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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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Impact of the Vpr mutations on HIV-1 replication in monocyte-derived macrophages. A) Packaging assay of the wt and mutated HA-tagged HIV-1 vpr into virus like particles. 293T cells were transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the vpr gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the vpr-defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean.
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Figure 6: Impact of the Vpr mutations on HIV-1 replication in monocyte-derived macrophages. A) Packaging assay of the wt and mutated HA-tagged HIV-1 vpr into virus like particles. 293T cells were transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the vpr gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the vpr-defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean.

Mentions: Finally, the relationship between the Vpr docking at the NE and HIV-1 replication in non-dividing cells was explored by analyzing the impact of the hCG1-binding deficient Vpr-L23F and -K27M mutations on viral replication in primary macrophages. The requirement of Vpr for early stages of the virus life cycle, including nuclear transport of the viral DNA (for review, see Ref. [17]), has been associated with its packaging into virions and the resultant presence in the cytoplasm of newly infected cells. Using a transient Vpr packaging assay in which HA-tagged Vpr is expressed in trans in virus producing cells [32], we therefore analyzed whether the two Vpr mutants were incorporated into virions. As evidenced in Fig. 6A, both Vpr-L23F and -K27M were efficiently packaged into purified virions, but a slight difference in the level of incorporation was repeatedly observed.


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)

Impact of the Vpr mutations on HIV-1 replication in monocyte-derived macrophages. A) Packaging assay of the wt and mutated HA-tagged HIV-1 vpr into virus like particles. 293T cells were transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the vpr gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the vpr-defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean.
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Figure 6: Impact of the Vpr mutations on HIV-1 replication in monocyte-derived macrophages. A) Packaging assay of the wt and mutated HA-tagged HIV-1 vpr into virus like particles. 293T cells were transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the vpr gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the vpr-defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean.
Mentions: Finally, the relationship between the Vpr docking at the NE and HIV-1 replication in non-dividing cells was explored by analyzing the impact of the hCG1-binding deficient Vpr-L23F and -K27M mutations on viral replication in primary macrophages. The requirement of Vpr for early stages of the virus life cycle, including nuclear transport of the viral DNA (for review, see Ref. [17]), has been associated with its packaging into virions and the resultant presence in the cytoplasm of newly infected cells. Using a transient Vpr packaging assay in which HA-tagged Vpr is expressed in trans in virus producing cells [32], we therefore analyzed whether the two Vpr mutants were incorporated into virions. As evidenced in Fig. 6A, both Vpr-L23F and -K27M were efficiently packaged into purified virions, but a slight difference in the level of incorporation was repeatedly observed.

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