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Title: 1
HIV-1 Vpr oligomerization and not that of Gag directs the interaction between Vpr and 2
Gag. 3
4
Authors : Joëlle V. Fritz1, Denis Dujardin
1, Julien Godet
1, Pascal Didier
1, Jan De Mey
1, 5
Jean-Luc Darlix2, Yves Mély
1 and Hugues de Rocquigny
1* 6
7
1 Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de 8
Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France. 9
2 LaboRétro Unité de Virologie Humaine INSERM 758, IFR 128 Ecole Normale 10
Supérieure de Lyon, 46 allée d'Italie, 69364 LYON, France 11
12
13
* Corresponding author 14
Tel : +33 (0)3 90 24 41 03 Fax : +33 (0)3 90 24 43 12 15
e-mail: [email protected] 16
17
18
Running title: Gag-Vpr interaction visualized by fluorescence imaging 19
20
Word count for abstract: 217 21
Word count for the text: 5535 22
23
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.01691-09 JVI Accepts, published online ahead of print on 18 November 2009
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Abstract 24
During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral 25
particles via an interaction with the C terminal domain of the Gag polyprotein precursor 26
Pr55Gag
. Vpr has been implicated in the nuclear import of the newly made viral DNA and 27
subsequently in its transcription. In addition, Vpr can impact on the cell physiology by 28
causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers but their roles have not 29
yet been investigated. We have developed FLIM-FRET based assays to monitor the 30
interaction between Pr55Gag
and Vpr in HeLa cells. To that end we used eGFP-Vpr that can be 31
incorporated into the virus and a tetracysteine (TC) tagged Pr55Gag
-TC. This TC motif is 32
tethered to the C terminus of Pr55Gag
and does not interfere with Pr55Gag
trafficking and virus 33
like particle assembly. Results show that the Pr55Gag
-Vpr complexes mainly accumulated at 34
the plasma membrane. In addition, results with Pr55Gag
-TC mutants confirm that the 35
41LXXLF domain of Gag-p6 is essential for Pr55
Gag-Vpr interaction. We also report that Vpr 36
oligomerization is crucial for Pr55Gag
recognition and its accumulation at the plasma 37
membrane. On the other hand, Pr55Gag
-Vpr complexes are still formed when Pr55Gag
carries 38
mutations impairing its multimerization. These findings suggest that Pr55Gag
-Vpr recognition 39
and complex formation probably occur early during the Pr55Gag
assembly. 40
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Introduction 41
The Gag polyprotein precursor Pr55Gag
plays a central role in the assembly and production of 42
HIV-1 particles. Pr55Gag
on its own is necessary and sufficient for the production of viral like 43
particles (VLP) (26) but the genomic RNA, the Pol enzymes and Env glycoproteins are 44
necessary for the production of infectious viruses (1). Pr55Gag
consists of four structural 45
domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two small spacer 46
sequences SP1 and SP2 flanking the NC domain. The N terminal myristic acid of matrix 47
together with a cluster of basic residues promotes the anchoring of the Gag precursor into the 48
inner leaflet of the plasma membrane (PM). The CA and NC domains are involved in Pr55Gag
49
and Pr160Gag-Pol
oligomerization concomitant with NC-mediated selection of the genomic 50
RNA. 51
Gag multimerization has been extensively studied for HIV-1 and RSV both in vitro and in 52
cells (for reviews: (1, 9). Several studies carried out notably in macrophages have reported 53
that the assembly of Gag and the budding of infectious particles can occur in intracellular 54
vesicles referred to as late endosomes (29, 30, 54-56, 63). However, more recent observations 55
of fluorescent Gag and quantitative imaging suggest that the assembly occurs at the plasma 56
membrane, while the presence of viral particles in endosomal vesicles could be the result of 57
endocytosis following a budding failure (21, 22, 31, 33, 34, 59). 58
In addition to its role in HIV-1 assembly, Pr55Gag
is involved in the incorporation of cellular 59
and viral proteins such as Vpr (27)). Vpr, is a small basic protein of 96 amino acids with a 60
three dimensional structure composed of three amphipathic α-helices mutually oriented to 61
form a central hydrophobic core surrounded by flexible sequences (49). This hydrophobic 62
core promotes the formation of Vpr oligomers in HeLa cells and their targeting at the nuclear 63
envelope (24). Vpr plays a pivotal role in viral pathogenesis since it displays several activities 64
in the host cell, such as its implication in the nuclear import of the HIV-1 pre-integration 65
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complex (PIC) in non-dividing cells, transactivation of HIV-1 long terminal repeat (LTR), 66
cell cycle arrest at the G2/M transition and induction of apoptosis (reviewed in (2, 42, 74)). 67
Virion incorporation of Vpr was shown to be mediated through interactions between the NC 68
and p6 domains of Gag precursor and a least the two first α helices of Vpr (4, 18, 35, 41). 69
However little is known on the mechanism of their mutual recognition since Pr55Gag
70
accumulates at the PM while Vpr on its own is mainly located at the nuclear rim and in the 71
nucleus (70). In addition, the role of protein oligomerization in Pr55Gag
-Vpr interaction 72
remains to be determined. 73
In order to characterize more deeply the Pr55Gag
-Vpr complex, we performed confocal 74
microscopy and two photon fluorescence lifetime imaging microscopy (FLIM) using HeLa 75
cells expressing wild type or mutant forms of HIV-1 Pr55Gag
and Vpr proteins. To this end, 76
eGFP or mCherry was tethered to the N-terminus of Vpr while Pr55Gag
was labelled by the 77
biarsenical-tetracysteine method (59). We visualized Pr55Gag
-Vpr complexes in the 78
cytoplasm, mainly at the plasma membrane and not in the nucleus. Thus, this interaction 79
caused Vpr accumulation at the cell periphery. Moreover, we show that Vpr oligomerization 80
is essential for its interaction with Pr55Gag
precursor, as well as for its relocation mediated by 81
the Pr55Gag
. In contrast, the correct oligomerization and plasma membrane targeting of 82
Pr55Gag
were not required for Vpr recruitment. 83
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84
Material and Methods 85
Cell culture and transfections 86
105 HeLa cells (unless otherwise noted) were cultured on 35 mm coverslips (µ-Dish IBIDI, 87
Biovalley, France) in Dulbecco’s modified eagle medium supplemented with 10% fetal calf 88
serum (Invitrogen Corporation, Cergy Pontoise, France) and 1% of an antibiotic mixture 89
(penicillin/streptomycin: Invitrogen Corporation Pontoise, France) at 37°C in a 5% CO2 90
atmosphere. HeLa cells were transfected using jetPEITM
(PolyPlus transfection, Illkirch, 91
France) according to supplier’s recommendations. To keep a constant amount of 1µg of 92
transfected DNA, each transfection assay was supplemented with pcDNA3 (Invitrogen 93
Corporation, Cergy Pontoise, France). 94
95
Plasmids 96
Construction of eGFP-Vpr, mCherry-Vpr and HA-Vpr were previously described (20, 24, 97
62). The human codon-optimized Pr55Gag-TC
encoding plasmid and pNL4-3∆Env∆Vpr were 98
kindly provided by David E. Ott (National Cancer Institute at Frederick, Maryland) and J-C. 99
Paillart (Institut de Biologie Moléculaire et Cellulaire, Strasbourg), respectively. Deletion or 100
substitution mutants were constructed by PCR based site-directed mutagenesis on the eGFP-101
Vpr, mCherry-Vpr or the Pr55Gag-TC
expressing vector following the supplier’s protocol 102
(Stratagene). The integrity of all constructs was confirmed by DNA sequencing. 103
104
Biarsenical labelling 105
HeLa cells cultured on 35 mm coverslips (µ-Dish IBIDI, Biovalley, France), were transfected 106
with eGFP, eGFP-Vpr wild type or eGFP-Vpr mutants encoding plasmids alone or with 107
Pr55Gag-TC
wild type or its cognate mutants. Biarsenical labelling was achieved 24 hours post-108
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transfection and adapted from the published protocol (25). Briefly, a biarsenical solution was 109
prepared by mixing 0.33 µl of 2 mM ReAsH (Invitrogen) with 0.33 µl of 25 mM 1,2-110
ethaneditiol (EDT; Fluka) and 0.33 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) and 111
incubated for 15 min at room temperature in the dark followed by 10 min incubation in 112
Hanks’ balanced salt solution (HBSS; Invitrogen) supplemented by 1 g of D (+)-glucose/litre 113
(Sigma). The biarsenical solution was applied to each cover slip followed by 1h incubation at 114
37°C. After labelling, cells were rinsed extensively with HBSS/glucose, followed by three 115
separate 10 min incubations with 300 µM EDT in HBSS/glucose. The last washing step 116
consisted in the replacement of the EDT solution by the HBSS/glucose solution. Live cells 117
were imaged immediately after labelling. 118
119
Immunofluorescence detection of HA-Vpr and Pr55Gag-TC
120
HeLa cells were transfected with 0.25 µg of HA-Vpr construct with either 0.25 µg human 121
codon-optimized Pr55Gag
or 1.75 µg pNL4-3∆Env∆Vpr DNA vectors. At 24 h 122
posttransfection, the cells were fixed with a 4% paraformaldehyde/PBS solution, 123
permeabilized with 0.2% triton/PBS pH7.4 and blocked for 45 min with a PBS blocking 124
buffer composed by 10% of horse serum, 1% of BSA, 0.02% of NaN3. Then cells were 125
incubated with an anti-HA (Ozyme) and after successive washings, with an antibody fused to 126
Alexa 568 (Invitrogen Corporation, Cergy Pontoise, France). Next, the cells were analysed by 127
confocal microscopy (Bio-Rad 1024, Kr/Ar laser 488/568). For the immunodetection of 128
Pr55Gag-TC
expressing cells, the same protocol was used with a polyclonal antibody anti-Gag 129
(kind gift of P. Boulanger, Medical University, Lyon, France) and an anti-rabbit antibody 130
coupled to Alexa 568. 131
132
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Immunodetection of Pr55Gag-TC
, eGFP-Vpr and of their corresponding mutants 133
5 105
HeLa cells transfected with 2.5 µg of plasmid expressing either eGFP, eGFP-Vpr wild 134
type or mutant eGFP-Vpr, Pr55Gag-TC
or mutant Pr55Gag-TC
were treated with trypsin and 135
resuspended in ice cold lysis buffer (1% Triton X-100, 100 mM NaF, 10 mM NaPPi, 1 mM 136
Na3VO4 in PBS pH 7.4 supplemented with a complete anti-protease cocktail from Roche, 137
Meylan, France). After sonication and centrifugation, protein concentration was assessed by a 138
Bradford assay (Bio-Rad). 25 µg of total proteins were reduced with 10 mM DTT containing 139
loading buffer (Laemmli, Bio-Rad), heat denaturated and electrophoresed on 12% SDS-140
PAGE gel. Subsequently, proteins were transferred onto a polyvinylidene difluoride (PVDF) 141
membrane (Amersham, Orsay, France) and blots were probed either with a monoclonal 142
mouse antibody directed against the GFP protein (Clontech) or with a polyclonal rabbit anti 143
Pr55Gag
protein. After several washings, secondary anti-mouse or anti-rabbit antibodies 144
conjugated to horseradish peroxidase were added to the membrane and visualization of 145
proteins was carried out using the chemiluminescent ECL system (Amersham). 146
147
Fluorescence Lifetime Imaging Microscopy (FLIM) 148
The FLIM methodology allows monitoring the Fluorescence Resonance Energy Transfer 149
(FRET) between a fluorescent donor and an acceptor when they are less than 10 nm apart, a 150
distance corresponding to intermolecular protein-protein interactions (7, 17, 69). The FRET 151
phenomenon causes a decrease in the fluorescent lifetime (τ) of the donor, which can be 152
measured by the FLIM technique at each pixel or group of pixels. Based on these lifetime 153
values, the FRET efficiency can be calculated using equation 1. 154
D
DAE
τ
τ
−= 1 155 (1)
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where τDA is the lifetime of the donor in the presence of the acceptor and τD is the lifetime of 156
the donor in the absence of the acceptor. 157
A more detailed description of FLIM imaging and analysis is given in the supplementary 158
figure S1. 159
The experimental setup for FLIM measurements has already been described (24). 160
Briefly, time-correlated single-photon counting FLIM measurements were performed on a 161
home-made two-photon excitation scanning microscope based on an Olympus IX70 inverted 162
microscope with an Olympus 60× 1.2NA water immersion objective operating in the 163
descanned fluorescence collection mode (3, 16). Two-photon excitation at 900 nm was 164
provided by a mode-locked titanium-saphire laser (Tsunami, Spectra Physics). Photons were 165
collected using a set of two filters: a short pass filter with a cut-off wavelength of 680 nm 166
(F75-680, AHF, Germany), and a band-pass filter of 520 ± 17 nm (F37-520, AHF, Germany). 167
The fluorescence was directed to a fiber coupled APD (SPCM-AQR-14-FC, Perkin Elmer), 168
which was connected to a time-correlated single photon counting (TCSPC) module (SPC830, 169
Becker & Hickl, Germany). 170
Typically, the samples were scanned continuously for about 30 s to achieve appropriate 171
photon statistics to analyze the fluorescence decays. Data were analyzed using a commercial 172
software package (SPCImage V2.8, Becker & Hickl, Germany). FLIM images are constructed 173
through an arbitrary color scale, ranging from blue (short lifetime) to red (long lifetime) 174
corresponding to the different lifetimes of the donor. 175
176
Confocal fluorescence microscopy 177
Fluorescence confocal images of Vpr tagged proteins in living cells in presence or absence of 178
Pr55Gag
were taken 24 h post-transfection using a confocal microscope (Bio-Rad MRC 1024) 179
equipped with a Nikon 60x 1.2NA water immersion objective and an Ar/Kr laser. The eGFP 180
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images were obtained by scanning the cells with a 488 nm laser line and filtering the emission 181
with a 500 to 550 nm band-pass. For the mCherry or ReAsH images, a 568 nm or 594 lasers 182
line was used and the emission used filtered from 580 to 700 nm or 604 to 700 nm 183
respectively. The confocal images of tagged Vpr’s taken in presence or absence of the non 184
replicative HIV-1 clone pNL4-3∆Env∆Vpr were done with cells fixed in 4% of 185
paraformaldehyde. 186
187
Statistical analysis 188
Multifactorial ANOVA and post-hoc Dunnett or Tukey tests for pairwise multiple 189
comparisons were performed with the R software (version 2.8.0), from Comprehensive R 190
Archive Network (67). The type I error was set at 5 %. 191
192
Results 193
Chimeras of HIV-1 proteins used in the FLIM assays 194
To monitor Vpr-Vpr, Pr55Gag
-Vpr and Pr55Gag
- Pr55Gag
interactions, we used the FLIM- 195
FRET intermolecular approach. A scheme of all the donor-acceptor pairs used in this work is 196
presented in figure 1. 197
Figure 1 198
Vpr-Vpr interaction was monitored using the eGFP-Vpr fluorescence lifetime (donor) in the 199
presence of the mCherry-Vpr (acceptor). These recombinant tagged Vpr proteins were 200
preferred over Vpr-eGFP/Vpr-mCherry since tagging the Vpr N-terminus with a reporter tag 201
does not hamper Vpr incorporation into HIV-1 particles (14, 39, 48). 202
To monitor the Pr55Gag
-Vpr interaction, we used the biarsenical-tetracysteine labeling 203
approach (47) with a codon optimized plasmid encoding for the Pr55Gag
tagged at its C-204
terminus with the small tetracysteine (TC) motif (referred to as Pr55Gag-TC
herein). In contrast 205
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to Pr55Gag
-eGFP, Pr55Gag-TC
can efficiently traffic to the plasma membrane and form correctly 206
assembled VLPs (59). Cells co-expressing eGFP-Vpr and Pr55Gag-TC
were treated with the 207
membrane-permeable biarsenical dye ReAsH prior to FLIM measurements. Under these 208
experimental conditions, the eGFP-Vpr was the donor and Pr55Gag-TC
-ReAsH
was the acceptor. 209
Finally, Pr55Gag
-Pr55Gag
interaction was monitored in HeLa cells expressing Pr55Gag-TC
210
labeled with the FlAsH biarsenical dye. Pr55Gag
polymerization was expected to cluster the 211
FlAsH chromophores, resulting in a decrease of the fluorescence lifetime due to self 212
quenching and/or exciton coupling (36, 37). Self quenching of fluorescein derivatives (FlAsH 213
is a fluorescein derivative) was already successfully used to study protein-protein and protein-214
lipid interaction (19, 38, 60). 215
216
Pr55Gag
causes an accumulation of Vpr at the plasma membrane in HeLa cells 217
Since the N-terminal tagging of Vpr with eGFP or mCherry could possibly affect Vpr 218
localization (70), we used confocal microscopy to compare the cellular distribution of eGFP-219
Vpr and HA-Vpr. As depicted in figure 2a and 2b, eGFP-Vpr and HA-Vpr accumulate in the 220
nucleus with a clear exclusion of the nucleoli (70). For both constructions, a second 221
phenotype (≈ 40%) with a stronger labeling at the nuclear envelope was obtained (figure 2a 222
and 2b, compare right with left cells). In agreement with the report of Waldhuber et al using 223
YFP-Vpr, the eGFP-Vpr protein differs from HA-Vpr, mainly by its comparatively higher 224
expression in the cytoplasm (70). The cellular staining pattern of mCherry-Vpr and its 225
expression was comparable with that of eGFP-Vpr (data not shown). 226
In the presence of the Pr55Gag
precursor, eGFP-Vpr (figure 2c) and HA-Vpr (figure 2d) form a 227
punctuate staining delineating the PM with almost no signal in the nucleus and only a 228
moderate intracytoplasmic dotted pattern. Such a plasma membrane localization of eGFP-Vpr 229
was not driven by eGFP since eGFP fluorescence was found all over the cell when only eGFP 230
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protein was co-expressed with Pr55Gag
(data not shown). Thus, both eGFP- and HA-tagged 231
Vpr are directed at the PM in the presence of Pr55Gag
. 232
To examine if this Pr55Gag
-mediated redistribution of Vpr to the plasma membrane also 233
occurs in the presence of the other viral proteins, eGFP- and HA-tagged Vpr were transiently 234
co-expressed with an HIV-1 molecular clone unable to self replicate (pNL4-3∆Env∆Vpr). As 235
shown in figures 2e and 2f, eGFP-Vpr and HA-Vpr presented the same punctuate staining at 236
the PM. 237
Taken together our data show that Pr55Gag
causes the accumulation of Vpr at the PM. In 238
addition, results indicate that the eGFP-tagged Vpr could be a tool of choice to analyze 239
Pr55Gag
-Vpr complex formation during virus assembly. 240
241
Imaging Pr55Gag
-Vpr complexes in cells 242
During virion assembly, Pr55Gag
was found to recruit Vpr into newly made particles (35, 41). 243
The Pr55Gag
domain that recognizes Vpr was mapped to the 15
FRFG and
41LXXLF motifs of 244
p6 (4, 46, 75). However little is known on complex formation in cells and where Pr55Gag
-Vpr 245
interactions are taking place. 246
For a more comprehensive view of Pr55Gag
-Vpr complex formation, we first performed 247
confocal microscopy imaging on cells co-expressing Pr55Gag-TC
stained with ReAsH and 248
eGFP-Vpr. These images were compared with those obtained on cells co-expressing Pr55Gag-
249
TC stained with FlAsH and HA-Vpr immunostained by the red Alexa 568 dye. 250
Figure 3 251
As depicted in figure 3 column 1, Pr55Gag
either detected by ReAsH or FlAsH accumulated 252
mainly at or near to the plasma membrane with little, if any, fluorescence in the cytoplasm 253
(59). Moreover, eGFP-Vpr and HA-Vpr (column 2) were almost completely absent from the 254
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nucleus and the nuclear envelope in agreement with the images presented in figure 2. Thus, 255
merge images show the co-localization of the two partners mainly at the plasma membrane. 256
Figure 4 257
To further demonstrate that the co-localization of the two partners results from their direct 258
interaction, we combined confocal microscopy for Pr55Gag
localization together with FLIM 259
based FRET to monitor Pr55Gag-TC
/ eGFP-Vpr interaction
in HeLa cells. When eGFP-Vpr was 260
expressed alone and visualized by FLIM after ReAsH staining, the fluorescent lifetime (τ) of 261
eGFP-Vpr was ≈ 2.50 ns (figure 4A, image a). This value corresponds to the fluorescent 262
lifetime of free eGFP, indicating an absence of FRET between the eGFP-Vpr and unbound 263
ReAsH (24, 57). Interestingly, cells observed by the FLIM technique also show an 264
accumulation of eGFP-Vpr in the nucleus. In sharp contrast, when eGFP-Vpr and Pr55Gag-TC
265
were co-expressed and monitored by FLIM after ReAsH staining, a decrease of eGFP lifetime 266
to ≈ 2.11 ns was measured and symbolized by the blue color distribution (figure 4A, image c). 267
This image corresponds to the main observed phenotype (>90%) when equivalent amount of 268
plasmid were co transfected (0.5µg/0.5µg) but could vary depending on the plasmid ratio 269
(data not shown). This fluorescent lifetime drop (from 2.5ns to 2.1ns) corresponds to an 270
average FRET efficiency (E) of 16% in the whole cell which is significantly different from 271
control using multifactorial ANOVA statistical tests (figure 4C). Interestingly, higher transfer 272
efficiency (21%) was observed at/near the PM (black bars), while no significant FRET was 273
observed in the nucleus (white dotted black bars). This experiment is, to our knowledge, the 274
first to visualize a direct interaction between Pr55Gag
and Vpr notably at the level of the 275
plasma membrane. 276
To further correlate the Pr55Gag
/Vpr interaction with the cellular redistribution of Vpr, 277
mutations in 15
FRFG or/and 41
LXXLF sequences of p6 were constructed. All three mutants, 278
Pr55Gag(p6)F15A-TC
, Pr55Gag(p6)L44A-TC
and Pr55Gag(p6)F15AL44A-TC
were efficiently expressed in 279
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cells as shown by western blot analysis (figure 4B). Moreover, confocal analysis of cells 280
expressing these Pr55Gag
mutants stained with ReAsH (figures 4, images d, f and h) revealed 281
that these mutations have a poor effect on the cellular distribution of Pr55Gag
. 282
FLIM measurements on cells co-expressing eGFP-Vpr and Pr55Gag(p6)F15A-TC
-ReAsH showed 283
a decreased Vpr relocation to the PM (figure 4A, image e) associated with a decrease of 284
FRET efficiency at this level (15%) and in the cytoplasm (9%) (figure 4C). An even weaker 285
redirection of eGFP-Vpr to the PM was observed upon changing leucine 44 for an alanine 286
(figure 4A, image g). With this mutant, no FRET was observed in the cytoplasm and only a 287
limited FRET was measured at the PM, highlighting a correlation between the PM 288
localization of Pr55Gag
and Pr55Gag
-Vpr interaction. Finally, this correlation was confirmed 289
with the double mutant Pr55Gag(p6)F15AL44A-TC
(figure 4A, image i) which showed no significant 290
FRET with eGFP-Vpr (figure 4C) and a distribution of eGFP-Vpr mainly in the nucleus, as 291
observed in the absence of Pr55Gag
(figure 4A, image a). 292
Taken together, our data reveals a clear correlation between the interaction of Pr55Gag
with 293
Vpr and the accumulation of Vpr at the PM and confirm the major role of the 41
LXXLF 294
sequence of Pr55Gag
for Vpr recognition. 295
296
Pr55Gag
interacts with Vpr oligomers to promote Vpr accumulation at the plasma 297
membrane. 298
Vpr can form oligomers in vitro and in cells as recently observed (4, 10, 62, 65, 73). This 299
oligomerization is mediated by the Vpr hydrophobic core but not by the flexible N- and C- 300
terminal domains. Indeed, a disruption of the hydrophobic core by point mutations in the first 301
helix (L23F), in the second (∆Q44) and the third one (L67A) results in a loss of Vpr-Vpr 302
interaction (24). The role of this oligomerization in Vpr functions remains to be determined 303
because it does not appear to be required for either Vpr-mediated apoptosis or the G2/M cell 304
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cycle arrest (10, 24). However, since VprL23F, Vpr∆Q44 and VprL67A mutants were not or 305
very weakly incorporated into nascent particles (64, 65, 72), Vpr oligomerization could be 306
needed for its recruitment by Pr55Gag
. 307
Figure 5 308
To establish a correlation between Vpr oligomerization and its interaction with Pr55Gag
, we 309
carried out FLIM experiments on cells expressing eGFP-Vpr and mCherry-Vpr with and 310
without non labeled Pr55Gag
. When the two fusion proteins were co-expressed in HeLa cells, 311
the FRET between the eGFP end the mCherry moiety reflects the presence of Vpr oligomers 312
at the nuclear envelope, in the cytoplasm and in the nucleus (Figure 5A, image b and Figure 313
5B). These data are in agreement with previous studies showing that Vpr can oligomerize in a 314
cellular context (10, 24). To verify our hypothesis that Vpr oligomerization could be required 315
for its interaction with Pr55Gag
, the two Vpr chimeras were co-expressed with non labeled 316
Pr55Gag
. Under this condition, the FRET signal provided by Vpr oligomerization was mainly 317
observed at the plasma membrane (Figure 5A, image d). Interestingly, this Pr55Gag
-promoted 318
relocation of Vpr oligomers resulted in a statistically significant (p< 10-3
) increased FRET 319
between Vpr species (note the darker blue on figure 5A, image d; figure 5B), suggesting a 320
Pr55Gag
– induced compaction of Vpr oligomers or alternatively, a structural rearrangement of 321
Vpr oligomers. 322
Figure 6 323
To confirm that Vpr oligomerization is needed for interaction with Pr55Gag
, Vpr was mutated 324
in its non structured N- and C- termini (Q3R, R77Q) or in its hydrophobic core (L23F, ∆Q44, 325
L67A) (24). The resulting eGFP-Vpr mutants were transfected and imaged in the absence 326
(figure 6, column A) or in the presence of their mCherry-Vpr counterparts (column B) and 327
compared with cells co-expressing eGFP-Vpr derivatives and Pr55GagTC
-ReAsH (column C). 328
As with the non mutated eGFP-Vpr (Figure 6A, images A1, B1, C1), we visualized for eGFP-329
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VprQ3R (Fig. 6A, images A2, B2, C2), a high transfer efficiency when co-expressed with 330
mCherry-VprQ3R (Figure 6C) or with Pr55Gag-TC
-ReAsH (figure 6D). Similar results were 331
obtained with VprR77Q (data not shown). Thus, these mutations did not abolish Vpr 332
oligomerization and interaction with Pr55Gag
. 333
In sharp contrast, mutations in the three helices (L23F, ∆Q44, L67A) abolished Vpr 334
oligomerization and localization at the nuclear envelope (figure 6A, images B3-5 and figure 335
6C), as previously shown (24). Furthermore, co-expression of such mutated Vpr with Pr55Gag-
336
TC, labeled with ReAsH, (figure 6, images C3-5 and figure 6D) resulted in a loss of Vpr 337
interaction with Pr55Gag
and in its redistribution at the PM. These phenotypes cannot be 338
accounted for by a poor expression or degradation of the Vpr proteins since an immunoblot 339
analysis (figure 6B) reveals the sustained expression of all fusion proteins. 340
Taken together, these results show that Pr55Gag
interacts with Vpr oligomers promoting their 341
redistribution at the PM and probably their incorporation into nascent viral particles. 342
Mutations in Vpr helices which prevent oligomerization also inhibit Pr55Gag
-Vpr complex 343
formation. 344
345
Pr55Gag
multimerization and anchoring into the PM are not necessary for its interaction 346
with Vpr. 347
Figure 7 348
Pr55Gag
directs retroviral assembly by multimer formation upon binding to the genomic RNA 349
via the NC domain, and simultaneously through its interaction with the PM by the MA 350
domain (for reviews see (1, 15)). To investigate if Pr55Gag assembly is required for Vpr 351
incorporation into virions, multimerization of Pr55Gag-TC
derivatives was investigated by 352
monitoring the fluorescence lifetime of FlAsH (figure 7A, column A and figure 7B). Results 353
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were compared with those obtained on cells expressing eGFP-Vpr and either the wild type or 354
a mutant form of Pr55Gag-TC
-ReAsH (figure 7A, column B and figure 7C). 355
As a negative control, FlAsH was added to non transfected HeLa cells and imaged by FLIM. 356
A homogeneous staining of the cells with an average fluorescence lifetime of 3.52 ns was 357
measured (figure 7A, image A1 and figure 7B). In contrast, the fluorescence lifetime of 358
FlAsH bound to Pr55Gag-TC
decreases to 2.57 ns at the PM (figure 7A, image A2 and figure 359
7B), reflecting the multimerization of Pr55Gag
. 360
Interestingly, green labeled dotted structures with a lifetime of 2.82 ns were also detected 361
(white arrows), in line with the presence of truncated Pr55Gag
or low molecular weight Pr55Gag
362
complexes in sub-cellular compartments (61, 68). In agreement with the data of figure 6A, 363
image c1 and figure 6D, when eGFP-Vpr was co-expressed with Pr55Gag-TC
and cells were 364
incubated with ReAsH, an interaction between Vpr and Pr55Gag
was found at the PM with an 365
efficiency transfer of 20.6% (Figure 7A, image B2 and figure 7C). 366
Next, to establish a correlation between Pr55Gag
assembly and Pr55Gag
mediated accumulation 367
of Vpr at the PM, a series of mutations was made in Pr55Gag-TC
. All these mutants were 368
expressed in HeLa cells as revealed by western blot (figure S2). First, methionine 369 in the 369
SP1 spacer was substituted for alanine (Pr55GagM369A-TC
). This mutant presents a severe defect 370
in Pr55Gag
assembly in spite of its localization at the PM (21, 31, 40, 45 ). Confocal 371
microscopy confirmed the localization of this mutant at the PM (figure S2b). When cells 372
expressing this mutant were stained with FlAsH and monitored by FLIM, the fluorescent 373
lifetime value of the chromophore was 3.08 ns (figure 7A3, figure 7B). This value is 374
intermediate between those obtained for free FlAsH (3.52 ns) and for Pr55Gag-TC
-FlAsH (2.57 375
ns), indicating that the M369A mutation caused a multimerization defect of Pr55Gag
. When 376
this mutant was labeled with ReAsH and co-expressed with eGFP-Vpr (figure 7A, image B3), 377
an interaction between the two proteins was observed at the PM (E=18.8%, figure 7C), 378
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showing that eGFP-Vpr can interact with an assembly defective Pr55Gag
mutant and be 379
directed to the PM. 380
Then, we checked if Pr55Gag
localization at the PM was required for its interaction with Vpr, 381
since Pr55Gag
-Vpr complexes accumulated at this site. To that end, the myristoyl-acceptor 382
glycine in position 2 was substituted for alanine in the Pr55GagG2A
mutant. This substitution 383
abolished the stable association of Pr55Gag
with the PM and caused a diffuse cellular 384
distribution (figure S2c) (12, 21, 28). Using FLIM, we found a fluorescence lifetime of 2.87 385
ns, homogenously distributed throughout the cytoplasm (figure 7A, image A4 and figure 7B). 386
This value is in between that of Pr55Gag-TC
-FlAsH and that of free FlAsH, and is in line with 387
previous reports indicating that myristylation-defective Pr55Gag
can oligomerize but cannot 388
form high molecular weight complexes (21, 43, 44, 51). Moreover, we observed Pr55GagG2A
389
with a fluorescence lifetime of 3.16 ns within the nucleus suggesting that Pr55Gag
mutants 390
could undergo a nuclear import. When co-transfected with eGFP-Vpr, the Pr55GagG2A-TC
391
mutant redistributed Vpr throughout the cytoplasm (figure 7A, image B4). However, the 392
FRET efficiency found in the cytoplasm for eGFP-Vpr/Pr55GagG2A-TC-ReAsH
(E=21%, figure 393
7C) fully matches that determined for the wild type eGFP-Vpr/Pr55Gag
complex at the PM, 394
indicating that Pr55Gag
/Vpr interaction is independent from Pr55Gag
anchoring into the PM. 395
To further highlight the role of Pr55Gag
on the intracellular distribution of Vpr, K30E and 396
K32E substitutions were inserted in the basic stretch of the MA domain since these mutations 397
have been shown to redirect Pr55Gag
to intracellular vesicles, such as MVB (Multi Vesicular 398
Bodies) or MVB like structures (29, 55). In agreement with this, we observed Pr55GagK30EK32E-
399
TC mainly in vesicles and only slightly at the PM (figure S2d). Interestingly, the fluorescence 400
lifetime of Pr55GagK30EK32E-TC
-FlAsH on the vesicles was 2.64 ns (figure 7A, image A5), close 401
to that obtained for wild type Pr55Gag-TC
at the PM (figure 7B). Thus, these two mutations 402
target the precursor to intracellular vesicles where Pr55Gag
multimerization still takes place. 403
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When co-expressed with eGFP-Vpr, the FRET efficiency between eGFP-Vpr and 404
Pr55GagK30EK32E-TC
-ReAsH monitored in vesicles (E=17.2%, figure 7C) indicates that this 405
Pr55Gag
mutant strongly interacts with eGFP-Vpr (figure 7A, image B5). 406
Taken together, these data suggest that neither the Pr55Gag
localization at the PM nor the 407
Pr55Gag
multimers formation are required for the interaction with Vpr oligomers and the 408
redirection of Vpr to the PM. Moreover, we show here that the cellular localization of Vpr 409
depends upon the cellular localization of Pr55Gag
. 410
411
Discussion 412
The aim of this work was to investigate whether Vpr oligomerization and Pr55Gag
413
multimerization were required for their mutual recognition during virus assembly. To this 414
end, we designed a series of FLIM-FRET measurements to investigate the interaction 415
between Pr55Gag-TC
and eGFP-Vpr in HeLa cells. These proteins were selected because the N-416
terminal tagged Vpr is incorporated into nascent viral particles and Pr55Gag-TC
assembles in 417
VLPs similarly to the wild-type Pr55Gag
(20, 59, 70). As the wild-type Vpr, the fluorescently 418
tagged-Vpr proteins accumulated at the level of the PM, upon co-expression with Pr55Gag
419
alone or in the viral context (figures 2 and 3) indicating that Pr55Gag
directs Vpr at this 420
cellular site. Interestingly, a residual staining was observed in the nucleus when Vpr was co-421
expressed in the viral context (figure 2e). This suggests that an optimal Vpr recruitment 422
requires a correct Pr55Gag
/Vpr ratio (53, 66) or that other viral components compete with Vpr 423
for binding to Pr55Gag
(6). 424
A Gag-mediated redistribution of eGFP-Vpr has been previously observed in 293T cells (13), 425
but in contrast to our results, eGFP-Vpr was found to be directed into the cytoplasm. This 426
discrepancy could be related to the more efficient PM targeting of our codon optimized 427
Pr55Gag
protein (figures 3 and 4, (59)). Nevertheless, the Gag-induced redistribution observed 428
in both cases suggests that Pr55Gag
-Vpr recognition is not dependent on the Pr55Gag
429
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intracellular distribution. In line with this conclusion, both the Pr55GagG2A
mutant which has 430
lost its ability to stably interact with cellular membranes, and the Pr55GagK30E/K32E
mutant 431
which is mainly located in MVBs or MVB-like structures (29, 55) still interact with Vpr 432
(figure 7A). Thus, the Vpr protein likely follows the intracellular Pr55Gag
trafficking. The two 433
viral proteins probably interact early after their synthesis, as further supported by the common 434
intracellular trafficking signals identified on Pr55Gag
and Vpr encoding mRNAs (8, 52). 435
Similarly, the recruitment of the Vif protein by Pr55Gag
was also found to be independent 436
from the anchoring of Pr55Gag
to the PM (5). This emphasizes that Pr55Gag
-mediated 437
packaging of co-factors is not the result of a simple co-localization at the PM, but probably 438
takes place at the site of their synthesis. 439
In line with the interaction of Vpr with the C-terminal domain of Pr55Gag
(4, 18, 35, 46, 62, 440
75), we observed a strong decrease of the FRET signal when Vpr was expressed with 441
Pr55Gag(p6)L44A
and to a lesser extend with Pr55Gag(p6)F15A
, confirming the critical role played 442
by the 41
LXXLF and
15FRFG motifs. Moreover, these domains likely act in concert in the 443
interaction with Pr55Gag
since the double mutant failed to interact with Pr55Gag
. 444
Though Vpr protein self oligomerizes in vitro (11, 65, 71, 73) and in cells (10, 24), the 445
functional role of this oligomerization remains elusive. Interestingly, co-expression of eGFP-446
Vpr and mCherry-Vpr with non labelled Pr55Gag
(figure 5A) clearly show an interaction of 447
Pr55Gag
with Vpr oligomers. This correlation between Vpr oligomerization and Pr55Gag
448
interaction was further confirmed using Vpr mutants (figures 6A, 6C, 6D), though a direct 449
involvement of the mutated residues in Pr55Gag
recognition or in the localization of the 450
mutated Vpr proteins in sub-cellular compartments inaccessible to Pr55Gag
cannot be fully 451
excluded. Interestingly, the inability of L23F, ∆Q44 and L67A Vpr mutants to oligomerize 452
and thus to interact with Pr55Gag
could explain their poor incorporation into viral particles (32, 453
64, 65, 72). 454
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The interaction of Vpr oligomers with Pr55Gag
is likely mediated through hydrophobic 455
contacts between the two hydrophobic 15
FRFG and 41
LXXLF motifs of the p6 domain, which 456
are thought to be in close proximity (23), and the hydrophobic core formed by the 457
neighboring amphipathic α helices in Vpr oligomers (50). This would explain the resistance 458
of Pr55Gag
-Vpr complexes to high salt concentrations (4, 18). 459
In line with previous data (9, 21, 31, 33, 40), we found that Pr55Gag-TC
polymerizes mainly at 460
the level of the PM and to a smaller extent in the cytoplasm (30, 54, 56, 58, 63). Indeed, a 461
lower FRET efficiency was observed in the cytoplasm as compared to the PM. This may 462
reflect an increased distance between Pr55Gag
molecules, as a result for instance of an 463
architecture defect of the polymer or an increased curvature of the lipid bilayer of the MVBs 464
as compared to the PM, or a lower density of Gag polyproteins at the MVB surface. Rather 465
low FRET efficiencies were also observed using the TC-coupled Pr55GagM369A
and Pr55GagG2A
, 466
mutants in line with their ability to form low-order Pr55Gag
oligomers (43, 45). Surprisingly, 467
these two mutants were able to interact with Vpr as efficiently as the wild type protein 468
Pr55Gag
, despite their misfolding. Thus, these data show that in contrast to Vpr, Pr55Gag
469
multimerization is not required per se for its interaction with Vpr. 470
471
Conclusion 472
We report here that Vpr oligomerization is required for its recruitment at the PM by Pr55Gag
473
while Pr55Gag
-Vpr interaction does not need an extensive multimerization of Pr55Gag
. This 474
suggests that Pr55Gag
assembly and Vpr interaction are probably separated events. 475
476
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700
701
Figure legends 702
Figure 1: A schematic view of the chromophore pairs used for the FLIM experiments. 703
Vpr is represented as a small wavy black stick and Pr55Gag
as black bars corresponding to 704
MA, CA, NC and p6. The red and green circles represent mCherry and eGFP proteins, 705
respectively, while the red and the green hooks correspond to ReAsH and FlAsH 706
chromophores. The spatial proximity between the proteins of interest (blue arrows) induces a 707
decrease of either eGFP or FlAsH fluorescence lifetime. 708
709
Figure 2: Pr55Gag
promotes Vpr recruitment at the plasma membrane. 710
A: Fluorescence confocal microscopy was performed 24 hours posttransfection on HeLa cells 711
expressing either eGFP-Vpr or HA-Vpr proteins (panels a and b) alone or together with 712
Pr55Gag
(panels c and d) or pNL4-3∆Env∆Vpr (panels e and f). The cellular distribution of 713
HA-Vpr was revealed by a primary mouse anti-HA while the intracellular localization of 714
eGFP-Vpr was detected by the eGFP fluorescence. Cell nucleus is represented by dashed 715
lines. Note the redistribution of Vpr from the nucleus to the plasma membrane in the presence 716
of Pr55Gag
or pNL4-3∆Env∆Vpr. 717
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B: Western blot of eGFP-Vpr and Pr55Gag
. Plasmids encoding eGFP-Vpr or Pr55Gag
were 718
transfected and 24 hours later, proteins were extracted and analyzed by SDS-PAGE and 719
immunoblotted using an anti-eGFP or an anti-Gag antibody. 720
721
Figure 3: Visualization of the intracellular co-expression of N-tagged Vpr fusion 722
proteins and biarsenical labeled Pr55Gag
. 723
Plasmid DNA expressing the Vpr fusion proteins were co-transfected with plasmid DNA 724
encoding Pr55Gag-TC
. Cells were observed by confocal microscopy 24h post transfection. Each 725
panel shows the major phenotype. 726
Panels A1-A3: Images were recorded on non fixed cells to monitor the biarsenical dye ReAsH 727
and eGFP-Vpr expression. 728
Panels B1-B3: 24h post transfection, Pr55Gag-TC
was labeled by the biarsenical dye FlAsH. 729
The cells were fixed and HA-Vpr was immunodetected using specific HA antibodies and 730
Alexa568 coupled secondary antibodies. Note the co-localization of the Vpr fusion proteins 731
and Pr55Gag
at or close to the plasma membrane. 732
733
Figure 4: Imaging the interaction between HIV-1 Pr55Gag
and Vpr. 734
A: Panels b, d, f and h show confocal microscopy images of HeLa cells incubated with 735
ReAsH and expressing either Pr55Gag-TC
, Pr55Gag(p6)F15A-TC
, Pr55Gag(p6)L44A-TC
or 736
Pr55Gag(p6)F15AL44A-TC
. Panels a, c, e, g and i represent FLIM images of HeLa cells labeled with 737
ReAsH and a: eGFP-Vpr alone, c: eGFP-Vpr and Pr55Gag-TC
, e : eGFP-Vpr and Pr55Gag(p6)F15A-
738
TC, g : eGFP-Vpr and Pr55
Gag(p6)L44A-TC and i : eGFP-Vpr and Pr55
Gag(p6)F15AL44A-TC. The 739
fluorescence lifetime τ of eGFP-Vpr was measured, in the presence or the absence of the 740
acceptor protein in each pixel of the image and converted using a color scale ranging from 741
blue (1.5 ns = short fluorescence lifetime) to red (3.0 ns = long fluorescence lifetime). A 742
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significant drop of eGFP-Vpr fluorescence lifetime, caused by FRET, gives a blue color. Note 743
that eGFP-Vpr/Pr55Gag-TC
-ReAsH complexes predominantly accumulated at the plasma 744
membrane. 745
B: Western blot analysis of Pr55Gag-TC
derivatives: Pr55Gag-TC
derivatives were deposited on 746
SDS PAGE and revealed by a polyclonal anti-Gag antibody. The level of expression was 747
compared with GADPH proteins. All mutants migrate to the expected molecular weight, 748
reflecting their integrity. 749
C: Histograms representing the FRET efficiencies between eGFP-Vpr and Pr55Gag-TC-ReAsH
750
derivatives in different cellular compartments. The FRET efficiencies of eGFP-Vpr in the 751
presence of the acceptor ReAsH were measured in the cytoplasm, at the nuclear envelope, at 752
the plasma membrane, in the nucleus and over the whole cell. The FRET efficiencies were 753
calculated using the average lifetime values from at least 30 cells in three independent 754
experiments. Multifactorial ANOVA and post-hoc Dunnett test were performed to compare 755
the FRET efficiencies (*: p<0.05, ***: p<10-3
). Note that the FRET efficiency is the highest at 756
the PM, whereas no significant FRET is observed in the nucleus. 757
758
Figure 5: Pr55Gag
interacts with Vpr oligomers 759
A: FLIM of HeLa cells expressing eGFP-Vpr alone (a), eGFP-Vpr and mCherry-Vpr (b), 760
eGFP-Vpr and Pr55Gag
(c) and both eGFP-Vpr/mCherry-Vpr and Pr55Gag
(d). 761
B: Histograms representing the FRET efficiencies between eGFP-Vpr and mCherry-Vpr in 762
the absence or the presence of none labeled Pr55Gag
. The FRET efficiency was calculated 763
using the average lifetime values from at least 30 cells. Note that the darker blue in image d 764
compared to image b corresponds to a significantly higher FRET efficiency between 765
fluorescent proteins as determined by the use of one-way ANOVA and Tukey’s HSD test 766
(***: p<10-3
). 767
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768
Figure 6: Vpr oligomerization is essential for Pr55Gag
–Vpr interaction. 769
A: FLIM experiments were carried out on cells expressing: Column A: wild type or mutant 770
eGFP-Vpr alone; Column B: wild type or mutant eGFP-Vpr and their equivalent counterparts 771
fused to mCherry; Column C: wild type or mutant eGFP-Vpr and Pr55Gag-TC
bound to ReAsH. 772
Note that only the oligomerization prone Vpr proteins (light blue color, column B) were 773
found to interact with Pr55Gag-TC
(dark blue, column C). 774
B: Western blot analysis of eGFP-Vpr derivatives. The eGFP-Vpr proteins were deposited on 775
SDS/PAGE and detected using monoclonal anti-eGFP. All mutants migrate to the expected 776
molecular weight, which reflects their integrity. Nevertheless, a weak band (0.5-2%) of Vpr 777
fusion proteins, was observed at a lower molecular weight and may thus correspond to a 778
partly truncated eGFP-Vpr (13) 779
C and D: Histograms representing the FRET efficiencies between eGFP-Vpr and mCherry-780
Vpr derivatives (C) and between eGFP-Vpr derivatives and Pr55Gag-TC-ReAsH
(D) in the 781
cytoplasm, at the nuclear envelope, at the plasma membrane, in the nucleus and over the 782
whole cell. The FRET efficiency was calculated using the average lifetime values from at 783
least 30 cells in three independent experiments. Multifactorial ANOVA and post-hoc Dunnett 784
test were performed to compare the FRET efficiencies (*: p<0.05, ***: p<10-3
). 785
786
Figure 7: Patterns of Pr55Gag
-Vpr interaction as a function of Pr55Gag
multimerization 787
and localization at the plasma membrane. 788
Figure 7A: FLIM experiments using two different donor-acceptor pairs (FIAsH/FIAsH and 789
eGFP/ReAsH). The lifetime scales are shown at the top of the two first panels. Column A: 790
Cellular distribution of the FlAsH chromophore alone (A1) or covalently bound to Pr55Gag-TC
791
(A2), Pr55GagM369A-TC
(A3), Pr55GagG2A-TC
(A4) and Pr55GagK30EK32E-TC
(A5). Cells containing 792
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the FlAsH chromophore (A1) alone gave a fluorescent background signal, due to association 793
with endogenous cystein motifs. Column B: FLIM images on HeLa cells expressing either 794
eGFP-Vpr alone (B1) or eGFP-Vpr and Pr55Gag-TC
wild type (B2) or eGFP-Vpr and the 795
mutant proteins Pr55GagM369A-TC
(B3), Pr55GagG2A-TC
(B4), Pr55GagK30EK32E-TC
(B5). The strong 796
drop of eGFP-Vpr fluorescence lifetime when co-expressed with wild type or mutant Pr55Gag-
797
TC–ReAsH suggests that Pr55
Gag-Vpr interaction can take place in the absence of proper 798
Pr55Gag
multimerization and PM localization. 799
Figure 7B and 7C: Box and whiskers plots of the fluorescence lifetime of FlAsH for the 800
different FlAsH-labeled Pr55Gag
derivatives (B) and the FRET efficiencies between eGFP-Vpr 801
and the TC-ReAsH-labeled Pr55Gag
derivatives (C). The boxes define the interquartile range 802
which extends from the 25th
to the 75th
percentile, whereas the horizontal lines show the 803
median values. The whiskers correspond to 1.5 times the interquartile range. Outliers are 804
shown as circles. 805
806
Figure S1: Scheme of the FLIM imaging and analysis. 807
First, HeLa cells were co-transfected with the fluorescent acceptor and donor pairs described 808
in figure 1. 24 hours post transfection, cells were chosen under a mercury lamp for their 809
comparable expression of green (eGFP-Vpr) and red (mCherry-Vpr or Pr55Gag-TC-ReAsH
) 810
fluorescence. These cells were then imaged individually by the 2-photon laser scan of the 811
FLIM setup. The resulting intensity image was analyzed with the SPC image software 812
(Becker and Hickl), that calculates the fluorescence lifetime from the fluorescence decay 813
curves measured in each pixel (left part of this scheme). Using an arbitrary color scale, a 814
convolution between the intensity image and the lifetime image can thus give rise to a colored 815
FLIM image (right part of this scheme). On this colored FLIM image, different regions of 816
interest (plasma membrane, cytoplasm and nucleus) were selected and the corresponding 817
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