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Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages.

Jacquot G, Le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, Benichou S - Retrovirology (2007)

Bottom Line: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr.In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages.These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. jacquot@cochin.inserm.fr

ABSTRACT

Background: HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages.

Results: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first alpha-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors.

Conclusion: These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

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G2-arrest and pro-apoptotic activities of the Vpr mutants. HPB-ALL T cells were transfected with the HA-tagged Vpr (wt or mutated) expression vectors in combination with the GFP expression vector. A) G2-arrest activity. The DNA content was analyzed 48 h after transfection by flow cytometry on GFP-positive cells after staining with propidium iodide. Results are expressed as the percentage of the G2M/G1 ratio relative to that of the wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. B) Pro-apoptotic activity. Cell surface PS exposure was analyzed 72 h after transfection by flow cytometry on GFP-positive cells after staining with phycoerythrin-labelled Annexin V. Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure relative to that measured with wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. C) Expression of wt and mutated HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP (upper panels) and anti-HA antibodies (lower panels).
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Figure 4: G2-arrest and pro-apoptotic activities of the Vpr mutants. HPB-ALL T cells were transfected with the HA-tagged Vpr (wt or mutated) expression vectors in combination with the GFP expression vector. A) G2-arrest activity. The DNA content was analyzed 48 h after transfection by flow cytometry on GFP-positive cells after staining with propidium iodide. Results are expressed as the percentage of the G2M/G1 ratio relative to that of the wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. B) Pro-apoptotic activity. Cell surface PS exposure was analyzed 72 h after transfection by flow cytometry on GFP-positive cells after staining with phycoerythrin-labelled Annexin V. Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure relative to that measured with wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. C) Expression of wt and mutated HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP (upper panels) and anti-HA antibodies (lower panels).

Mentions: Since a functional link was reported between the targeting at the NE and the Vpr-induced cell cycle arrest [36,37], the G2-arrest activity of the Vpr-L23F and Vpr-K27M mutants was first assessed in T lymphocytes. HPB-ALL T lymphoid cells were transfected with wt or mutated HA-tagged Vpr expression vector together with a GFP expression vector (see Fig. 4C), and the DNA content was analyzed 48 h later by flow cytometry on GFP-positive cells after staining with propidium iodide. The results of four independent experiments are recapitulated on Fig. 4A. The Vpr-L23F mutant was affected but retained about 50% of the activity measured for the wt protein, while the Vpr-K27M mutant was more severely affected leading to a residual G2-arrest activity. Consistent with previous observations, the Vpr-F34I mutant was partially altered for the G2-arrest activity [25], while the Vpr-A30L mutant was completely defective [20,36] (Fig. 4A). As controls, the Vpr-R80A and -R90K variants, which still accumulated at the NE (Fig. 3C), were unable to induce a G2-arrest (Fig. 4A and Refs. [31,37]). The pro-apoptotic activity of the wt Vpr protein and the mutants was also assayed, 72 h after transfection, by flow cytometry analysis of the cell surface exposure of phosphatidylserine (PS) after staining with phycoerythrin-labeled Annexin V (Fig. 4B). Interestingly, the Vpr-induced pro-apoptotic activity of all the Vpr mutants, including Vpr-L23F and -K27M, strictly paralleled the results obtained in the cell cycle experiments (compare Fig. 4A and 4B), suggesting that induction of G2-arrest and apoptosis by HIV-1 Vpr are functionally related. As evidenced on Fig. 4C, the reduction in G2-arrest and cell death induction observed with the Vpr mutants could not be explained by important differences in their expression levels, since all mutants were correctly expressed in HPB-ALL T lymphoid cells.


Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages.

Jacquot G, Le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, Benichou S - Retrovirology (2007)

G2-arrest and pro-apoptotic activities of the Vpr mutants. HPB-ALL T cells were transfected with the HA-tagged Vpr (wt or mutated) expression vectors in combination with the GFP expression vector. A) G2-arrest activity. The DNA content was analyzed 48 h after transfection by flow cytometry on GFP-positive cells after staining with propidium iodide. Results are expressed as the percentage of the G2M/G1 ratio relative to that of the wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. B) Pro-apoptotic activity. Cell surface PS exposure was analyzed 72 h after transfection by flow cytometry on GFP-positive cells after staining with phycoerythrin-labelled Annexin V. Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure relative to that measured with wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. C) Expression of wt and mutated HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP (upper panels) and anti-HA antibodies (lower panels).
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: G2-arrest and pro-apoptotic activities of the Vpr mutants. HPB-ALL T cells were transfected with the HA-tagged Vpr (wt or mutated) expression vectors in combination with the GFP expression vector. A) G2-arrest activity. The DNA content was analyzed 48 h after transfection by flow cytometry on GFP-positive cells after staining with propidium iodide. Results are expressed as the percentage of the G2M/G1 ratio relative to that of the wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. B) Pro-apoptotic activity. Cell surface PS exposure was analyzed 72 h after transfection by flow cytometry on GFP-positive cells after staining with phycoerythrin-labelled Annexin V. Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure relative to that measured with wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. C) Expression of wt and mutated HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP (upper panels) and anti-HA antibodies (lower panels).
Mentions: Since a functional link was reported between the targeting at the NE and the Vpr-induced cell cycle arrest [36,37], the G2-arrest activity of the Vpr-L23F and Vpr-K27M mutants was first assessed in T lymphocytes. HPB-ALL T lymphoid cells were transfected with wt or mutated HA-tagged Vpr expression vector together with a GFP expression vector (see Fig. 4C), and the DNA content was analyzed 48 h later by flow cytometry on GFP-positive cells after staining with propidium iodide. The results of four independent experiments are recapitulated on Fig. 4A. The Vpr-L23F mutant was affected but retained about 50% of the activity measured for the wt protein, while the Vpr-K27M mutant was more severely affected leading to a residual G2-arrest activity. Consistent with previous observations, the Vpr-F34I mutant was partially altered for the G2-arrest activity [25], while the Vpr-A30L mutant was completely defective [20,36] (Fig. 4A). As controls, the Vpr-R80A and -R90K variants, which still accumulated at the NE (Fig. 3C), were unable to induce a G2-arrest (Fig. 4A and Refs. [31,37]). The pro-apoptotic activity of the wt Vpr protein and the mutants was also assayed, 72 h after transfection, by flow cytometry analysis of the cell surface exposure of phosphatidylserine (PS) after staining with phycoerythrin-labeled Annexin V (Fig. 4B). Interestingly, the Vpr-induced pro-apoptotic activity of all the Vpr mutants, including Vpr-L23F and -K27M, strictly paralleled the results obtained in the cell cycle experiments (compare Fig. 4A and 4B), suggesting that induction of G2-arrest and apoptosis by HIV-1 Vpr are functionally related. As evidenced on Fig. 4C, the reduction in G2-arrest and cell death induction observed with the Vpr mutants could not be explained by important differences in their expression levels, since all mutants were correctly expressed in HPB-ALL T lymphoid cells.

Bottom Line: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr.In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages.These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. jacquot@cochin.inserm.fr

ABSTRACT

Background: HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages.

Results: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first alpha-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors.

Conclusion: These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

Show MeSH
Related in: MedlinePlus