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

Mapping of Vpr-Vpr interaction by FLIM. HeLa cells were co transfected with mutated Vpr-eGFP and its own counterpart fused to mCherry. FLIM was carried out 24 h posttransfection (see methods). Column A corresponds to the FLIM images of the Vpr-eGFP mutants alone, column B to the FLIM images of cells co expressing the mutant Vpr-eGFP and the mutant Vpr-mCherry. FRET efficiency (E) expressed in percentage represents the average value calculated over the entire cell (column C). The color scale used to create theses images is the same than the one used for figure 4. Note the drastic reduction of Vpr-Vpr interaction and the loss of Vpr nuclear envelope accumulation upon mutating residues L23, Q44, I60 and L67 (column B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Mapping of Vpr-Vpr interaction by FLIM. HeLa cells were co transfected with mutated Vpr-eGFP and its own counterpart fused to mCherry. FLIM was carried out 24 h posttransfection (see methods). Column A corresponds to the FLIM images of the Vpr-eGFP mutants alone, column B to the FLIM images of cells co expressing the mutant Vpr-eGFP and the mutant Vpr-mCherry. FRET efficiency (E) expressed in percentage represents the average value calculated over the entire cell (column C). The color scale used to create theses images is the same than the one used for figure 4. Note the drastic reduction of Vpr-Vpr interaction and the loss of Vpr nuclear envelope accumulation upon mutating residues L23, Q44, I60 and L67 (column B and C).

Mentions: Mutated proteins were expressed in HeLa cells. Immunodetection by Western Blots revealed that none of the point mutations impeded expression of the Vpr fusion proteins (data not shown). The fluorescence lifetime images were recorded and compared with those of the two wild type Vpr fusion proteins. Figure 5 shows the lifetime images of the Vpr-eGFP mutants expressed in the absence (Column A) and in the presence of the corresponding Vpr-mCherry mutant (Column B). The mean values obtained for the entire cell are reported on the right of the figure. Among the eight mutants, four of them, namely Q3R, W54G, R77Q and R90K, showed a staining pattern similar to that of the wild type fusion proteins with an accumulation at the nuclear rim (compare with Figure 4, panel A2). Oligomers of these mutant proteins were found in the cytoplasm, the nucleus and at the nuclear envelope. The transfer efficiency in the whole cell for these mutants was respectively 19%, 16%, 22% and 18%, similar to the value obtained for the wild type fusion protein (23%). Thus, the Q3, W54, R77 and R90 residues located outside the α-helices are probably not critical for the intracellular localization and oligomerization of Vpr.


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)

Mapping of Vpr-Vpr interaction by FLIM. HeLa cells were co transfected with mutated Vpr-eGFP and its own counterpart fused to mCherry. FLIM was carried out 24 h posttransfection (see methods). Column A corresponds to the FLIM images of the Vpr-eGFP mutants alone, column B to the FLIM images of cells co expressing the mutant Vpr-eGFP and the mutant Vpr-mCherry. FRET efficiency (E) expressed in percentage represents the average value calculated over the entire cell (column C). The color scale used to create theses images is the same than the one used for figure 4. Note the drastic reduction of Vpr-Vpr interaction and the loss of Vpr nuclear envelope accumulation upon mutating residues L23, Q44, I60 and L67 (column B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Mapping of Vpr-Vpr interaction by FLIM. HeLa cells were co transfected with mutated Vpr-eGFP and its own counterpart fused to mCherry. FLIM was carried out 24 h posttransfection (see methods). Column A corresponds to the FLIM images of the Vpr-eGFP mutants alone, column B to the FLIM images of cells co expressing the mutant Vpr-eGFP and the mutant Vpr-mCherry. FRET efficiency (E) expressed in percentage represents the average value calculated over the entire cell (column C). The color scale used to create theses images is the same than the one used for figure 4. Note the drastic reduction of Vpr-Vpr interaction and the loss of Vpr nuclear envelope accumulation upon mutating residues L23, Q44, I60 and L67 (column B and C).
Mentions: Mutated proteins were expressed in HeLa cells. Immunodetection by Western Blots revealed that none of the point mutations impeded expression of the Vpr fusion proteins (data not shown). The fluorescence lifetime images were recorded and compared with those of the two wild type Vpr fusion proteins. Figure 5 shows the lifetime images of the Vpr-eGFP mutants expressed in the absence (Column A) and in the presence of the corresponding Vpr-mCherry mutant (Column B). The mean values obtained for the entire cell are reported on the right of the figure. Among the eight mutants, four of them, namely Q3R, W54G, R77Q and R90K, showed a staining pattern similar to that of the wild type fusion proteins with an accumulation at the nuclear rim (compare with Figure 4, panel A2). Oligomers of these mutant proteins were found in the cytoplasm, the nucleus and at the nuclear envelope. The transfer efficiency in the whole cell for these mutants was respectively 19%, 16%, 22% and 18%, similar to the value obtained for the wild type fusion protein (23%). Thus, the Q3, W54, R77 and R90 residues located outside the α-helices are probably not critical for the intracellular localization and oligomerization of Vpr.

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