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During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.
The Gag polyprotein precursor Pr55Gag plays a central role in the assembly and production of HIV-1 particles. Pr55Gag on its own is necessary and sufficient for the production of virus-like particles (VLP) (26), while the genomic RNA, the Pol enzymes, and Env glycoproteins are necessary for the production of infectious viruses (1). Pr55Gag consists of four structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two small spacer sequences, SP1 and SP2, flanking the NC domain. The N-terminal myristic acid of matrix together with a cluster of basic residues promotes the anchoring of the Gag precursor into the inner leaflet of the plasma membrane (PM). The CA and NC domains are involved in Pr55Gag and Pr160Gag-Pol oligomerization concomitantly with the NC-mediated selection and packing of the genomic RNA.
Gag multimerization has been studied extensively for HIV-1 and Rous sarcoma virus both in vitro and in cells (for reviews see references 1 and 9). Several notable studies carried out in macrophages have reported that the assembly of Gag and the budding of infectious particles can occur in intracellular vesicles that are referred to as late endosomes (29, 30, 54-56, 64). However, more recent observations of fluorescent Gag and quantitative imaging suggest that the assembly occurs at the plasma membrane, while the presence of viral particles in endosomal vesicles could be the result of endocytosis following a budding failure (20, 22, 31, 33, 34).
In addition to its role in HIV-1 assembly, Pr55Gag is involved in the incorporation of cellular and viral proteins such as Vpr (27). Vpr is a small basic protein of 96 amino acids with a three-dimensional structure composed of three amphipathic α-helices mutually oriented to form a central hydrophobic core surrounded by flexible sequences (49). This hydrophobic core promotes the formation of Vpr oligomers in HeLa cells and their targeting to the nuclear envelope (24). Vpr plays a pivotal role in viral pathogenesis, since it displays several activities in the host cell, such as its implication in the nuclear import of the HIV-1 preintegration complex (PIC) in nondividing cells, the transactivation of the HIV-1 long terminal repeat (LTR), cell cycle arrest at the G2/M transition, and the induction of apoptosis (reviewed in references 2, 43, and 74).
The virion incorporation of Vpr was shown to be mediated through interactions between the NC and p6 domains of Gag precursor and at least the first two α helices of Vpr (4, 21, 35, 41). However, little is known about the mechanism of their mutual recognition, since Pr55Gag accumulates at the PM, while Vpr on its own is located mainly at the nuclear rim and in the nucleus (70). In addition, the role of protein oligomerization in Pr55Gag-Vpr interaction remains to be determined.
To characterize more deeply the Pr55Gag-Vpr complex, we performed confocal microscopy and two-photon fluorescence lifetime imaging microscopy (FLIM) using HeLa cells expressing wild-type or mutant forms of HIV-1 Pr55Gag and Vpr proteins. To this end, enhanced green fluorescent protein (eGFP) or mCherry was tethered to the N terminus of Vpr, while Pr55Gag was labeled by the biarsenical-tetracysteine (TC) method (47). We visualized Pr55Gag-Vpr complexes in the cytoplasm mainly at the plasma membrane and not in the nucleus. Thus, this interaction caused Vpr accumulation at the cell periphery. Moreover, we show that Vpr oligomerization is essential for its interaction with Pr55Gag precursor, as well as for its relocation mediated by Pr55Gag. In contrast, the correct multimerization and plasma membrane targeting of Pr55Gag were not required for Vpr recruitment.
HeLa cells (105) (unless otherwise noted) were cultured on 35-mm coverslips (μ-Dish IBIDI; Biovalley, France) in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (Invitrogen Corporation, Cergy Pontoise, France) and a 1% antibiotic mixture (penicillin-streptomycin; Invitrogen Corporation, Pontoise, France) at 37°C in a 5% CO2 atmosphere. HeLa cells were transfected using jetPEI (PolyPlus Transfection; Illkirch, France) according to the supplier's recommendations. To keep a constant amount of 1 μg of transfected DNA, each transfection assay was supplemented with pcDNA3 (Invitrogen Corporation, Cergy Pontoise, France).
The construction of eGFP-Vpr, mCherry-Vpr, and hemagglutinin-Vpr (HA-Vpr) were described previously (19, 24, 63). The human codon-optimized Pr55Gag-TC-encoding plasmid and pNL4-3ΔEnvΔVpr were kindly provided by David E. Ott (National Cancer Institute, Frederick, Maryland) and J.-C. Paillart (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France), respectively. Deletion or substitution mutants were constructed by PCR-based site-directed mutagenesis on the eGFP-Vpr-, mCherry-Vpr-, or Pr55Gag-TC-expressing vector by following the supplier's protocol (Stratagene). The integrity of all constructs was confirmed by DNA sequencing.
HeLa cells cultured on 35-mm coverslips (μ-Dish IBIDI; Biovalley) were transfected with plasmids encoding eGFP, wild-type eGFP-Vpr, or eGFP-Vpr mutants alone or with wild-type Pr55Gag-TC or its cognate mutants. Biarsenical labeling was achieved 24 h posttransfection and adapted from the published protocol (25). Briefly, a biarsenical solution was prepared by mixing 0.33 μl of 2 mM ReAsH (Invitrogen) with 0.33 μl of 25 mM 1,2-ethaneditiol (EDT; Fluka) and 0.33 μl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) and was incubated for 15 min at room temperature in the dark, followed by a 10-min incubation in Hanks' balanced salt solution (HBSS; Invitrogen) supplemented with 1 g of D-(+)-glucose/liter (Sigma). The biarsenical solution was applied to each coverslip, followed by 1 h of incubation at 37°C. After being labeled, cells were rinsed extensively with HBSS-glucose, followed by three separate 10-min incubations with 300 μM EDT in HBSS-glucose. The last washing step consisted of the replacement of the EDT solution with the HBSS-glucose solution. Live cells were imaged immediately after labeling.
HeLa cells were transfected with 0.25 μg of HA-Vpr construct with either 0.25 μg human codon-optimized Pr55Gag or 1.75 μg pNL4-3ΔEnvΔVpr DNA vector. At 24 h posttransfection, the cells were fixed with a 4% paraformaldehyde-phosphate-buffered saline (PBS) solution, permeabilized with 0.2% Triton-PBS, pH 7.4, and blocked for 45 min with a PBS blocking buffer composed of 10% horse serum, 1% bovine serum albumin (BSA), 0.02% NaN3. Cells then were incubated with an anti-HA (Ozyme) and, after successive washings, with an antibody fused to Alexa 568 (Invitrogen Corporation, Cergy Pontoise, France). The cells next were analyzed by confocal microscopy (Kr/Ar laser 488/568; MRC 1024; Bio-Rad). For the immunodetection of Pr55Gag-TC-expressing cells, the same protocol was used with anti-Gag polyclonal antibody (a kind gift of P. Boulanger, Medical University, Lyon, France) and an anti-rabbit antibody coupled to Alexa 568.
HeLa cells (5 × 105) transfected with 2.5 μg of plasmid expressing either eGFP, wild-type eGFP-Vpr, mutant eGFP-Vpr, Pr55Gag-TC, or mutant Pr55Gag-TC were treated with trypsin and resuspended in ice-cold lysis buffer (1% Triton X-100, 100 mM NaF, 10 mM tetrasodium diphosphate decahydrate, 1 mM Na3VO4 in PBS, pH 7.4, supplemented with a complete anti-protease cocktail from Roche, Meylan, France). After sonication and centrifugation, the protein concentration was assessed by a Bradford assay (Bio-Rad). Twenty-five micrograms of total proteins was reduced with 10 mM dithiothreitol containing loading buffer (Laemmli; Bio-Rad), heat denatured, and electrophoresed on a SDS-PAGE (12%) gel. Subsequently, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham, Orsay, France), and blots were probed either with a monoclonal mouse antibody directed against the GFP protein (Clontech) or with a polyclonal rabbit anti-Pr55Gag protein. After several washings, secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase were added to the membrane, and the visualization of proteins was carried out using the ECL system (Amersham).
The FLIM methodology allows for monitoring the fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor when they are less than 10 nm apart, a distance corresponding to intermolecular protein-protein interactions (7, 17, 69). The FRET phenomenon causes a decrease in the fluorescent lifetime (τ) of the donor, which can be measured by the FLIM technique at each pixel or group of pixels. Based on these lifetime values, the FRET efficiency can be calculated using equation 1.
Where τDA is the lifetime of the donor in the presence of the acceptor and τD is the lifetime of the donor in the absence of the acceptor.
A more detailed description of FLIM imaging and analysis is given in Fig. S1 in the supplemental material.
The experimental setup for FLIM measurements has been described already (24). Briefly, time-correlated single-photon-counting (TCSPC) FLIM measurements were performed on a home-made two-photon excitation scanning microscope based on an Olympus IX70 inverted microscope with an Olympus water immersion objective (60× magnification, 1.2 numeric aperture [NA]) operating in the descanned fluorescence collection mode (3, 16). Two-photon excitation at 900 nm was provided by a mode-locked titanium-sapphire laser (Tsunami; Spectra Physics). Photons were collected using a set of two filters: a short-pass filter with a cutoff wavelength of 680 nm (F75-680; AHF, Germany) and a band pass filter of 520 ± 17 nm (F37-520; AHF, Germany). The fluorescence was directed to a fiber-coupled APD (SPCM-AQR-14-FC; Perkin Elmer), which was connected to a TCSPC module (SPC830; Becker & Hickl, Germany).
Typically, the samples were scanned continuously for about 30 s to achieve appropriate photon statistics to analyze the fluorescence decays. Data were analyzed using a commercial software package (SPCImage V2.8; Becker & Hickl, Germany). FLIM images are constructed through an arbitrary color scale, ranging from blue (short lifetime) to red (long lifetime), corresponding to the different lifetimes of the donor.
Fluorescence confocal images of Vpr-tagged proteins in living cells in the presence or absence of Pr55Gag were taken 24 h posttransfection using a confocal microscope (MRC 1024; Bio-Rad) equipped with a Nikon water immersion objective (60× magnification, 1.2 NA) and an Ar/Kr laser. The eGFP images were obtained by scanning the cells with a 488-nm laser line and filtering the emission with a 500- to 550-nm band pass. For the mCherry or ReAsH images, a 568- or 594-nm laser line was used, and the emission was filtered from 580 to 700 nm or 604 to 700 nm, respectively. The confocal images of tagged Vpr taken in the presence or absence of the nonreplicative HIV-1 clone pNL4-3ΔEnvΔVpr were done with cells fixed in 4% paraformaldehyde.
Multifactorial analysis of variance (ANOVA) and a post hoc Dunnett or Tukey test for pairwise multiple comparisons were performed with R software (version 2.8.0) from the Comprehensive R Archive Network (59). The type I error was set at 5%.
To monitor Vpr-Vpr, Pr55Gag-Vpr, and Pr55Gag-Pr55Gag interactions, we used the FLIM-FRET intermolecular approach. A scheme of all of the donor-acceptor pairs used in this work is presented in Fig. Fig.11.
Vpr-Vpr interaction was monitored using the eGFP-Vpr fluorescence lifetime (donor) in the presence of mCherry-Vpr (acceptor). These recombinant-tagged Vpr proteins were preferred over Vpr-eGFP/Vpr-mCherry, since tagging the Vpr N terminus with a reporter tag does not hamper Vpr incorporation into HIV-1 particles (14, 39, 48).
To monitor the Pr55Gag-Vpr interaction, we used the biarsenical-TC labeling approach (47) with a codon-optimized plasmid coding for Pr55Gag tagged at its C terminus with the small TC motif (referred to as Pr55Gag-TC herein). In contrast to Pr55Gag-eGFP, Pr55Gag-TC can efficiently traffic to the plasma membrane and form correctly assembled VLPs (60). Cells coexpressing eGFP-Vpr and Pr55Gag-TC were treated with the membrane-permeable biarsenical dye ReAsH prior to FLIM measurements. Under these experimental conditions, eGFP-Vpr was the donor and Pr55Gag-TC-ReAsH was the acceptor.
Finally, Pr55Gag-Pr55Gag interaction was monitored in HeLa cells expressing Pr55Gag-TC labeled with the FlAsH biarsenical dye. Pr55Gag polymerization was expected to cluster the FlAsH chromophores, resulting in a decrease of the fluorescence lifetime due to self quenching and/or exciton coupling (36, 37). The self quenching of fluorescein derivatives (FlAsH is a fluorescein derivative) was already used successfully to study protein-protein and protein-lipid interactions (18, 38, 61).
Since the N-terminal tagging of Vpr with eGFP or mCherry could affect Vpr localization (70), we used confocal microscopy to compare the cellular distribution of eGFP-Vpr and HA-Vpr. As depicted in Fig. 2a and b, eGFP-Vpr and HA-Vpr accumulate in the nucleus with a clear exclusion of the nucleoli (70). For both constructions, a second phenotype (≈40%) with stronger labeling at the nuclear envelope was obtained (Fig. 2a and b, compare cells on the right to those on the left). In agreement with the report of Waldhuber et al., who used yellow fluorescent protein-Vpr, the eGFP-Vpr protein differs from HA-Vpr mainly by its comparatively higher expression in the cytoplasm (70). The cellular staining pattern of mCherry-Vpr and its expression was similar to that of eGFP-Vpr (data not shown).
In the presence of the Pr55Gag precursor, eGFP-Vpr (Fig. (Fig.2c)2c) and HA-Vpr (Fig. (Fig.2d)2d) form a punctate staining delineating the PM with almost no signal in the nucleus and only a moderate intracytoplasmic dotted pattern. Such a plasma membrane localization of eGFP-Vpr was not driven by eGFP, since eGFP fluorescence was found all over the cell when only eGFP protein was coexpressed with Pr55Gag (data not shown). Thus, both eGFP- and HA-tagged Vpr are directed at the PM in the presence of Pr55Gag.
To examine if this Pr55Gag-mediated redistribution of Vpr to the plasma membrane also occurs in the presence of the other viral proteins, eGFP- and HA-tagged Vpr were transiently coexpressed with an HIV-1 molecular clone that was unable to self replicate (pNL4-3ΔEnvΔVpr). As shown in Fig. 2e and f, eGFP-Vpr and HA-Vpr presented the same punctate staining at the PM.
Taken together, our data show that Pr55Gag causes the accumulation of Vpr at the PM. In addition, results indicate that the eGFP-tagged Vpr is a tool of choice to analyze Pr55Gag-Vpr complex formation during virus assembly.
During virion assembly, Pr55Gag was found to recruit Vpr into newly made particles (35, 41). The Pr55Gag domain that recognizes Vpr was mapped to the 15FRFG and 41LXXLF motifs of p6 (4, 46, 75). However, little is known about complex formation in cells and where Pr55Gag-Vpr interactions are taking place.
For a more comprehensive view of Pr55Gag-Vpr complex formation, first we performed confocal microscopy imaging on cells coexpressing Pr55Gag-TC stained with ReAsH and eGFP-Vpr. These images were compared to those obtained from cells coexpressing Pr55Gag-TC stained with FlAsH and HA-Vpr immunostained by the red Alexa 568 dye.
As depicted in Fig. Fig.3,3, column 1, Pr55Gag detected by ReAsH or FlAsH accumulated mainly at or near the plasma membrane with little, if any, fluorescence in the cytoplasm (60). Moreover, eGFP-Vpr and HA-Vpr (column 2) were almost completely absent from the nucleus and the nuclear envelope, which is in agreement with the images presented in Fig. Fig.2.2. Thus, merge images show the colocalization of the two partners mainly at the plasma membrane.
To further show that the colocalization of the two partners results from their direct interaction, we combined confocal microscopy for Pr55Gag localization together with FLIM-based FRET to monitor Pr55Gag-TC-eGFP-Vpr interaction in HeLa cells. When eGFP-Vpr was expressed alone and visualized by FLIM after ReAsH staining, the fluorescent lifetime (τ) of eGFP-Vpr was ≈2.50 ns (Fig. (Fig.4A,4A, image a). This value corresponds to the fluorescence lifetime of free eGFP, indicating an absence of FRET between eGFP-Vpr and unbound ReAsH (24, 57). Interestingly, cells observed by the FLIM technique also show an accumulation of eGFP-Vpr in the nucleus. In sharp contrast, when eGFP-Vpr and Pr55Gag-TC were coexpressed and monitored by FLIM after ReAsH staining, a decrease of the eGFP lifetime to ≈2.11 ns was measured and is symbolized by the distribution of blue color (Fig. (Fig.4A,4A, image c). This image corresponds to the main observed phenotype (>90%) when an equivalent amount of plasmid was cotransfected (0.5 μg/0.5 μg) but varied depending on the plasmid ratio (data not shown). This fluorescence lifetime drop (from 2.5 to 2.1 ns) corresponds to an average FRET efficiency (E) of 16% in the whole cell, which is significantly different from that of the control using multifactorial ANOVA statistical tests (Fig. (Fig.4C).4C). Interestingly, higher transfer efficiency (21%) was observed at or near the PM (black bars), while no significant FRET was observed in the nucleus (data not shown). This experiment is, to our knowledge, the first to visualize notably a direct interaction between Pr55Gag and Vpr at the level of the plasma membrane.
To further correlate the Pr55Gag-Vpr interaction with the cellular redistribution of Vpr, mutations in 15FRFG and/or 41LXXLF sequences of p6 were constructed. All three mutants, Pr55Gag(p6)F15A-TC, Pr55Gag(p6)L44A-TC, and Pr55Gag(p6)F15AL44A-TC, were efficiently expressed in cells as shown by Western blot analysis (Fig. (Fig.4B).4B). Moreover, the confocal analysis of cells expressing these Pr55Gag mutants stained with ReAsH (Fig. (Fig.4A,4A, images d, f, and h) revealed that these mutations have a poor effect on the cellular distribution of Pr55Gag.
FLIM measurements on cells coexpressing eGFP-Vpr and Pr55Gag(p6)F15A-TC-ReAsH showed a decreased Vpr relocation to the PM (Fig. (Fig.4A,4A, image e) associated with a decrease of FRET efficiency at this level (15%) and in the cytoplasm (9%) (Fig. (Fig.4C).4C). An even weaker redirection of eGFP-Vpr to the PM was observed upon changing leucine 44 to alanine (Fig. (Fig.4A,4A, image g). With this mutant, no FRET was observed in the cytoplasm and only a limited FRET was measured at the PM, highlighting a correlation between the PM localization of Pr55Gag and Pr55Gag-Vpr interaction. Finally, this correlation was confirmed with the double mutant Pr55Gag(p6)F15AL44A-TC (Fig. (Fig.4A,4A, image i), which showed no significant FRET with eGFP-Vpr (Fig. (Fig.4C)4C) and a distribution of eGFP-Vpr mainly in the nucleus, as observed in the absence of Pr55Gag (Fig. (Fig.4A,4A, image a).
Taken together, our data reveal a clear correlation between the interaction of Pr55Gag with Vpr and the accumulation of Vpr at the PM and confirm the major role of the 41LXXLF sequence of Pr55Gag for Vpr recognition (4).
Vpr can form oligomers in vitro and in cells as recently observed (4, 10, 63, 66, 73). This oligomerization is mediated by the Vpr hydrophobic core but not by the flexible N- and C-terminal domains. Indeed, a disruption of the hydrophobic core by point mutations in the first (L23F), second (ΔQ44), and third helix (L67A) results in a loss of Vpr-Vpr interaction (24). The role of this oligomerization in Vpr functions remains to be determined, because it does not appear to be required for either Vpr-mediated apoptosis or G2/M cell cycle arrest (10, 24). However, since VprL23F, VprΔQ44, and VprL67A mutants were not or were very poorly incorporated into nascent particles (65, 66, 72), Vpr oligomerization could be needed for its recruitment by Pr55Gag.
To establish a correlation between Vpr oligomerization and its interaction with Pr55Gag, we carried out FLIM experiments on cells expressing eGFP-Vpr and mCherry-Vpr with and without nonlabeled Pr55Gag. When the two fusion proteins were coexpressed in HeLa cells, the FRET between the eGFP and the mCherry moiety reflects the presence of Vpr oligomers at the nuclear envelope in the cytoplasm and in the nucleus (Fig. (Fig.5A,5A, image b, and B). These data are in agreement with previous studies showing that Vpr can oligomerize in a cellular context (10, 24). To verify that Vpr oligomerization could be required for its interaction with Pr55Gag, the two Vpr chimeras were coexpressed with nonlabeled Pr55Gag. Under this condition, the FRET signal provided by Vpr oligomerization was observed mainly at the plasma membrane (Fig. (Fig.5A,5A, image d). Interestingly, this Pr55Gag-promoted relocation of Vpr oligomers resulted in a statistically significant (P < 10−3) increased FRET between Vpr species (Fig. (Fig.5A5A [note the darker blue in image d] and B), suggesting a Pr55Gag-induced compaction of Vpr oligomers or, alternatively, a structural rearrangement of Vpr oligomers.
To confirm that Vpr oligomerization is a prerequisite for PrGag interaction, Vpr was mutated at the level of its nonstructured N and C termini (Q3R and R77Q) or in its hydrophobic core (L23F, ΔQ44, and L67A) (24). The resulting eGFP-Vpr mutants were transfected and imaged in the absence (Fig. (Fig.6,6, column A) or in the presence of their mCherry-Vpr counterparts (column B) and compared to cells coexpressing eGFP-Vpr derivatives and Pr55GagTC-ReAsH (column C). As with the nonmutated eGFP-Vpr (Fig. (Fig.6A,6A, images A1, B1, and C1), we visualized for eGFP-VprQ3R (Fig. (Fig.6A,6A, images A2, B2, and C2) a high transfer efficiency when coexpressed with mCherry-VprQ3R (Fig. (Fig.6C)6C) or with Pr55Gag-TC-ReAsH (Fig. (Fig.6D).6D). Similar results were obtained with VprR77Q (data not shown). Thus, these mutations did not abolish Vpr oligomerization and interaction with Pr55Gag.
In sharp contrast, mutations in the three helices (L23F, ΔQ44, and L67A) abolished Vpr oligomerization and localization at the nuclear envelope (Fig. (Fig.6A,6A, images B3 to B5, and C), as previously shown (24). Furthermore, the coexpression of such a mutated Vpr with Pr55Gag-TC labeled with ReAsH (Fig. (Fig.6,6, images C3 to C5, and D) resulted in a loss of Vpr interaction with Pr55Gag and in its redistribution at the PM. These phenotypes cannot be accounted for by poor expression or degradation of the Vpr proteins, since an immunoblot analysis (Fig. (Fig.6B)6B) reveals the sustained expression of all fusion proteins.
Taken together, these results show that Pr55Gag interacts with Vpr oligomers, promoting their redistribution at the PM and probably their incorporation into nascent viral particles. Mutations in Vpr helices that prevent oligomerization also inhibit Pr55Gag-Vpr complex formation.
Pr55Gag directs retroviral assembly by multimer formation upon binding to the genomic RNA via the NC domain and simultaneously through its interaction with the PM by the MA domain (for reviews, see references 1 and 15). To investigate if Pr55Gag assembly is required for Vpr incorporation into virions, the multimerization of Pr55Gag-TC derivatives was investigated by monitoring the fluorescence lifetime of FlAsH (Fig. (Fig.7A,7A, column A, and B). Results were compared to those obtained with cells expressing eGFP-Vpr and either the wild type or a mutant form of Pr55Gag-TC-ReAsH (Fig. (Fig.7A,7A, column B, and C).
As a negative control, FlAsH was added to nontransfected HeLa cells and imaged by FLIM. A homogeneous staining of the cells with an average fluorescence lifetime of 3.52 ns was measured (Fig. (Fig.7A,7A, image A1, and B). In contrast, the fluorescence lifetime of FlAsH bound to Pr55Gag-TC decreases to 2.57 ns at the PM (Fig. (Fig.7A,7A, image A2, and B), reflecting the multimerization of Pr55Gag.
Interestingly, green-labeled dotted structures with a lifetime of 2.82 ns also were detected (white arrows), which is in line with the presence of truncated Pr55Gag or low-molecular-weight Pr55Gag complexes in subcellular compartments (62, 68). In agreement with the data shown in Fig. Fig.6A,6A, image c1, and Fig. Fig.6D,6D, when eGFP-Vpr was coexpressed with Pr55Gag-TC and cells were incubated with ReAsH, an interaction between Vpr and Pr55Gag was found at the PM with a transfer efficiency of 20.6% (Fig. (Fig.7A,7A, image B2, and C).
To establish a correlation between Pr55Gag assembly and the Pr55Gag-mediated accumulation of Vpr at the PM, a series of mutations was made in Pr55Gag-TC. All of these mutants were expressed in HeLa cells as revealed by Western blotting (see Fig. S2 in the supplemental material). First, methionine 369 in the SP1 spacer was substituted for alanine (Pr55GagM369A-TC). This mutant presents a severe defect in Pr55Gag assembly in spite of its localization at the PM (20, 31, 40, 45). Confocal microscopy confirmed the localization of this mutant at the PM (see Fig. S2b in the supplemental material). When cells expressing this mutant were stained with FlAsH and monitored by FLIM, the fluorescence lifetime value of the chromophore was 3.08 ns (Fig. (Fig.7A,7A, image A3, and B). This value is intermediate between those obtained for free FlAsH (3.52 ns) and for Pr55Gag-TC-FlAsH (2.57 ns), indicating that the M369A mutation caused a multimerization defect of Pr55Gag. When this mutant was labeled with ReAsH and coexpressed with eGFP-Vpr (Fig. (Fig.7A,7A, image B3), an interaction between the two proteins was observed at the PM (E = 18.8%) (Fig. (Fig.7C),7C), showing that eGFP-Vpr can interact with an assembly-defective Pr55Gag mutant and can be directed to the PM.
We then checked if Pr55Gag localization at the PM was required for its interaction with Vpr, since Pr55Gag-Vpr complexes accumulated at this site. To that end, the myristoyl-acceptor glycine in position 2 was substituted for alanine in the Pr55GagG2A mutant. This substitution abolished the stable association of Pr55Gag with the PM and caused a diffuse cellular distribution (see Fig. S2c in the supplemental material) (12, 20, 28). Using FLIM, we found a fluorescence lifetime of 2.87 ns, and it was evenly distributed throughout the cytoplasm (Fig. (Fig.7A,7A, image A4, and B). This value is in between that of Pr55Gag-TC-FlAsH and that of free FlAsH and is in line with previous reports indicating that myristylation-defective Pr55Gag can oligomerize but cannot form high-molecular-weight complexes (20, 42, 44, 51). Moreover, we observed Pr55GagG2A with a fluorescence lifetime of 3.16 ns within the nucleus, suggesting that Pr55Gag mutants undergo nuclear import. When cotransfected with eGFP-Vpr, the Pr55GagG2A-TC mutant redistributed Vpr throughout the cytoplasm (Fig. (Fig.7A,7A, image B4). In addition, the FRET efficiency found in the cytoplasm for eGFP-Vpr/Pr55GagG2A-TC-ReAsH (E = 21%) (Fig. (Fig.7C)7C) fully matches that determined for the wild-type eGFP-Vpr/Pr55Gag complex at the PM, indicating that Pr55Gag-Vpr interaction is independent from Pr55Gag anchoring into the PM.
To further highlight the role of Pr55Gag on the intracellular distribution of Vpr, K30E and K32E substitutions were inserted in the basic stretch of the MA domain, since these mutations have been shown to redirect Pr55Gag to intracellular vesicles, such as MVB (multivesicular bodies) or MVB-like structures (29, 55). In agreement with this, we observed Pr55GagK30EK32E-TC mainly in vesicles and only slightly at the PM (see Fig. S2d in the supplemental material). Interestingly, the fluorescence lifetime of Pr55GagK30EK32E-TC-FlAsH on the vesicles was 2.64 ns (Fig. (Fig.7A,7A, image A5), which is close to that obtained for wild-type Pr55Gag-TC at the PM (Fig. (Fig.7B).7B). Thus, these two mutations target the precursor to intracellular vesicles, where Pr55Gag multimerization still takes place. When coexpressed with eGFP-Vpr, the FRET efficiency between eGFP-Vpr and Pr55GagK30EK32E-TC-ReAsH monitored in vesicles (E = 17.2%) (Fig. (Fig.7C)7C) indicates that this Pr55Gag mutant strongly interacts with eGFP-Vpr (Fig. (Fig.7A,7A, image B5).
Taken together, these data suggest that neither the Pr55Gag localization at the PM nor Pr55Gag multimer formation are required for the interaction with Vpr oligomers and the redistribution of Vpr to the PM. Moreover, we show here that the cellular localization of Vpr depends upon that of Pr55Gag.
The aim of this work was to investigate whether Vpr oligomerization and Pr55Gag multimerization were required for their mutual recognition during virus assembly. To this end, we designed a series of FLIM-FRET measurements to investigate the interaction between Pr55Gag-TC and eGFP-Vpr in HeLa cells. These proteins were selected because the N-terminal-tagged Vpr is incorporated into nascent viral particles, and the assembly of Pr55Gag-TC takes place in a manner similar to that of wild-type Pr55Gag (19, 60). As the wild-type Vpr, the fluorescently tagged Vpr proteins accumulated at the level of the PM upon coexpression with Pr55Gag alone or in the viral context (Fig. (Fig.22 and and3),3), indicating that Pr55Gag directs Vpr at this cellular site. Interestingly, a residual staining was observed in the nucleus when Vpr was coexpressed in the viral context (Fig. (Fig.2e).2e). This suggests that an optimal Vpr recruitment requires a correct Pr55Gag/Vpr ratio (53, 67), or that other viral components compete with Vpr for binding to Pr55Gag (6).
A Gag-mediated redistribution of eGFP-Vpr has been observed previously in 293T cells (13), but in contrast to our results, eGFP-Vpr was found to be directed to the cytoplasm. This discrepancy could be related to the more efficient PM targeting of our codon-optimized Pr55Gag protein (Fig. (Fig.33 and and4)4) (60). Nevertheless, the Gag-induced redistribution observed in both cases suggests that Pr55Gag-Vpr recognition is not dependent on the Pr55Gag intracellular distribution. In line with this conclusion, both the Pr55GagG2A mutant, which has lost its ability to stably interact with cellular membranes, and the Pr55GagK30E/K32E mutant, which is located mainly in MVBs or MVB-like structures (29, 55), still interact with Vpr (Fig. (Fig.7A).7A). Thus, the Vpr protein likely follows intracellular Pr55Gag trafficking. The two viral proteins probably interact early after their synthesis, as further supported by the common intracellular trafficking signals identified on Pr55Gag- and Vpr-encoding mRNAs (8, 52). Similarly, the recruitment of the Vif protein by Pr55Gag also was found to be independent from the anchoring of Pr55Gag to the PM (5). This emphasizes that the Pr55Gag-mediated packaging of cofactors is not the result of a simple colocalization at the PM, but probably takes place at the site of their synthesis.
In line with the interaction of Vpr with the C-terminal domain of Pr55Gag (4, 21, 35, 46, 63, 75), we observed a strong decrease of the FRET signal when Vpr was expressed with Pr55Gag(p6)L44A and, to a lesser extent, with Pr55Gag(p6)F15A, confirming the critical role played by the 41LXXLF and 15FRFG motifs. Moreover, these domains probably act in concert in the interaction with Pr55Gag, since the double mutant failed to interact with Pr55Gag.
Although Vpr protein self oligomerizes in vitro (11, 66, 71, 73) and in cells (10, 24), the functional role of this oligomerization remains elusive. Interestingly, the coexpression of eGFP-Vpr and mCherry-Vpr with nonlabeled Pr55Gag (Fig. (Fig.5A)5A) clearly show that Pr55Gag interacts with Vpr oligomers. This correlation between Vpr oligomerization and Pr55Gag interaction was confirmed further using Vpr mutants (Fig. (Fig.6A,6A, C, and D), although a direct involvement of the mutated residues in Pr55Gag recognition or in the localization of the mutated Vpr proteins in subcellular compartments inaccessible to Pr55Gag cannot be excluded fully. Interestingly, the inability of L23F, ΔQ44, and L67A Vpr mutants to oligomerize and, thus, to interact with Pr55Gag could explain their poor virion incorporation (32, 65, 66, 72).
The interaction of Vpr oligomers with Pr55Gag is likely mediated through hydrophobic contacts between the two hydrophobic 15FRFG and 41LXXLF motifs of the p6 domain, which are thought to be in close proximity (23), and the hydrophobic core formed by the neighboring amphipathic α helices in Vpr oligomers (50). This would explain the resistance of Pr55Gag-Vpr complexes to high salt concentrations (4, 21).
In line with previous data (9, 20, 31, 33, 40), we found that Pr55Gag-TC polymerizes mainly at the level of the PM and, to a smaller extent, in the cytoplasm (30, 54, 56, 58, 64). Indeed, a lower FRET efficiency was observed in the cytoplasm compared to that of the PM. This may reflect an increased distance between Pr55Gag molecules as a result of, for instance, an architecture defect of the polymer or an increased curvature of the lipid bilayer of the MVBs compared to that of the PM or a lower density of Gag polyproteins at the MVB surface. Rather, low FRET efficiencies also were observed using TC-coupled Pr55GagM369A and Pr55GagG2A mutants, which is in line with their ability to form low-order Pr55Gag oligomers (42, 45). Surprisingly, these two mutants were able to interact with Vpr as efficiently as the wild-type protein Pr55Gag despite their misfolding. Thus, these data show that in contrast to Vpr, Pr55Gag multimerization is not required per se for its interaction with Vpr.
We report here that Vpr oligomerization is required for Vpr accumulation at the PM mediated by Pr55Gag. At the same time, Pr55Gag-Vpr interaction does not need an extensive multimerization of Pr55Gag. This suggests that Pr55Gag assembly and Vpr interaction probably are separate events.
Thanks are due to David E. Ott for providing Gas-TC expressing plasmid (NCI-Frederick), Gabrielle Mengus for HeLa cells (IGBMC), Pierre Boulanger for anti-Gag antibodies (IFR62, Lyon Est), and Serge Bouaziz (UMR8151, Paris) for fruitful discussions.
J.V.F. is supported by a fellowship of Fonds national de la Recherche (Luxembourg). This work was supported by ANRS, FRM, and Sidaction.
Published ahead of print on 18 November 2009.
†Supplemental material for this article may be found at http://jvi.asm.org/.