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Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging.

Fritz JV, Didier P, Clamme JP, Schaub E, Muriaux D, Cabanne C, Morellet N, Bouaziz S, Darlix JL, Mély Y, de Rocquigny H - Retrovirology (2008)

Bottom Line: Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus.Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect.The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7175 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Illkirch Cedex, France. joelle.fritz@pharma.u-strasbg.fr

ABSTRACT

Background: The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.

Results: Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the alpha-helices could perturb the leucine zipper like motifs. The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.

Conclusion: We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three alpha helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

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Pro-apoptotic properties of the Vpr-eGFP mutants. Cells expressing either the wild type Vpr-eGFP or mutant Vpr-eGFP were selected by fluorescence cytometry, using the eGFP fluorescence. The percentage of cells undergoing apoptosis was assessed by the number of cells labeled with cells with Cy5 alone, or with both Cy5 and PI. Statistical analysis was achieved using the multi-factorial ANOVA test and the Dunnett analysis. Three independent measurements were performed for each assay.
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Figure 7: Pro-apoptotic properties of the Vpr-eGFP mutants. Cells expressing either the wild type Vpr-eGFP or mutant Vpr-eGFP were selected by fluorescence cytometry, using the eGFP fluorescence. The percentage of cells undergoing apoptosis was assessed by the number of cells labeled with cells with Cy5 alone, or with both Cy5 and PI. Statistical analysis was achieved using the multi-factorial ANOVA test and the Dunnett analysis. Three independent measurements were performed for each assay.

Mentions: Vpr can induce apoptosis of infected cells and probably of bystander cells [5,6]. In order to evaluate the role of Vpr oligomerization on its pro-apoptotic activity, FACS analyses were carried out. To this end, annexin V and propidium iodide staining of HeLa cells expressing eGFP, Vpr-eGFP or Vpr-eGFP mutants were performed 72 hours after transfection (see methods). Results show that 6% of mock transfected cells (data not shown) and 16% of cells expressing eGFP were apoptotic (Figure. 7). The percentages of apoptotic cells expressing either Vpr-eGFP or one mutant varied from 45 to 70% as compared to the 43% obtained with wt Vpr (data not shown) [12,47]. Thus, no significant reduction of apoptosis was monitored for the Vpr-eGFP mutants examined here. As a consequence there is no clear correlation between the intracellular oligomerization of Vpr and its pro-apoptotic properties.


Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging.

Fritz JV, Didier P, Clamme JP, Schaub E, Muriaux D, Cabanne C, Morellet N, Bouaziz S, Darlix JL, Mély Y, de Rocquigny H - Retrovirology (2008)

Pro-apoptotic properties of the Vpr-eGFP mutants. Cells expressing either the wild type Vpr-eGFP or mutant Vpr-eGFP were selected by fluorescence cytometry, using the eGFP fluorescence. The percentage of cells undergoing apoptosis was assessed by the number of cells labeled with cells with Cy5 alone, or with both Cy5 and PI. Statistical analysis was achieved using the multi-factorial ANOVA test and the Dunnett analysis. Three independent measurements were performed for each assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562391&req=5

Figure 7: Pro-apoptotic properties of the Vpr-eGFP mutants. Cells expressing either the wild type Vpr-eGFP or mutant Vpr-eGFP were selected by fluorescence cytometry, using the eGFP fluorescence. The percentage of cells undergoing apoptosis was assessed by the number of cells labeled with cells with Cy5 alone, or with both Cy5 and PI. Statistical analysis was achieved using the multi-factorial ANOVA test and the Dunnett analysis. Three independent measurements were performed for each assay.
Mentions: Vpr can induce apoptosis of infected cells and probably of bystander cells [5,6]. In order to evaluate the role of Vpr oligomerization on its pro-apoptotic activity, FACS analyses were carried out. To this end, annexin V and propidium iodide staining of HeLa cells expressing eGFP, Vpr-eGFP or Vpr-eGFP mutants were performed 72 hours after transfection (see methods). Results show that 6% of mock transfected cells (data not shown) and 16% of cells expressing eGFP were apoptotic (Figure. 7). The percentages of apoptotic cells expressing either Vpr-eGFP or one mutant varied from 45 to 70% as compared to the 43% obtained with wt Vpr (data not shown) [12,47]. Thus, no significant reduction of apoptosis was monitored for the Vpr-eGFP mutants examined here. As a consequence there is no clear correlation between the intracellular oligomerization of Vpr and its pro-apoptotic properties.

Bottom Line: Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus.Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect.The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7175 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Illkirch Cedex, France. joelle.fritz@pharma.u-strasbg.fr

ABSTRACT

Background: The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.

Results: Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the alpha-helices could perturb the leucine zipper like motifs. The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.

Conclusion: We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three alpha helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

Show MeSH
Related in: MedlinePlus