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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-L23F and -K27M substitutions on the three-dimensional structure of Vpr. A) 3D structure of HIV-1 Vpr [10], showing the three α-helices (residues 17–33, 38–50 and 54–77) represented in light blue, yellow and purple, respectively. The L23, K27, A30 and F34 residues are colored in red. The unstructured N- and C-terminal domains are represented in dark blue. B) CPK representation of Vpr. Residues are colored according to their hydrophobicity, except for L23 and K27 which are colored in yellow. The yellow box is enlarged in C), and this region shows a pocket that is organized around the L23 and K27 residues within the first α-helix and may represent a site for hCG1 binding. D) Helical-wheel diagram of the first α-helix of Vpr extending from a.a. D17 to F34. Residues L23, K27, A30 and F34 which have been mutated in the present study are indicated. Hydrophilic residues are in blue, whereas hydrophobic residues are in red.
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Figure 2: Impact of the Vpr-L23F and -K27M substitutions on the three-dimensional structure of Vpr. A) 3D structure of HIV-1 Vpr [10], showing the three α-helices (residues 17–33, 38–50 and 54–77) represented in light blue, yellow and purple, respectively. The L23, K27, A30 and F34 residues are colored in red. The unstructured N- and C-terminal domains are represented in dark blue. B) CPK representation of Vpr. Residues are colored according to their hydrophobicity, except for L23 and K27 which are colored in yellow. The yellow box is enlarged in C), and this region shows a pocket that is organized around the L23 and K27 residues within the first α-helix and may represent a site for hCG1 binding. D) Helical-wheel diagram of the first α-helix of Vpr extending from a.a. D17 to F34. Residues L23, K27, A30 and F34 which have been mutated in the present study are indicated. Hydrophilic residues are in blue, whereas hydrophobic residues are in red.

Mentions: As deduced from the 3D structure organization of Vpr resolved by NMR (see on Fig. 2A), the Leu23 and Lys27 residues are located in the first N-terminal α-helix H1 (residues 17–33) of Vpr which has amphipathic properties. Leu23 and Lys27 are separated by 3 residues and are thus located on the same face of the first α-helix (Fig. 2D). The connection between these two residues is favored by the formation of a hydrogen-bonding network through the O19/NH23, O23/NH27 and O27/NH31 atoms maintaining the structure of the α-helix. Moreover, the Corey, Pauling, and Koltun (CPK) representation, indicates that the Leu23 and Lys27 residues are located at the bottom of a pocket that is easily accessible to the solvent (Fig. 2B) and could constitute a binding site for hCG1. In addition, the Leu23 residue is hydrophobic and is surrounded by rather hydrophobic residues (Leu20, Trp54, Gly51 and Tyr47) that border one edge of the pocket (Fig. 2C), whereas the Lys27 residue is hydrophilic, positively charged and bordered by hydrophilic residues (Gln44, His40, Asn28 and Glu24) that constitute the second edge of the pocket. The potential structural modifications induced by substitution of Leu23 and Lys27 in Phe and Met, respectively, have been calculated by homology with the wild type Vpr protein using the Swiss-Model program [33-35]. The analysis indicated that the structure of the first α-helix (residues 17–33) is conserved as well as the hydrogen-bonding network allowing the stabilization of the 3 helices of HIV-1 Vpr. This supports the notion that the global 3D structure of the protein is not modified in these two Vpr mutants, as suggested from the yeast two-hybrid analysis.


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-L23F and -K27M substitutions on the three-dimensional structure of Vpr. A) 3D structure of HIV-1 Vpr [10], showing the three α-helices (residues 17–33, 38–50 and 54–77) represented in light blue, yellow and purple, respectively. The L23, K27, A30 and F34 residues are colored in red. The unstructured N- and C-terminal domains are represented in dark blue. B) CPK representation of Vpr. Residues are colored according to their hydrophobicity, except for L23 and K27 which are colored in yellow. The yellow box is enlarged in C), and this region shows a pocket that is organized around the L23 and K27 residues within the first α-helix and may represent a site for hCG1 binding. D) Helical-wheel diagram of the first α-helix of Vpr extending from a.a. D17 to F34. Residues L23, K27, A30 and F34 which have been mutated in the present study are indicated. Hydrophilic residues are in blue, whereas hydrophobic residues are in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Impact of the Vpr-L23F and -K27M substitutions on the three-dimensional structure of Vpr. A) 3D structure of HIV-1 Vpr [10], showing the three α-helices (residues 17–33, 38–50 and 54–77) represented in light blue, yellow and purple, respectively. The L23, K27, A30 and F34 residues are colored in red. The unstructured N- and C-terminal domains are represented in dark blue. B) CPK representation of Vpr. Residues are colored according to their hydrophobicity, except for L23 and K27 which are colored in yellow. The yellow box is enlarged in C), and this region shows a pocket that is organized around the L23 and K27 residues within the first α-helix and may represent a site for hCG1 binding. D) Helical-wheel diagram of the first α-helix of Vpr extending from a.a. D17 to F34. Residues L23, K27, A30 and F34 which have been mutated in the present study are indicated. Hydrophilic residues are in blue, whereas hydrophobic residues are in red.
Mentions: As deduced from the 3D structure organization of Vpr resolved by NMR (see on Fig. 2A), the Leu23 and Lys27 residues are located in the first N-terminal α-helix H1 (residues 17–33) of Vpr which has amphipathic properties. Leu23 and Lys27 are separated by 3 residues and are thus located on the same face of the first α-helix (Fig. 2D). The connection between these two residues is favored by the formation of a hydrogen-bonding network through the O19/NH23, O23/NH27 and O27/NH31 atoms maintaining the structure of the α-helix. Moreover, the Corey, Pauling, and Koltun (CPK) representation, indicates that the Leu23 and Lys27 residues are located at the bottom of a pocket that is easily accessible to the solvent (Fig. 2B) and could constitute a binding site for hCG1. In addition, the Leu23 residue is hydrophobic and is surrounded by rather hydrophobic residues (Leu20, Trp54, Gly51 and Tyr47) that border one edge of the pocket (Fig. 2C), whereas the Lys27 residue is hydrophilic, positively charged and bordered by hydrophilic residues (Gln44, His40, Asn28 and Glu24) that constitute the second edge of the pocket. The potential structural modifications induced by substitution of Leu23 and Lys27 in Phe and Met, respectively, have been calculated by homology with the wild type Vpr protein using the Swiss-Model program [33-35]. The analysis indicated that the structure of the first α-helix (residues 17–33) is conserved as well as the hydrogen-bonding network allowing the stabilization of the 3 helices of HIV-1 Vpr. This supports the notion that the global 3D structure of the protein is not modified in these two Vpr mutants, as suggested from the yeast two-hybrid analysis.

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