Title: Authors : Joëlle V. Fritz , Denis Dujardin...

1

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

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Word count for abstract: 217 21

Word count for the text: 5535 22

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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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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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