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PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins.

Godet AN, Guergnon J, Croset A, Cayla X, Falanga PB, Colle JH, Garcia A - PLoS ONE (2010)

Bottom Line: We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis.In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire E3 Phosphatases, Unité Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, Paris, France.

ABSTRACT

Background: The hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal findings: Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.

Conclusion: Together our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

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Effect of DPT-Vpr peptides on cell penetration and intracellular delivery of streptavidin-peroxydase.(A) For penetration and localization analysis, cells were incubated with 150 µM of peptides for 2 h at 37°C. After fixation, the presence of biotinynlated peptides is revealed by incubation of permeabilized cells with streptavidin-peroxydase. The sequence of non penetrating peptide used as negative control is GVIFYLRDK. The sequence of positive Tat control is YGRKKRRQRR. (B) Intracellular delivery of streptavidin-peroxydase by biotinylated-DPT-Vpr and Tat peptides in HeLa cells. Streptavidin-peroxydase coupled with biotinylated peptides were incubated for 6 h at 37°C and internalized complexes were visualized by a colorimetric test. Statistical analysis was carried out using Anova's test and significance was assessed at p<0.0001.
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pone-0013760-g002: Effect of DPT-Vpr peptides on cell penetration and intracellular delivery of streptavidin-peroxydase.(A) For penetration and localization analysis, cells were incubated with 150 µM of peptides for 2 h at 37°C. After fixation, the presence of biotinynlated peptides is revealed by incubation of permeabilized cells with streptavidin-peroxydase. The sequence of non penetrating peptide used as negative control is GVIFYLRDK. The sequence of positive Tat control is YGRKKRRQRR. (B) Intracellular delivery of streptavidin-peroxydase by biotinylated-DPT-Vpr and Tat peptides in HeLa cells. Streptavidin-peroxydase coupled with biotinylated peptides were incubated for 6 h at 37°C and internalized complexes were visualized by a colorimetric test. Statistical analysis was carried out using Anova's test and significance was assessed at p<0.0001.

Mentions: It is well established that Tat peptides derived from the cell penetrating protein HIV-1 Tat are cell penetrating sequences that allow intracellular delivery of proteins. Tat transduction depends on various factors, and current protocols permit the transduction into many cells [10]. Since Vpr is a known as cell penetrating protein, we hypothesized that DPT-Vpr peptides containing the Vpr77–92 sequence could also display a transduction activity. To test whether DPT-Vpr peptides display similar activity, biotinylated DPT-Vpr peptides were assessed to penetrate and to deliver a protein marker (Streptavidin-HRP) into HeLa cells. As shown in figure 2A, Tat, DPT-Vpr1, DPT-Vpr2 and DPT-Vpr3 peptides penetrate into HeLa cells. However, Tat and DPT-Vpr peptides showed subtle cell staining differences concerning their intra-cellular localization. While Tat, DPT-Vpr1 and DPT-Vpr3 mostly provided a homogeneous cytoplasm labeling, DPT-Vpr2 provided also nuclear/nucleolar labeling. Then the capacity of the biotinylated DPT-Vpr peptides to cargo protein into cell was analyzed. The amounts of HRP internalized by incubating the cells for five hours with three different biotinylated DPT-Vpr Streptavidin-HRP complexes were shown in figure 2B. DPT-Vpr1 and DPT-Vpr3 peptides (50 µM) resulted in higher levels of HRP internalization than Tat-peptide, the positive reference of the assay (16.7 and 45.3 fold increase respectively). Although DPT-Vpr2, was found able to permeate cells (Fig. 2A), surprisingly no cargo effect could be detected with cells incubated with Streptavidin-HRP linked to biotinylated-DPT-Vpr2, the signal recorded remained as low as the cells control incubated with the media plus Streptavidin-HRP alone. Thus the lack of cargo effect with DPT-Vpr2 peptide correlated with its inability of binding to PP2A1 from cell extracts.


PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins.

Godet AN, Guergnon J, Croset A, Cayla X, Falanga PB, Colle JH, Garcia A - PLoS ONE (2010)

Effect of DPT-Vpr peptides on cell penetration and intracellular delivery of streptavidin-peroxydase.(A) For penetration and localization analysis, cells were incubated with 150 µM of peptides for 2 h at 37°C. After fixation, the presence of biotinynlated peptides is revealed by incubation of permeabilized cells with streptavidin-peroxydase. The sequence of non penetrating peptide used as negative control is GVIFYLRDK. The sequence of positive Tat control is YGRKKRRQRR. (B) Intracellular delivery of streptavidin-peroxydase by biotinylated-DPT-Vpr and Tat peptides in HeLa cells. Streptavidin-peroxydase coupled with biotinylated peptides were incubated for 6 h at 37°C and internalized complexes were visualized by a colorimetric test. Statistical analysis was carried out using Anova's test and significance was assessed at p<0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2967473&req=5

pone-0013760-g002: Effect of DPT-Vpr peptides on cell penetration and intracellular delivery of streptavidin-peroxydase.(A) For penetration and localization analysis, cells were incubated with 150 µM of peptides for 2 h at 37°C. After fixation, the presence of biotinynlated peptides is revealed by incubation of permeabilized cells with streptavidin-peroxydase. The sequence of non penetrating peptide used as negative control is GVIFYLRDK. The sequence of positive Tat control is YGRKKRRQRR. (B) Intracellular delivery of streptavidin-peroxydase by biotinylated-DPT-Vpr and Tat peptides in HeLa cells. Streptavidin-peroxydase coupled with biotinylated peptides were incubated for 6 h at 37°C and internalized complexes were visualized by a colorimetric test. Statistical analysis was carried out using Anova's test and significance was assessed at p<0.0001.
Mentions: It is well established that Tat peptides derived from the cell penetrating protein HIV-1 Tat are cell penetrating sequences that allow intracellular delivery of proteins. Tat transduction depends on various factors, and current protocols permit the transduction into many cells [10]. Since Vpr is a known as cell penetrating protein, we hypothesized that DPT-Vpr peptides containing the Vpr77–92 sequence could also display a transduction activity. To test whether DPT-Vpr peptides display similar activity, biotinylated DPT-Vpr peptides were assessed to penetrate and to deliver a protein marker (Streptavidin-HRP) into HeLa cells. As shown in figure 2A, Tat, DPT-Vpr1, DPT-Vpr2 and DPT-Vpr3 peptides penetrate into HeLa cells. However, Tat and DPT-Vpr peptides showed subtle cell staining differences concerning their intra-cellular localization. While Tat, DPT-Vpr1 and DPT-Vpr3 mostly provided a homogeneous cytoplasm labeling, DPT-Vpr2 provided also nuclear/nucleolar labeling. Then the capacity of the biotinylated DPT-Vpr peptides to cargo protein into cell was analyzed. The amounts of HRP internalized by incubating the cells for five hours with three different biotinylated DPT-Vpr Streptavidin-HRP complexes were shown in figure 2B. DPT-Vpr1 and DPT-Vpr3 peptides (50 µM) resulted in higher levels of HRP internalization than Tat-peptide, the positive reference of the assay (16.7 and 45.3 fold increase respectively). Although DPT-Vpr2, was found able to permeate cells (Fig. 2A), surprisingly no cargo effect could be detected with cells incubated with Streptavidin-HRP linked to biotinylated-DPT-Vpr2, the signal recorded remained as low as the cells control incubated with the media plus Streptavidin-HRP alone. Thus the lack of cargo effect with DPT-Vpr2 peptide correlated with its inability of binding to PP2A1 from cell extracts.

Bottom Line: We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis.In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire E3 Phosphatases, Unité Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, Paris, France.

ABSTRACT

Background: The hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal findings: Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.

Conclusion: Together our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

Show MeSH
Related in: MedlinePlus