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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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NMR based structure of Vpr. The NMR-based 3D- structure of Vpr (1–96) is characterised by three α helices in close vicinity surrounded by flexible N and C termini [22]. Helices are presented in dark blue (17–33), green (38–50) and orange (54–77). Mutated amino acids Q3R, L23A, ΔQ44, W54G, I60A, L67A, R77Q and R90K are represented in CPK mode. Noticeably, the NMR studies were carried out on the Vpr sequence of the HIV-1 pNL43 strain with a Leucine at the position 60 instead of an Isoleucine for the HIV-1LAI strain used here. Nevertheless, a predictive study on I60 Vpr showed that the third α helix was not altered compared to L60 Vpr (data not shown).
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Figure 1: NMR based structure of Vpr. The NMR-based 3D- structure of Vpr (1–96) is characterised by three α helices in close vicinity surrounded by flexible N and C termini [22]. Helices are presented in dark blue (17–33), green (38–50) and orange (54–77). Mutated amino acids Q3R, L23A, ΔQ44, W54G, I60A, L67A, R77Q and R90K are represented in CPK mode. Noticeably, the NMR studies were carried out on the Vpr sequence of the HIV-1 pNL43 strain with a Leucine at the position 60 instead of an Isoleucine for the HIV-1LAI strain used here. Nevertheless, a predictive study on I60 Vpr showed that the third α helix was not altered compared to L60 Vpr (data not shown).

Mentions: The 3D structure of Vpr peptides and of full length Vpr in hydrophobic solvents or in the presence of micelles was solved by NMR [20,21]. As illustrated in Figure 1, Vpr is composed of three amphipathic α helices spanning residues (17–33), (38–50) and (54–77), surrounded by flexible N- and C-terminal sequences [22]. Two loops spanning residues (34–37) and (51–53) allow a mutual orientation of these helices, conferring a globular conformation to the protein and promoting the formation of a hydrophobic core with numerous hydrophobic amino acids scattered throughout Vpr. The difficulties encountered to solve the Vpr 3D structure might be explained by its ability to oligomerize via the formation of leucine zipper like motifs [14,23-26].


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)

NMR based structure of Vpr. The NMR-based 3D- structure of Vpr (1–96) is characterised by three α helices in close vicinity surrounded by flexible N and C termini [22]. Helices are presented in dark blue (17–33), green (38–50) and orange (54–77). Mutated amino acids Q3R, L23A, ΔQ44, W54G, I60A, L67A, R77Q and R90K are represented in CPK mode. Noticeably, the NMR studies were carried out on the Vpr sequence of the HIV-1 pNL43 strain with a Leucine at the position 60 instead of an Isoleucine for the HIV-1LAI strain used here. Nevertheless, a predictive study on I60 Vpr showed that the third α helix was not altered compared to L60 Vpr (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: NMR based structure of Vpr. The NMR-based 3D- structure of Vpr (1–96) is characterised by three α helices in close vicinity surrounded by flexible N and C termini [22]. Helices are presented in dark blue (17–33), green (38–50) and orange (54–77). Mutated amino acids Q3R, L23A, ΔQ44, W54G, I60A, L67A, R77Q and R90K are represented in CPK mode. Noticeably, the NMR studies were carried out on the Vpr sequence of the HIV-1 pNL43 strain with a Leucine at the position 60 instead of an Isoleucine for the HIV-1LAI strain used here. Nevertheless, a predictive study on I60 Vpr showed that the third α helix was not altered compared to L60 Vpr (data not shown).
Mentions: The 3D structure of Vpr peptides and of full length Vpr in hydrophobic solvents or in the presence of micelles was solved by NMR [20,21]. As illustrated in Figure 1, Vpr is composed of three amphipathic α helices spanning residues (17–33), (38–50) and (54–77), surrounded by flexible N- and C-terminal sequences [22]. Two loops spanning residues (34–37) and (51–53) allow a mutual orientation of these helices, conferring a globular conformation to the protein and promoting the formation of a hydrophobic core with numerous hydrophobic amino acids scattered throughout Vpr. The difficulties encountered to solve the Vpr 3D structure might be explained by its ability to oligomerize via the formation of leucine zipper like motifs [14,23-26].

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