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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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Direct Vpr-Vpr interaction in HeLa cells visualized by FLIM. Cells were transfected with the DNA construct encoding eGFP or eGFP-Vpr alone or in combination with mCherry-Vpr. In the FLIM images, the lifetimes are represented using an arbitrary color scale ranging from blue to red for short and long lifetimes in nanoseconds (right bottom), respectively. The Vpr-eGFP or eGFP-Vpr with short lifetime fluorescence symbolized by the blue color were mainly localized at the nuclear envelope and also in other cell compartments when co transfected with mCherry tagged Vpr. Panels A1 to A3 show the lifetime images of cells expressing eGFP or eGFP-tagged Vpr alone. Panels B1 and B2 represent cells coexpressing eGFP-tagged Vpr and mCherry; Panels B3 and C1-C3 show the lifetime images of cells coexpressing eGFP-tagged Vpr and mCherry-tagged Vpr. Note the accumulation of Vpr fusion proteins at or near the nuclear envelope.
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Figure 4: Direct Vpr-Vpr interaction in HeLa cells visualized by FLIM. Cells were transfected with the DNA construct encoding eGFP or eGFP-Vpr alone or in combination with mCherry-Vpr. In the FLIM images, the lifetimes are represented using an arbitrary color scale ranging from blue to red for short and long lifetimes in nanoseconds (right bottom), respectively. The Vpr-eGFP or eGFP-Vpr with short lifetime fluorescence symbolized by the blue color were mainly localized at the nuclear envelope and also in other cell compartments when co transfected with mCherry tagged Vpr. Panels A1 to A3 show the lifetime images of cells expressing eGFP or eGFP-tagged Vpr alone. Panels B1 and B2 represent cells coexpressing eGFP-tagged Vpr and mCherry; Panels B3 and C1-C3 show the lifetime images of cells coexpressing eGFP-tagged Vpr and mCherry-tagged Vpr. Note the accumulation of Vpr fusion proteins at or near the nuclear envelope.

Mentions: HeLa cells were transfected and FLIM measurements were monitored at 24, 48 and 72 hours but since no time dependant effect was monitored; only measurements at 24 h are presented. Experiments were performed first on cells expressing eGFP or Vpr eGFP fusion protein as a control (Figure 4, panels A1-3) and next on cells co-expressing Vpr-eGFP and mCherry fusion proteins (Figure 4, panels B1-3 and C1-3). An arbitrary color scale, ranging from blue to red, illustrates short to long lifetimes. The Vpr-eGFP fluorescence was mainly localized at the nuclear envelope and also in other cell compartments, where FLIM measurements can be carried out. We focused on three distinct regions, namely the nuclear rim, the cytoplasm and the nucleus (Table 1). For the cytoplasm and the nuclear region, care was taken to exclude pixels with contribution from the nuclear envelope. Moreover, due to the thickness of the nuclear envelope, the pixels used to calculate the lifetime values of the nuclear envelope involved contributions from cytoplasmic and nuclear Vpr. Nevertheless, due to the strong accumulation of Vpr at the nuclear membrane, we assumed that the lifetimes mainly reflected the behaviour of the Vpr fusion proteins at this site (see Table 1). FLIM measurements were carried out


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)

Direct Vpr-Vpr interaction in HeLa cells visualized by FLIM. Cells were transfected with the DNA construct encoding eGFP or eGFP-Vpr alone or in combination with mCherry-Vpr. In the FLIM images, the lifetimes are represented using an arbitrary color scale ranging from blue to red for short and long lifetimes in nanoseconds (right bottom), respectively. The Vpr-eGFP or eGFP-Vpr with short lifetime fluorescence symbolized by the blue color were mainly localized at the nuclear envelope and also in other cell compartments when co transfected with mCherry tagged Vpr. Panels A1 to A3 show the lifetime images of cells expressing eGFP or eGFP-tagged Vpr alone. Panels B1 and B2 represent cells coexpressing eGFP-tagged Vpr and mCherry; Panels B3 and C1-C3 show the lifetime images of cells coexpressing eGFP-tagged Vpr and mCherry-tagged Vpr. Note the accumulation of Vpr fusion proteins at or near the nuclear envelope.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Direct Vpr-Vpr interaction in HeLa cells visualized by FLIM. Cells were transfected with the DNA construct encoding eGFP or eGFP-Vpr alone or in combination with mCherry-Vpr. In the FLIM images, the lifetimes are represented using an arbitrary color scale ranging from blue to red for short and long lifetimes in nanoseconds (right bottom), respectively. The Vpr-eGFP or eGFP-Vpr with short lifetime fluorescence symbolized by the blue color were mainly localized at the nuclear envelope and also in other cell compartments when co transfected with mCherry tagged Vpr. Panels A1 to A3 show the lifetime images of cells expressing eGFP or eGFP-tagged Vpr alone. Panels B1 and B2 represent cells coexpressing eGFP-tagged Vpr and mCherry; Panels B3 and C1-C3 show the lifetime images of cells coexpressing eGFP-tagged Vpr and mCherry-tagged Vpr. Note the accumulation of Vpr fusion proteins at or near the nuclear envelope.
Mentions: HeLa cells were transfected and FLIM measurements were monitored at 24, 48 and 72 hours but since no time dependant effect was monitored; only measurements at 24 h are presented. Experiments were performed first on cells expressing eGFP or Vpr eGFP fusion protein as a control (Figure 4, panels A1-3) and next on cells co-expressing Vpr-eGFP and mCherry fusion proteins (Figure 4, panels B1-3 and C1-3). An arbitrary color scale, ranging from blue to red, illustrates short to long lifetimes. The Vpr-eGFP fluorescence was mainly localized at the nuclear envelope and also in other cell compartments, where FLIM measurements can be carried out. We focused on three distinct regions, namely the nuclear rim, the cytoplasm and the nucleus (Table 1). For the cytoplasm and the nuclear region, care was taken to exclude pixels with contribution from the nuclear envelope. Moreover, due to the thickness of the nuclear envelope, the pixels used to calculate the lifetime values of the nuclear envelope involved contributions from cytoplasmic and nuclear Vpr. Nevertheless, due to the strong accumulation of Vpr at the nuclear membrane, we assumed that the lifetimes mainly reflected the behaviour of the Vpr fusion proteins at this site (see Table 1). FLIM measurements were carried out

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