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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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Subcellular localization of wild type Vpr and Vpr mutants in human monocyte-derived-macrophages. MDMs expressing either GFP, wt Vpr-GFP, or the indicated Vpr-GFP mutants were fixed and analyzed by wide-field microscopy. Z stacks of fluorescent images were acquired using a piezo with a 0.2 μm increment and one medial section is shown (left panels). Phase contrast images of the same cells were acquired to identify the nucleus (right panels). Scale bar, 5 μm.
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Figure 5: Subcellular localization of wild type Vpr and Vpr mutants in human monocyte-derived-macrophages. MDMs expressing either GFP, wt Vpr-GFP, or the indicated Vpr-GFP mutants were fixed and analyzed by wide-field microscopy. Z stacks of fluorescent images were acquired using a piezo with a 0.2 μm increment and one medial section is shown (left panels). Phase contrast images of the same cells were acquired to identify the nucleus (right panels). Scale bar, 5 μm.

Mentions: In order to confirm that Vpr also accumulated at the nuclear envelope in target cells relevant for HIV-1 replication, the distribution of both wt and mutated Vpr proteins was then analyzed in primary macrophages derived from monocytes (MDMs) isolated from buffy coats of healthy donors. As previously shown in HeLa cells (see Fig. 3), the wt Vpr-GFP fusion localized in the nucleus of MDMs but also concentrated at the NE as a punctuate staining likely corresponding to NPC structures (Fig. 5). A similar punctuate staining at the NE was observed in a myeloid cell line, such as THP-1 cells, expressing the Vpr-GFP fusion (not shown). Again, both Vpr-L23F and -K27M mutants failed to concentrate at the NE and predominantly localized in the cytoplasm as a diffuse staining. These data confirm that Vpr mutants deficient for hCG1-binding also fail to accumulate at the NE in primary macrophages.


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)

Subcellular localization of wild type Vpr and Vpr mutants in human monocyte-derived-macrophages. MDMs expressing either GFP, wt Vpr-GFP, or the indicated Vpr-GFP mutants were fixed and analyzed by wide-field microscopy. Z stacks of fluorescent images were acquired using a piezo with a 0.2 μm increment and one medial section is shown (left panels). Phase contrast images of the same cells were acquired to identify the nucleus (right panels). Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211753&req=5

Figure 5: Subcellular localization of wild type Vpr and Vpr mutants in human monocyte-derived-macrophages. MDMs expressing either GFP, wt Vpr-GFP, or the indicated Vpr-GFP mutants were fixed and analyzed by wide-field microscopy. Z stacks of fluorescent images were acquired using a piezo with a 0.2 μm increment and one medial section is shown (left panels). Phase contrast images of the same cells were acquired to identify the nucleus (right panels). Scale bar, 5 μm.
Mentions: In order to confirm that Vpr also accumulated at the nuclear envelope in target cells relevant for HIV-1 replication, the distribution of both wt and mutated Vpr proteins was then analyzed in primary macrophages derived from monocytes (MDMs) isolated from buffy coats of healthy donors. As previously shown in HeLa cells (see Fig. 3), the wt Vpr-GFP fusion localized in the nucleus of MDMs but also concentrated at the NE as a punctuate staining likely corresponding to NPC structures (Fig. 5). A similar punctuate staining at the NE was observed in a myeloid cell line, such as THP-1 cells, expressing the Vpr-GFP fusion (not shown). Again, both Vpr-L23F and -K27M mutants failed to concentrate at the NE and predominantly localized in the cytoplasm as a diffuse staining. These data confirm that Vpr mutants deficient for hCG1-binding also fail to accumulate at the NE in primary macrophages.

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