Limits...
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.

Show MeSH

Related in: MedlinePlus

Subcellular localization of eGFP or mCherry tagged Vpr by confocal microscopy. HeLa cells were co-transfected with 0.5 μg of each plasmid and 0.5 μg pcDNA3. Cells were observed by confocal microscopy 24 h post transfection. Each panel shows the major phenotype. (A) mCherry images with excitation at 568 nm and emission at 580 to 700 nm. (B) eGFP images with excitation at 488 nm and emission at 500 to 550 nm. Note the intracellular redistribution of eGFP and mCherry upon fusion with Vpr.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2562391&req=5

Figure 2: Subcellular localization of eGFP or mCherry tagged Vpr by confocal microscopy. HeLa cells were co-transfected with 0.5 μg of each plasmid and 0.5 μg pcDNA3. Cells were observed by confocal microscopy 24 h post transfection. Each panel shows the major phenotype. (A) mCherry images with excitation at 568 nm and emission at 580 to 700 nm. (B) eGFP images with excitation at 488 nm and emission at 500 to 550 nm. Note the intracellular redistribution of eGFP and mCherry upon fusion with Vpr.

Mentions: Four labelled Vpr proteins were obtained by fusing eGFP or mCherry to Vpr either to its N- or C-terminus. Since both eGFP and mCherry are large with respect to Vpr, we first checked whether the fusion affects the intracellular localization of Vpr. To this end, we analyzed by confocal microscopy at 24 h post transfection the expression of both mCherry- (Figure 2, panels A2-3) and eGFP Vpr fusions in HeLa cells (Figure 2, panels B 2-3). Both Vpr-eGFP and Vpr-mCherry showed a nuclear rim staining coincident with the nuclear envelope (NE) (Figure 2, panels A2 and B2) in agreement with the localization of HA-Vpr (additional file 1, [12]). This localization of Vpr at the NE is not driven by the eGFP and mCherry proteins since both fluorescent proteins were found to be spread all over the cells when expressed in their free form (Figure 2 A1 and B1). Localization of HA-Vpr (additional file 1) or His-Vpr [12] confirms that these proteins are predominantly localized at the nuclear membrane and in the nucleus with some cytoplasmic localization. Thus, the fusion of either mCherry or eGFP to the C terminus of Vpr has a limited effect on Vpr localization in the cell even though the relative proportion of Vpr in the nucleus, at the nuclear envelope or in the cytoplasm was modified [10,12,13,24,32]. The distribution pattern of mCherry-Vpr was close to that of Vpr-mCherry except that a larger amount of protein diffused out in the cytoplasm, indicating a limited alteration of Vpr intracellular distribution by the mCherry fused to the N-terminus of Vpr. In contrast, eGFP-Vpr showed a diffuse distribution in both the cytoplasm and the nucleus (Figure 2, panel B 3) similar to the nuclear staining of eYFP-Vpr [10,12]. At least, it should be mentioned that Vpr distribution was not time dependent since the same pattern of localization was monitored at 48 and 72 h (data not shown).


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)

Subcellular localization of eGFP or mCherry tagged Vpr by confocal microscopy. HeLa cells were co-transfected with 0.5 μg of each plasmid and 0.5 μg pcDNA3. Cells were observed by confocal microscopy 24 h post transfection. Each panel shows the major phenotype. (A) mCherry images with excitation at 568 nm and emission at 580 to 700 nm. (B) eGFP images with excitation at 488 nm and emission at 500 to 550 nm. Note the intracellular redistribution of eGFP and mCherry upon fusion with Vpr.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Subcellular localization of eGFP or mCherry tagged Vpr by confocal microscopy. HeLa cells were co-transfected with 0.5 μg of each plasmid and 0.5 μg pcDNA3. Cells were observed by confocal microscopy 24 h post transfection. Each panel shows the major phenotype. (A) mCherry images with excitation at 568 nm and emission at 580 to 700 nm. (B) eGFP images with excitation at 488 nm and emission at 500 to 550 nm. Note the intracellular redistribution of eGFP and mCherry upon fusion with Vpr.
Mentions: Four labelled Vpr proteins were obtained by fusing eGFP or mCherry to Vpr either to its N- or C-terminus. Since both eGFP and mCherry are large with respect to Vpr, we first checked whether the fusion affects the intracellular localization of Vpr. To this end, we analyzed by confocal microscopy at 24 h post transfection the expression of both mCherry- (Figure 2, panels A2-3) and eGFP Vpr fusions in HeLa cells (Figure 2, panels B 2-3). Both Vpr-eGFP and Vpr-mCherry showed a nuclear rim staining coincident with the nuclear envelope (NE) (Figure 2, panels A2 and B2) in agreement with the localization of HA-Vpr (additional file 1, [12]). This localization of Vpr at the NE is not driven by the eGFP and mCherry proteins since both fluorescent proteins were found to be spread all over the cells when expressed in their free form (Figure 2 A1 and B1). Localization of HA-Vpr (additional file 1) or His-Vpr [12] confirms that these proteins are predominantly localized at the nuclear membrane and in the nucleus with some cytoplasmic localization. Thus, the fusion of either mCherry or eGFP to the C terminus of Vpr has a limited effect on Vpr localization in the cell even though the relative proportion of Vpr in the nucleus, at the nuclear envelope or in the cytoplasm was modified [10,12,13,24,32]. The distribution pattern of mCherry-Vpr was close to that of Vpr-mCherry except that a larger amount of protein diffused out in the cytoplasm, indicating a limited alteration of Vpr intracellular distribution by the mCherry fused to the N-terminus of Vpr. In contrast, eGFP-Vpr showed a diffuse distribution in both the cytoplasm and the nucleus (Figure 2, panel B 3) similar to the nuclear staining of eYFP-Vpr [10,12]. At least, it should be mentioned that Vpr distribution was not time dependent since the same pattern of localization was monitored at 48 and 72 h (data not shown).

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