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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 induces apoptosis in HIV-1–infected Jurkat cells. Uninfected Jurkat cells (a) or Jurkat cells infected with an m.o.i. of 5 with VSV-G-pseudotyped virus stocks of (b) wild-type (WT) NL4–3, (c) NL43-K1 (Env−), or (d) NL4–3/Udel (Vpu−) variants were used for this analysis. (A) Cultures were analyzed 48 h after infection for the presence of apoptotic cells by staining with 7-AAD and PE-conjugated annexin V, followed by flow cytometric analysis. Numbers represent the percentages of cells in the respective quadrants. (B) The same cultures were evaluated 24 h after infection for HIV-1 infection by intracellular p24 staining using PE-conjugated mouse mAb to HIV-1 p24 followed by flow cytometry. Numbers represent the percentages of p24-positive cells. The dotted lines in panels a–d represent p24-staining of mock-infected cells. The solid lines in panels a–d represent p24-staining of infected cells. The results shown are representative of three independent experiments.
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fig1: Vpu induces apoptosis in HIV-1–infected Jurkat cells. Uninfected Jurkat cells (a) or Jurkat cells infected with an m.o.i. of 5 with VSV-G-pseudotyped virus stocks of (b) wild-type (WT) NL4–3, (c) NL43-K1 (Env−), or (d) NL4–3/Udel (Vpu−) variants were used for this analysis. (A) Cultures were analyzed 48 h after infection for the presence of apoptotic cells by staining with 7-AAD and PE-conjugated annexin V, followed by flow cytometric analysis. Numbers represent the percentages of cells in the respective quadrants. (B) The same cultures were evaluated 24 h after infection for HIV-1 infection by intracellular p24 staining using PE-conjugated mouse mAb to HIV-1 p24 followed by flow cytometry. Numbers represent the percentages of p24-positive cells. The dotted lines in panels a–d represent p24-staining of mock-infected cells. The solid lines in panels a–d represent p24-staining of infected cells. The results shown are representative of three independent experiments.

Mentions: In the first set of experiments, Jurkat cells were single-cycle infected with VSV-G-pseudotyped HIV-1NL4–3 (NL4–3/G) at a multiplicity of infection (m.o.i.) of 5. Cells were analyzed 48 h after infection for the induction of apoptosis using annexin V and 7-AAD staining as markers (Fig. 1 A). In mock-infected cultures, only 1.7% of the cells were annexin V-positive with half of those cells also scoring positive for 7-AAD (Fig. 1 A, panel a). In contrast, in cultures infected with NL4–3/G, 51.1% of the cells were in the early phase of apoptosis (annexin V+ 7-AAD−) and 28.8% of the cells were already in the late phase of apoptosis (annexin V+ 7-AAD+; Fig. 1 A, panel b). To assess the relative impact of Env expression on the induction of apoptosis, Jurkat cells were infected with a VSV-G-pseudotyped Env-defective variant, HIV-1NL43-K1 (Fig. 1 A, panel c). The results showed that the proportion of cells found in the early (48.1%) or late phase of apoptosis (24.4%) was comparable to that in NL4–3/G-infected cells (compare panels b and c). This indicates that the expression of HIV-1 Env is not a major factor in the induction of apoptosis in our experimental system. Surprisingly, infection of Jurkat cells by the pseudotyped Vpu-defective variant, NL4–3/Udel/G (Fig. 1 A, panel d) showed a significant reduction of annexin V- and 7-AAD-positive cells. In fact, the total number of annexin V-positive cells was reduced by >40% in the absence of Vpu relative to wild-type virus (compare Fig. 1 panels b and d). These results suggest that Vpu may play a significant role in the induction of apoptosis in HIV-1-infected cells. To ascertain that the results from Fig. 1 A were not biased by different infection efficiencies, we determined the intracellular expression levels of Gag proteins for the cultures shown in Fig. 1 A by staining with a p24 capsid (CA) antibody followed by flow cytometry (Fig. 1 B). The results demonstrate that all cultures (except mock) were infected with similar efficiency and produced comparable levels of p24 CA antigen, as determined by fluorescence intensity.


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 induces apoptosis in HIV-1–infected Jurkat cells. Uninfected Jurkat cells (a) or Jurkat cells infected with an m.o.i. of 5 with VSV-G-pseudotyped virus stocks of (b) wild-type (WT) NL4–3, (c) NL43-K1 (Env−), or (d) NL4–3/Udel (Vpu−) variants were used for this analysis. (A) Cultures were analyzed 48 h after infection for the presence of apoptotic cells by staining with 7-AAD and PE-conjugated annexin V, followed by flow cytometric analysis. Numbers represent the percentages of cells in the respective quadrants. (B) The same cultures were evaluated 24 h after infection for HIV-1 infection by intracellular p24 staining using PE-conjugated mouse mAb to HIV-1 p24 followed by flow cytometry. Numbers represent the percentages of p24-positive cells. The dotted lines in panels a–d represent p24-staining of mock-infected cells. The solid lines in panels a–d represent p24-staining of infected cells. The results shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195969&req=5

fig1: Vpu induces apoptosis in HIV-1–infected Jurkat cells. Uninfected Jurkat cells (a) or Jurkat cells infected with an m.o.i. of 5 with VSV-G-pseudotyped virus stocks of (b) wild-type (WT) NL4–3, (c) NL43-K1 (Env−), or (d) NL4–3/Udel (Vpu−) variants were used for this analysis. (A) Cultures were analyzed 48 h after infection for the presence of apoptotic cells by staining with 7-AAD and PE-conjugated annexin V, followed by flow cytometric analysis. Numbers represent the percentages of cells in the respective quadrants. (B) The same cultures were evaluated 24 h after infection for HIV-1 infection by intracellular p24 staining using PE-conjugated mouse mAb to HIV-1 p24 followed by flow cytometry. Numbers represent the percentages of p24-positive cells. The dotted lines in panels a–d represent p24-staining of mock-infected cells. The solid lines in panels a–d represent p24-staining of infected cells. The results shown are representative of three independent experiments.
Mentions: In the first set of experiments, Jurkat cells were single-cycle infected with VSV-G-pseudotyped HIV-1NL4–3 (NL4–3/G) at a multiplicity of infection (m.o.i.) of 5. Cells were analyzed 48 h after infection for the induction of apoptosis using annexin V and 7-AAD staining as markers (Fig. 1 A). In mock-infected cultures, only 1.7% of the cells were annexin V-positive with half of those cells also scoring positive for 7-AAD (Fig. 1 A, panel a). In contrast, in cultures infected with NL4–3/G, 51.1% of the cells were in the early phase of apoptosis (annexin V+ 7-AAD−) and 28.8% of the cells were already in the late phase of apoptosis (annexin V+ 7-AAD+; Fig. 1 A, panel b). To assess the relative impact of Env expression on the induction of apoptosis, Jurkat cells were infected with a VSV-G-pseudotyped Env-defective variant, HIV-1NL43-K1 (Fig. 1 A, panel c). The results showed that the proportion of cells found in the early (48.1%) or late phase of apoptosis (24.4%) was comparable to that in NL4–3/G-infected cells (compare panels b and c). This indicates that the expression of HIV-1 Env is not a major factor in the induction of apoptosis in our experimental system. Surprisingly, infection of Jurkat cells by the pseudotyped Vpu-defective variant, NL4–3/Udel/G (Fig. 1 A, panel d) showed a significant reduction of annexin V- and 7-AAD-positive cells. In fact, the total number of annexin V-positive cells was reduced by >40% in the absence of Vpu relative to wild-type virus (compare Fig. 1 panels b and d). These results suggest that Vpu may play a significant role in the induction of apoptosis in HIV-1-infected cells. To ascertain that the results from Fig. 1 A were not biased by different infection efficiencies, we determined the intracellular expression levels of Gag proteins for the cultures shown in Fig. 1 A by staining with a p24 capsid (CA) antibody followed by flow cytometry (Fig. 1 B). The results demonstrate that all cultures (except mock) were infected with similar efficiency and produced comparable levels of p24 CA antigen, as determined by fluorescence intensity.

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