of antiapoptotic factors and induces caspase-8 activation. (A and B) HeLa-CD4U cells 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 cultures as indicated and incubation was continued for an additional 16 h. Cell lysates were analyzed by immunoblotting for the expression of Bcl-xL and A1/Bfl-1 (A) as well as TRAF1 and the active form of caspase-8 (B). Lysates were normalized for tubulin using an α-tubulin antibody. (C) Jurkat cells were single-cycle infected with VSV-G-pseudotyped NL43-K1, NL43-K1/Udel, or NL43-K1/U2/6. Cell lysates were analyzed 40 h after infection by immunoblotting to detect expression of Bcl-xL, A1/Bfl-1, TRAF1, p24 CA, Vpu, or α-tubulin.
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fig6: Vpu affects the expression of antiapoptotic factors and induces caspase-8 activation. (A and B) HeLa-CD4U cells 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 cultures as indicated and incubation was continued for an additional 16 h. Cell lysates were analyzed by immunoblotting for the expression of Bcl-xL and A1/Bfl-1 (A) as well as TRAF1 and the active form of caspase-8 (B). Lysates were normalized for tubulin using an α-tubulin antibody. (C) Jurkat cells were single-cycle infected with VSV-G-pseudotyped NL43-K1, NL43-K1/Udel, or NL43-K1/U2/6. Cell lysates were analyzed 40 h after infection by immunoblotting to detect expression of Bcl-xL, A1/Bfl-1, TRAF1, p24 CA, Vpu, or α-tubulin.

Mentions: There is a possibility that the expression levels of Bcl-xL and A1/Bfl-1, which are transcriptionally regulated by NF-κB, might be reduced in Vpu-expressing cells due to its inhibitory effect on NF-κB activity (16). To examine this possibility, we determined the levels of Bcl-xL and A1/Bfl-1 expression in the CD4U-HeLa cells by immunoblot analyses (Fig. 6 A). In uninduced CD4U cells (Fig. 6 A, lanes 3 and 4), both Bcl-xL and A1/Bfl-1 were expressed at considerable levels, reflecting the relatively high basal level of NF-κB activity in HeLa cells, and stimulation with TNF-α did not significantly augment their expression levels. After induction of CD4U expression, however, the steady-state levels of both factors were reduced to ∼60% of their levels in uninduced cultures (Fig. 6 A, lane 1 versus lane 3). Moreover, TNF-α treatment of the CD4U-induced cells further reduced the levels of Bcl-xL and A1/Bfl-1 to <20% of those in TNF-α–treated uninduced cells (Fig. 6 A, lane 2 versus lane 4). These results are significant considering that <10% of the cells shown in lane 1 and <20% of the cells shown in lane 2 were annexin V-positive at the time of the analysis (data not shown). Thus, Vpu indeed downregulated the steady-state levels of Bcl-xL and A1/Bfl-1. The further reduction of Bcl-xL and A1/Bfl-1 levels after TNF-α treatment is presumably a consequence of the concomitant activation of caspase-3 by TNF-α, which is known to proteolytically cleave Bcl-xL (42, 43).


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 affects the expression of antiapoptotic factors and induces caspase-8 activation. (A and B) HeLa-CD4U cells 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 cultures as indicated and incubation was continued for an additional 16 h. Cell lysates were analyzed by immunoblotting for the expression of Bcl-xL and A1/Bfl-1 (A) as well as TRAF1 and the active form of caspase-8 (B). Lysates were normalized for tubulin using an α-tubulin antibody. (C) Jurkat cells were single-cycle infected with VSV-G-pseudotyped NL43-K1, NL43-K1/Udel, or NL43-K1/U2/6. Cell lysates were analyzed 40 h after infection by immunoblotting to detect expression of Bcl-xL, A1/Bfl-1, TRAF1, p24 CA, Vpu, or α-tubulin.
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Related In: Results  -  Collection

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fig6: Vpu affects the expression of antiapoptotic factors and induces caspase-8 activation. (A and B) HeLa-CD4U cells 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 cultures as indicated and incubation was continued for an additional 16 h. Cell lysates were analyzed by immunoblotting for the expression of Bcl-xL and A1/Bfl-1 (A) as well as TRAF1 and the active form of caspase-8 (B). Lysates were normalized for tubulin using an α-tubulin antibody. (C) Jurkat cells were single-cycle infected with VSV-G-pseudotyped NL43-K1, NL43-K1/Udel, or NL43-K1/U2/6. Cell lysates were analyzed 40 h after infection by immunoblotting to detect expression of Bcl-xL, A1/Bfl-1, TRAF1, p24 CA, Vpu, or α-tubulin.
Mentions: There is a possibility that the expression levels of Bcl-xL and A1/Bfl-1, which are transcriptionally regulated by NF-κB, might be reduced in Vpu-expressing cells due to its inhibitory effect on NF-κB activity (16). To examine this possibility, we determined the levels of Bcl-xL and A1/Bfl-1 expression in the CD4U-HeLa cells by immunoblot analyses (Fig. 6 A). In uninduced CD4U cells (Fig. 6 A, lanes 3 and 4), both Bcl-xL and A1/Bfl-1 were expressed at considerable levels, reflecting the relatively high basal level of NF-κB activity in HeLa cells, and stimulation with TNF-α did not significantly augment their expression levels. After induction of CD4U expression, however, the steady-state levels of both factors were reduced to ∼60% of their levels in uninduced cultures (Fig. 6 A, lane 1 versus lane 3). Moreover, TNF-α treatment of the CD4U-induced cells further reduced the levels of Bcl-xL and A1/Bfl-1 to <20% of those in TNF-α–treated uninduced cells (Fig. 6 A, lane 2 versus lane 4). These results are significant considering that <10% of the cells shown in lane 1 and <20% of the cells shown in lane 2 were annexin V-positive at the time of the analysis (data not shown). Thus, Vpu indeed downregulated the steady-state levels of Bcl-xL and A1/Bfl-1. The further reduction of Bcl-xL and A1/Bfl-1 levels after TNF-α treatment is presumably a consequence of the concomitant activation of caspase-3 by TNF-α, which is known to proteolytically cleave Bcl-xL (42, 43).

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