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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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Related in: MedlinePlus

Visualization of the intracellular co-expression eGFP or mCherry tagged Vpr. Plasmid DNA (0.5 μg each) expressing the Vpr fusion proteins were cotransfected in HeLa cells. One day post transfection, images were recorded with an excitation at 488 nm and emission at 500–550 nm to monitor eGFP expression, and with an excitation at 568 nm and emission at 580–700 nm to monitor mCherry expression, respectively. In the merge images, co-localization of the two proteins is indicated in yellow. Each image is representative of the major phenotype. Note the accumulation of the Vpr fusion proteins at or close to the nuclear envelope.
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Figure 3: Visualization of the intracellular co-expression eGFP or mCherry tagged Vpr. Plasmid DNA (0.5 μg each) expressing the Vpr fusion proteins were cotransfected in HeLa cells. One day post transfection, images were recorded with an excitation at 488 nm and emission at 500–550 nm to monitor eGFP expression, and with an excitation at 568 nm and emission at 580–700 nm to monitor mCherry expression, respectively. In the merge images, co-localization of the two proteins is indicated in yellow. Each image is representative of the major phenotype. Note the accumulation of the Vpr fusion proteins at or close to the nuclear envelope.

Mentions: Co-localization of Vpr-eGFP and either mCherry-Vpr or Vpr-mCherry was visualized by confocal microscopy. As a control, Vpr-eGFP was first co-expressed with mCherry. Localization of Vpr-eGFP at the nuclear rim (Figure 3, panel A1) was similar to that in Figure 2 (panel B2), indicating that the expression of mCherry did not affect the intracellular distribution of Vpr-eGFP. When Vpr-eGFP was co-expressed with Vpr-mCherry, both green and red fluorescence emissions were localised at the rim of the nucleus and to a lesser extent in the cytoplasm and in the nucleus (Figure 3, panels B1-3). A full co-localization of the two Vpr fusion proteins in the same cellular compartments was further evidenced by the yellow color in Figure 3 (panel B3), that shows a nice superposition of the green and red emissions of the two Vpr fusion proteins. Interestingly, expression of Vpr-eGFP with mCherry-Vpr resulted in a partial redistribution of Vpr-eGFP from the nuclear rim toward the cytoplasm (compare Figure 3, panel C1 with Figure 2, panel B2). The overlap of their emissions all over the cell confirmed their similar intracellular distribution (Figure 3, panel C3).


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)

Visualization of the intracellular co-expression eGFP or mCherry tagged Vpr. Plasmid DNA (0.5 μg each) expressing the Vpr fusion proteins were cotransfected in HeLa cells. One day post transfection, images were recorded with an excitation at 488 nm and emission at 500–550 nm to monitor eGFP expression, and with an excitation at 568 nm and emission at 580–700 nm to monitor mCherry expression, respectively. In the merge images, co-localization of the two proteins is indicated in yellow. Each image is representative of the major phenotype. Note the accumulation of the Vpr fusion proteins at or close to the nuclear envelope.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Visualization of the intracellular co-expression eGFP or mCherry tagged Vpr. Plasmid DNA (0.5 μg each) expressing the Vpr fusion proteins were cotransfected in HeLa cells. One day post transfection, images were recorded with an excitation at 488 nm and emission at 500–550 nm to monitor eGFP expression, and with an excitation at 568 nm and emission at 580–700 nm to monitor mCherry expression, respectively. In the merge images, co-localization of the two proteins is indicated in yellow. Each image is representative of the major phenotype. Note the accumulation of the Vpr fusion proteins at or close to the nuclear envelope.
Mentions: Co-localization of Vpr-eGFP and either mCherry-Vpr or Vpr-mCherry was visualized by confocal microscopy. As a control, Vpr-eGFP was first co-expressed with mCherry. Localization of Vpr-eGFP at the nuclear rim (Figure 3, panel A1) was similar to that in Figure 2 (panel B2), indicating that the expression of mCherry did not affect the intracellular distribution of Vpr-eGFP. When Vpr-eGFP was co-expressed with Vpr-mCherry, both green and red fluorescence emissions were localised at the rim of the nucleus and to a lesser extent in the cytoplasm and in the nucleus (Figure 3, panels B1-3). A full co-localization of the two Vpr fusion proteins in the same cellular compartments was further evidenced by the yellow color in Figure 3 (panel B3), that shows a nice superposition of the green and red emissions of the two Vpr fusion proteins. Interestingly, expression of Vpr-eGFP with mCherry-Vpr resulted in a partial redistribution of Vpr-eGFP from the nuclear rim toward the cytoplasm (compare Figure 3, panel C1 with Figure 2, panel B2). The overlap of their emissions all over the cell confirmed their similar intracellular distribution (Figure 3, panel C3).

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