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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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Identification of Vpr mutants deficient for binding to the nucleoporin hCG1. A) Screening for Vpr mutants defective for the interaction with hCG1. The L40 yeast reporter strain expressing the wt or mutated (clones 11 and 35, and Vpr-R90K and -W54R single-point mutants) HIV-1 Vpr fused either to LexABD (upper panels) or to the Gal4 DNA binding domain (Gal4BD) (lower panels), in combination with each of the Gal4AD-hybrids indicated on the top was analyzed for histidine auxotrophy and β-Gal activity. Double transformants were patched on selective medium with histidine (+His) and then replica-plated on medium without histidine (-His) and on Whatman filters for β-Gal assay. Growth in the absence of histidine and expression of β-galactosidase indicated an interaction between hybrid proteins. B) Amino acid substitutions found in the hCG1-binding deficient Vpr mutants (clones 11 and 35). Mutants were derived by error prone PCR-mediated mutagenesis from the primary sequence of the VprLai strain that is shown at the top. C) Isolation of single-point Vpr mutants defective for the interaction with hCG1. Single-point mutants derived from Vpr clones 11 and 35 fused to LexABD were expressed in L40 strain in combination with each of the Gal4AD-hybrids indicated on the top. Double transformants were assessed as described in A).
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Figure 1: Identification of Vpr mutants deficient for binding to the nucleoporin hCG1. A) Screening for Vpr mutants defective for the interaction with hCG1. The L40 yeast reporter strain expressing the wt or mutated (clones 11 and 35, and Vpr-R90K and -W54R single-point mutants) HIV-1 Vpr fused either to LexABD (upper panels) or to the Gal4 DNA binding domain (Gal4BD) (lower panels), in combination with each of the Gal4AD-hybrids indicated on the top was analyzed for histidine auxotrophy and β-Gal activity. Double transformants were patched on selective medium with histidine (+His) and then replica-plated on medium without histidine (-His) and on Whatman filters for β-Gal assay. Growth in the absence of histidine and expression of β-galactosidase indicated an interaction between hybrid proteins. B) Amino acid substitutions found in the hCG1-binding deficient Vpr mutants (clones 11 and 35). Mutants were derived by error prone PCR-mediated mutagenesis from the primary sequence of the VprLai strain that is shown at the top. C) Isolation of single-point Vpr mutants defective for the interaction with hCG1. Single-point mutants derived from Vpr clones 11 and 35 fused to LexABD were expressed in L40 strain in combination with each of the Gal4AD-hybrids indicated on the top. Double transformants were assessed as described in A).

Mentions: Previous studies have established that the localization of HIV-1 Vpr to the NE is related to its ability to interact with components of the NPC [23,25,26], including the nucleoporin hCG1 [28]. In order to identify single-point mutations that altered the Vpr binding to hCG1, we generated a library of random Vpr mutants and used the yeast two-hybrid system to screen for hCG1-binding deficient Vpr mutants. Only mutants which retained the capacity to interact with UNG2 and HHR23A, two other known relevant host partners of Vpr [30,31] but failed to bind hCG1 were selected. Two Vpr mutants (clones 11 and 35) that still interacted with UNG2 and HHR23A were isolated (Fig. 1A and data not shown, respectively), as evidenced by growth of yeast-transformed cells on medium without histidine (-His) and β-gal activity. In contrast, these mutants did not bind to hCG1, since no growth on -His medium and β-gal activity was observed. Used as controls, the VprR90K mutant, which is known to abolish Vpr-induced G2-arrest [31], still bound both to hCG1 and UNG2, while the W54R mutant, which is deficient for binding to UNG2 [32], still interacted with hCG1 (Fig. 1A, lower panel). These results show that this yeast two-hybrid strategy is a powerful system to isolate specific hCG1-binding deficient Vpr mutants.


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)

Identification of Vpr mutants deficient for binding to the nucleoporin hCG1. A) Screening for Vpr mutants defective for the interaction with hCG1. The L40 yeast reporter strain expressing the wt or mutated (clones 11 and 35, and Vpr-R90K and -W54R single-point mutants) HIV-1 Vpr fused either to LexABD (upper panels) or to the Gal4 DNA binding domain (Gal4BD) (lower panels), in combination with each of the Gal4AD-hybrids indicated on the top was analyzed for histidine auxotrophy and β-Gal activity. Double transformants were patched on selective medium with histidine (+His) and then replica-plated on medium without histidine (-His) and on Whatman filters for β-Gal assay. Growth in the absence of histidine and expression of β-galactosidase indicated an interaction between hybrid proteins. B) Amino acid substitutions found in the hCG1-binding deficient Vpr mutants (clones 11 and 35). Mutants were derived by error prone PCR-mediated mutagenesis from the primary sequence of the VprLai strain that is shown at the top. C) Isolation of single-point Vpr mutants defective for the interaction with hCG1. Single-point mutants derived from Vpr clones 11 and 35 fused to LexABD were expressed in L40 strain in combination with each of the Gal4AD-hybrids indicated on the top. Double transformants were assessed as described in A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Identification of Vpr mutants deficient for binding to the nucleoporin hCG1. A) Screening for Vpr mutants defective for the interaction with hCG1. The L40 yeast reporter strain expressing the wt or mutated (clones 11 and 35, and Vpr-R90K and -W54R single-point mutants) HIV-1 Vpr fused either to LexABD (upper panels) or to the Gal4 DNA binding domain (Gal4BD) (lower panels), in combination with each of the Gal4AD-hybrids indicated on the top was analyzed for histidine auxotrophy and β-Gal activity. Double transformants were patched on selective medium with histidine (+His) and then replica-plated on medium without histidine (-His) and on Whatman filters for β-Gal assay. Growth in the absence of histidine and expression of β-galactosidase indicated an interaction between hybrid proteins. B) Amino acid substitutions found in the hCG1-binding deficient Vpr mutants (clones 11 and 35). Mutants were derived by error prone PCR-mediated mutagenesis from the primary sequence of the VprLai strain that is shown at the top. C) Isolation of single-point Vpr mutants defective for the interaction with hCG1. Single-point mutants derived from Vpr clones 11 and 35 fused to LexABD were expressed in L40 strain in combination with each of the Gal4AD-hybrids indicated on the top. Double transformants were assessed as described in A).
Mentions: Previous studies have established that the localization of HIV-1 Vpr to the NE is related to its ability to interact with components of the NPC [23,25,26], including the nucleoporin hCG1 [28]. In order to identify single-point mutations that altered the Vpr binding to hCG1, we generated a library of random Vpr mutants and used the yeast two-hybrid system to screen for hCG1-binding deficient Vpr mutants. Only mutants which retained the capacity to interact with UNG2 and HHR23A, two other known relevant host partners of Vpr [30,31] but failed to bind hCG1 were selected. Two Vpr mutants (clones 11 and 35) that still interacted with UNG2 and HHR23A were isolated (Fig. 1A and data not shown, respectively), as evidenced by growth of yeast-transformed cells on medium without histidine (-His) and β-gal activity. In contrast, these mutants did not bind to hCG1, since no growth on -His medium and β-gal activity was observed. Used as controls, the VprR90K mutant, which is known to abolish Vpr-induced G2-arrest [31], still bound both to hCG1 and UNG2, while the W54R mutant, which is deficient for binding to UNG2 [32], still interacted with hCG1 (Fig. 1A, lower panel). These results show that this yeast two-hybrid strategy is a powerful system to isolate specific hCG1-binding deficient Vpr mutants.

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