Rotavirus NSP3 is a translational surrogate of the PABP-poly(A

Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex

1

Matthieu Gratia1, Emeline Sarot2+, Patrice Vende1+, Annie Charpilienne1, Carolina 2

Hilma Baron1, Mariela Duarte3,1, Stephane Pyronnet2* and Didier Poncet1* 3

4

5

6

1-Institut de Biologie Integrative de la Cellule (I2BC), UMR 9198, département de 7

Virologie, USC INRA 1358, 1 avenue de la Terrasse 91198 Gif sur Yvette, France. 8

2-INSERM UMR-1037 - Université de Toulouse III-Paul Sabatier, Equipe labellisée 9

Ligue Contre le Cancer, Laboratoire d'Excellence Toulouse Cancer (TOUCAN), 2 10

avenue Hubert Curien, 31037 Toulouse Cedex 1, France 11

3-Université d’Evry Val d’Essonne, Département de Biologie, 91025 Evry Cedex, 12

France. 13

14

*corresponding authors: 15

Didier Poncet, [email protected] 16

Stephane Pyronnet, [email protected] 17

18

+ authors contributed equally to this work 19

running title: Rotavirus NSP3 enhances translation 20

Key words: translation, RNA 3' ends, rotavirus, NSP3.21

JVI Accepted Manuscript Posted Online 10 June 2015J. Virol. doi:10.1128/JVI.01402-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Rotavirus NSP3 enhances translation 2

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ABSTRACT 24

Through its interaction with the 5' translation initiation factor eIF4G, poly(A) 25

binding protein (PABP) facilitates the translation of 5'-capped and 3'-poly(A)-tailed 26

mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in 27

a 3' GACC motif that is recognized by the viral protein NSP3, which competes with 28

PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are 29

efficiently translated while host poly(A)-tailed mRNA translation is, in contrast, 30

severely impaired. To explore the roles of NSP3 in these two opposing events, the 31

translational capabilities of three capped mRNAs, distinguished by either a GACC, a 32

poly(A) or a non-GACC and non-poly(A) 3' end, have been monitored after 33

electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 34

(stably or transiently transfected cells). In infected cells, we found that the 35

magnitudes of translation induction (GACC-tailed mRNA) and translation reduction 36

(poly(A)-tailed mRNA) both depended on the rotavirus strain used but were not 37

genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was 38

sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to 39

slightly favor the translation of both poly(A)- and non-poly(A)-tailed mRNAs, likely 40

through by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is 41

a translational surrogate of the PABP-poly(A) complex and therefore cannot by itself 42

be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon 43

rotavirus infection. 44

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46

IMPORTANCE 47

To control host cell physiology and to circumvent innate immunity, many viruses have 48

evolved powerful mechanisms aimed at inhibiting host mRNA translation while 49

stimulating translation of their own mRNAs. How rotavirus tackles this challenge is 50

still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of 51

cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not 52

genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that 53

NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also 54

enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the 55

eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA 56

translation during rotavirus infection cannot be solely attributed to NSP3 and is more 57

likely the result of global competition between viral and host mRNAs for the cellular 58

translation machinery. 59

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61

INTRODUCTION. 62

When delivered into or synthesized by the host cell, viral mRNAs compete with 63

cellular mRNAs already present in the cytoplasm for access to the protein synthesis 64

machinery. Recruitment of the 40S ribosomal subunit onto mRNA (translation 65

initiation) is the rate-limiting and the most controlled step of eukaryotic protein 66

biosynthesis and, hence, highly competitive for both cellular and viral mRNAs. The 5’ 67

cap and 3’ poly(A) tail of most cellular mRNAs are joined by a protein complex 68

containing the cap binding protein eIF4E and the poly(A) binding protein PABP, 69

which are bound together by the scaffold protein eIF4G (1). This complex recruits the 70

pre-initiation complex (PIC), which comprises the 40S ribosomal subunit loaded with 71

the methionine initiator tRNA, eIF2, GTP and several other translation initiation 72

factors (2, 3). PABP is thought to stimulate translation initiation at least in part by 73

promoting cap-to-poly(A) circularization of mRNA (4, 5). This appears to be 74

especially true when mRNAs compete for ribosome binding. In this case, the 75

presence of either structure alone at mRNA extremities is not enough to drive 76

efficient translation, but together, they synergize and direct ribosome entry at the 5' 77

end (6-8). 78

Rotavirus mRNAs are capped but lack the poly(A) tail required for efficient 79

translation initiation. Instead, each rotavirus mRNA ends with the same 3’ GACC 80

sequence. Despite this apparent handicap, rotavirus mRNAs efficiently compete with 81

cellular mRNAs, and viral proteins are rapidly detectable in infected cells while the 82

synthesis of host proteins is shut off (9). 83

We and others have shown that the rotavirus non-structural protein NSP3 is 84

bound to the 3’ end of viral mRNAs during infection (10) and that NSP3 dimers 85


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specifically bind the 3’ GACC sequence (11, 12) and eIF4G (9, 13). The 86

simultaneous interactions of NSP3 with the viral mRNA 3’ end and with eIF4G have 87

been shown to enhance the translation of rotavirus-like reporter mRNAs, as does 88

PABP with cellular polyadenylated mRNAs (14, 15). 89

NSP3 dimers interact with eIF4G at the same position as PABP but with a 10-90

fold higher affinity (11). In fact, during rotavirus infection, PABP is evicted from eIF4G 91

by NSP3 (9) and relocalizes to the cell nucleus (16). These observations led to the 92

idea that the shutdown of cellular protein synthesis is due to the eviction of PABP 93

and, hence, to the displacement of cellular polyadenylated mRNAs from eIF4G. This 94

model has also been supported by in vivo experiments using recombinant vaccinia 95

virus expressing NSP3 (17), as well as by in vitro translation assays using 96

recombinant NSP3 or its eIF4G-binding domain (6, 15). 97

The opposite functions of NSP3 in viral versus cellular mRNA translation have 98

been challenged by data obtained in rotavirus-infected cells in which NSP3 levels 99

were down-regulated by RNA interference (18). The NSP3 RNA- and eIF4G-binding 100

domains (RNA-BD and 4G-BD, respectively) have been proposed to function 101

independently, with the enhancement of viral mRNA translation due to NSP3 instead 102

resulting from viral mRNA protection by the RNA-BD, whereas 4G-BD is involved in 103

inhibiting host poly(A) mRNA translation (18). In the same vein, the translation of 104

polyadenylated mRNAs introduced directly into the cytoplasm was not inhibited by 105

rotavirus infection, in contrast to mRNAs synthesized in the nucleus, suggesting that 106

a reduction in mRNA nuclear export rather than translation inhibition accounts for the 107

rotavirus-mediated extinction of host protein synthesis (19). However, using an in 108

vivo assay, we recently showed that the NSP3-dependent protection of viral mRNA 109

from degradation was not sufficient to enhance translation and that NSP3 RNA-BD 110


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and 4G-BD did not work separately (20). Here, using the same in vivo assay in cells 111

infected with either the RF or RRV rotavirus strain or in cells transfected solely with 112

NSP3, we show that both the extent of poly(A) mRNA translation inhibition and the 113

extent of rotavirus-like mRNA translation stimulation vary depending on the rotavirus 114

strain used. We also reveal that, independently of mRNA protection, the translation of 115

reporter mRNAs ending with the rotavirus GACC motif is hugely induced by even low 116

levels of NSP3 expression. Furthermore, we found that low levels of NSP3 similarly 117

enhance the translation of both polyadenylated and non-polyadenylated mRNAs and 118

favor eIF4E-eIF4G interaction, suggesting that NSP3 alone can substitute for the 119

PABP-poly(A) complex in translation initiation. 120

121

MATERIAL AND METHODS. 122

123

Cells and viruses. Embryonic rhesus monkey kidney MA104 and Baby 124

Hamster Kidney BSRT7 (21) cells were maintained in Eagle’s minimum essential 125

medium (Lonza) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 126

100 µg/ml streptomycin. Human HeLa-S3 cells were obtained from the American 127

Tissue Culture Collection and maintained in Dulbecco’s minimum essential medium 128

(Lonza) supplemented with 10% fetal bovine serum (Lonza) and 5 IU/ml penicillin-129

streptomycin (Lonza) in 5% CO2. 130

The bovine RF or simian RRV strains of group A rotavirus (RVA) were used to 131

infect MA104 cells. Viral infectivity was determined by plaque assay on MA104 cells 132

as described previously (22). Infections were performed at a multiplicity of infection 133

(MOI) of 10 PFU/cell in EMEM in the presence of trypsin (0.44 µg/ml) and antibiotics 134

but without serum. 135


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Plasmid construction and mutagenesis. The plasmid for in vitro 136

transcription of the Renilla luciferase rotavirus-like reporter mRNA (R-RNA) has been 137

described in detail (20). Briefly, the DNA template (pT7-RF-Rluc) comprises the 138

following from 5’ to 3’: the T7 promoter fused to the 5’ UTR of rotavirus RF gene 11, 139

the Renilla luciferase coding sequence, and the 3’ UTR of rotavirus RF gene 6 140

followed by a BsaI restriction site. The BsaI restriction site is positioned such that 141

run-off transcription of BsaI-linearized plasmids produces RNAs with the canonical 142

GACC rotavirus sequence at the 3’ end. Deletion by site-directed mutagenesis of the 143

last 3 nucleotides of the canonical sequence to generate pT7-RF-Rluc-nona for in 144

vitro synthesis of N-RNA, the addition of 66 adenosines (see below) at the 3’ end of 145

the 3' UTR to generate pT7-RF-Rluc-p(A) for in vitro synthesis of pA-RNA, and site-146

directed mutagenesis of the last nucleotides of the canonical 3' sequence to generate 147

pT7-RF-Rluc-GGCC and pT7-RF-Rluc-GAACC for in vitro synthesis of Nc-RNA, have 148

been described (20). The plasmid pEMCV-Fluc for in vitro transcription of the 149

standard RNA (S-RNA) has been described (20). S-RNA was obtained by in vitro 150

transcription of the EcoRI-linearized pEMCV-Fluc plasmid. 151

The construction of the NSP3 cytoplasmic-expression vector pT7-IRES NSP3 152

have also been described (20); this plasmid is composed of the T7 promoter fused to 153

the Encephalomyocarditis virus Internal Ribosome Entry Site (EMCV-IRES) cloned 154

upstream of the NSP3 ORF from either the RF or RRV rotavirus strain. A nonsense 155

codon was introduced via site-directed mutagenesis (23) using pfu DNA polymerase 156

at position 7 of NSP3 ORF to generate pT7-IRES-NSP3-KO, which was used as a 157

negative control. 158

The C-terminal half of RF-NSP3, either with (NSP3 150-313, hereafter named 159

cNSP3) or without (NSP3 206-313, hereafter named cΔRX) the RoXaN binding 160


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domain, was cloned into the peGFPC1 vector (Clontech) in frame with eGFP. The 161

eIF4G-binding domain was removed from cNSP3 by the addition of a stop codon at 162

position 240 via site-directed mutagenesis, yielding NSP3 150-239 (hereafter named 163

cΔ4G). 164

All plasmid constructs were verified by restriction enzyme mapping. PCR 165

fragments were entirely sequenced after cloning into the target plasmids. In the case 166

of site-directed mutagenesis, the whole functional unit (from the T7 promoter to the 167

T7 terminator) was sequenced. 168

169

In vitro transcription. Capped reporter RNAs were synthesized from BsaI-linearized 170

plasmid templates by in vitro transcription using the mMessage mMachine T7 ultra kit 171

(Ambion). S-RNA was synthesized using the T7 mega script kit (Ambion), which does 172

not contain the ARCA cap analog (24). DNA was removed by enzymatic treatment 173

(15 min at 37°C) with RNAse-free DNase, and RNAs were then purified on 174

MegaClear (Ambion) silica spin columns to eliminate non-incorporated cap analogue 175

and nucleotides prior to ethanol precipitation and washing with 70% ethanol. Purified 176

RNAs were quantified using a spectrophotometer (Nanodrop), confirmed on 177

denaturing agarose gels and stored in aliquots at –80°C. 178

179

180

Cell transfection. DNA was introduced into BSRT7 cells using Lipofectamine 2000 181

(Life Technologies) and into HeLa cells using ExGen 500 (EuroMedex) according to 182

manufacturers’ instructions. 183

RNA was introduced into BSRT7 or MA104 cells using a Neon electroporation device 184

(Life Technologies). Cells (106) were trypsinized and suspended in R buffer (Life 185


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Technologies) with 50 ng of reporter mRNAs (and 1 μg of standard RNA when 186

indicated). Conditions for optimal electroporation were determined for each cell line; 187

MA104 cells were electroporated by two 30-ms pulses of 1150 V, and BSRT7 cells 188

by two 20-ms pulses of 1400 V. After electroporation, cells were immediately 189

transferred to complete culture medium and incubated at 37°C. To remove RNA 190

remaining outside the cells, cells were incubated for 15 min at room temperature with 191

20 μg/ml of RNAse A before RNA purification. 192

193

MA104 retro-transduction and stable transfection. The MA104C20bis cell clone 194

(hereafter referred as C20b) was obtained by retro-transduction. The RF NSP3 195

coding sequence (amino acids 4 to 365) was amplified using the oligos 196

721033newdir CTATTCATAcgTATATTCATA and 721033newrev 197

gccaccATGGAGTCTACACAG and cloned into the pLenti 6.3 V5 topo vector (Life 198

Technologies) by TA cloning to generate pLenti 6.3 NSP3. VSV-pseudotyped 199

lentivirus particles were obtained by co-transfecting 293FT cells with pLenti 6.3 NSP3 200

and pLP1, pLP2, and pLP/VSVG (Virapower, Life Technologies). Cell culture medium 201

was then used to infect MA104 cells, and cells resistant to blasticidin (1 mg/ml) were 202

selected and cloned. A cell line expressing NSP3 (as detected by indirect 203

immunofluorescence) was subcloned by limited dilution and amplified. 204 The MA104 cell line constitutively expressing Renilla luciferase was obtained 205

by lipofection of the plasmid pcDNA3.1purodsRluc-4xJCV (25), which encodes a 206

destabilized Renilla luciferase under the control of the CMV promoter, into MA104 207

cells and selection with 10μg/ml of puromycin for 6 days. Cell clones were obtained 208

by limited dilution and amplified, and a clone expressing a moderate level of Renilla 209

was selected. 210


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211

RNA quantification by RT-qPCR 212

Primers for qPCR were selected using OLIGO 7 Primer analysis software (26). The 213

following primers were used: GGAATTATAATGCTTATCTACGTGC and 214

CAGTATTAGGAAACTTCTTGGC for Renilla reporters and 215

CAGCCTCAAGATCATCAGCA and TGTGGTCATGAGTCCTTCCA for GAPDH. 216

The PCR efficiency of each primer pair was established by measuring serial dilutions 217

of cDNA from transfected MA104 cells in triplicate. Only CT values <40 were used to 218

calculate the PCR efficiency from the given slope according to the equation PCR 219

efficiency= 10 (-1/slope) -1) x100. All PCRs displayed an efficiency between 98% and 220

100%. 221

RNA was extracted from cells using the Nucleospin RNA II (Macherey-Nagel) 222

protocol and precipitated using 5 μg of acrylamide (Ambion) as a carrier. RNA was 223

quantified using a Nanodrop spectrophotometer, and 100 ng of total RNA was 224

reverse transcribed by random priming using pdN6 oligonucleotides (10 μM) and 225

Superscript reverse transcriptase (Life Technologies). 226

q-PCR was performed on 5 μl of a 1:5 dilution of reverse-transcribed RNA (1:15 for 227

GAPDH) with MESA green qPCR MasterMix Plus (Eurogentec) using a MxPro3000 228

(Stratagene) apparatus. Primer efficiency was >98%. The results were analyzed 229

according to the 2-ΔΔct method using GAPDH mRNA as an endogenous reference 230

(27). A calibrator (MA104 cells transfected with Renilla and firefly luciferase mRNA) 231

was introduced on each plaque to compare results from plaque to plaque. Three 232

experiments with different in vitro synthesized RNA reporter preparations were 233

performed in triplicate. 234

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Luciferase activity. Cells were assayed using the Dual-Glo Luciferase Assay 236

System (Promega). 237

238

Immunoprecipitation and western blot. Immunoprecipitations from HeLa cells 239

were performed as previously described (28). For western blotting, proteins were 240

separated by SDS-PAGE, transferred to low-fluorescent PVDF membranes and 241

probed with a rabbit polyclonal antibody against NSP3 (4-150) (29), anti-GAPDH 242

monoclonal antibody (Abcam-ab8245), rabbit anti-eIF4GI (kindly provided by Dr. N. 243

Sonenberg), mouse anti-PABP (#10E10, Cell Signaling Technology), mouse anti-244

eIF4E (#9676, Santa Cruz) or rabbit anti-GFP (#6556, Abcam). Anti-rabbit and anti-245

mouse secondary antibodies coupled to Dyelight 800 (Perbio) and IRdye 700 (LI-246

COR) or coupled to horseradish peroxidase (Pierce) were used. Western blots were 247

visualized either using an OdysseyFC imager and quantified using Image Studio 248

software (LI-COR) or using SuperSignal West Pico Chemiluminescent Substrate 249

(Pierce) and exposed to Hyper films (GE Healthcare). 250

251

Generation and selection of RF-RRV monoreassortants. Reassortants between 252

the RF and RRV rotaviruses were obtained by coinfecting MA104 cells at an MOI of 253

5 for each virus. Progeny viruses were then plaque purified and amplified, and their 254

genomic dsRNA was extracted with TRIzol (Life Technologies) from 0.5 ml of cell 255

culture medium. The dsRNA profiles were determined via polyacrylamide gel 256

electrophoresis and silver staining (Silver Stain Plus kit; Bio-Rad). The parental 257

origins of segments with identical electrophoretic mobilities were further established 258

by RT-PCR using specific primers; genuine monoreassortants were plaque purified 259

twice and amplified. 260


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261

RESULTS 262

Translation of R-RNA, N-RNA and pA-RNA in mock- and rotavirus-infected 263

MA104 cells. 264

265

Three types of capped Renilla reporters differing only in their extreme 3' 266

terminal sequences were synthesized in vitro (figure 1A). The rotavirus-like mRNA 267

(R-RNA) contains the Renilla ORF cloned between the 5' and 3' UTRs of rotavirus 268

genes and ends with the canonical 3' sequence GACC. The non-rotavirus, non-269

polyadenylated mRNA (N-RNA) has an identical structure but, due to a deletion of 270

the last three nucleotides, ends with a UGUG sequence that is not recognized by 271

NSP3 (12). The polyadenylated mRNA (pA-RNA) that mimics a cellular mRNA is 272

identical to R-RNA but ends with a poly(A) sequence containing 65 adenosines. 273

R-RNA, N-RNA and pA-RNA were introduced via electroporation into MA104 274

cells that were either mock infected or infected for 2.5 h with the RF or RRV rotavirus 275

strain at an MOI of 10. The Renilla luciferase activity (Rluc) and the remaining 276

quantities of electroporated reporter RNA were measured in parallel in the same 277

lysates 6 h post electroporation (8.5 h post infection). 278

To establish whether GAPDH cellular mRNA could be used as a stable 279

reference gene over time for RT-qPCR quantification, RNA was purified from mock-, 280

RF- or RRV-infected cells at different times after infection. As illustrated in figure 1B, 281

the GAPDH Ct values were not significantly modified up to 9 h after infection with any 282

of the two strains used. This result validates the use of GAPDH as a reference gene 283

for RT-qPCR. 284


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Figure 2A shows the relative amount of the reporter RNAs still present in 285

mock- and rotavirus-infected cells as determined by RT-qPCR. Higher (2- to 3-fold) 286

amounts of reporter RNAs were present in rotavirus-infected cells, but no significant 287

difference was observed between the two strains used. This stabilization of steady-288

state RNA levels by infection was not more important for pA-RNA or N-RNA than for 289

the R-RNA, excluding a role of NSP3 in specifically protecting R-RNA from 290

degradation. 291

As the abundance of an mRNA does not always correlate with its translational 292

capability (30, 31), the relative Renilla activities measured in these different 293

conditions are presented in figure 2B without taking into account the amounts of RNA 294

detected; however, to ease comparison, the Rluc activity obtained with R-RNA 295

electroporated into mock-infected cells was set to 1. In mock-infected cells, the pA-296

RNA was the only mRNA that was efficiently translated, the R- and N- RNAs being 297

translated with several hundred fold lower efficiently (compare figures 2B and 2C). R-298

RNA was translated efficiently when cells were infected with either RF or RRV (figure 299

2B; of 29-fold and 46-fold stimulation for RF and RRV, respectively, compared with 300

mock-infected cells). However, R-RNA was translated 5-10- fold less efficiently in 301

rotavirus infected cells than pA-RNA in mock infected cells showing that a GACC 302

sequence at the 3' end of an mRNA is not as efficient as a poly(A) tail in stimulating 303

translation. A significant increase (x4-6) in the translation of N-RNA upon infection 304

with the RF or RRV strains was also detected. Infection with the RF or RRV rotavirus 305

strain differentially affected pA-RNA translation; a significant (5-fold) decrease was 306

observed when cells were infected with the RF strain, whereas RRV infection did not 307

significantly reduce pA-RNA translation relative to mock-infected cells (figure 2C). 308


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However, as the electroporated RNAs were 2 to 3-fold more abundant in 309

infected cells (figure 2A), when luciferase activities were normalized to RNA 310

abundance, R-RNA translation stimulation by infection was still substantial (10 and 311

34-fold for RF and RRV, respectively, figure 2D), but pA-RNA translation was 312

strongly diminished in both RF- (:19) and RRV- (:4) infected cells (figure 2E). 313

Furthermore, the translation stimulation of R-RNA was greatly reduced compared 314

with N-RNA (2 and 8-fold for RF and RRV, respectively: note that the large variations 315

observed with RRV render this difference not statistically significant). 316

These experiments showed that rotavirus infection did not preferentially 317

stabilize R-RNA and that the extents of rotavirus mRNA translation enhancement and 318

poly(A) mRNA translation inhibition both depended on the rotavirus strain used for 319

infection. 320

321

Translation of R-, pA- and N-mRNAs in MA104 cells expressing NSP3 322

323

The experiments described above indicated that rotavirus infection increased 324

R-RNA translation but decreased pA-RNA translation, two opposing functions that 325

are attributed to rotavirus NSP3 during infection (15). To determine if these 326

observations were reproducible in cells expressing only NSP3, cells constitutively 327

expressing NSP3-RF were generated. MA104 cells, which are readily infected with 328

rotavirus and can more accurately reflect what occurs in infected cells, were chosen 329

to express RF-NSP3 because this strain strongly inhibits poly(A) mRNA translation 330

(figures 2 and 4). The established cell line C20b was selected because it expresses a 331

low level of NSP3-RF (less than MA104 cells infected with the RF strain at an MOI of 332

10 for 3 h, as estimated by western blotting (figure 3A)). C20b and parent MA104 333


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cells were transfected with the reporter mRNAs, and RNAs were purified from the 334

transfected cells immediately (T0) or 6 h (T6) after electroporation. The amounts of 335

Rluc RNA present at T6 and T0 were quantified by RT-qPCR (figure 3B), and Rluc 336

activity was measured at T6 (figure 3 C-F). 337

The amounts of transfected RNA immediately after electroporation (T0) were 338

not significantly different in MA104 and C20b, showing that both cell types were 339

electroporated with similar efficiencies with the three types of mRNAs (figure 3B). Six 340

hours after electroporation, all the reporter mRNAs were diminished in both cell 341

types. Importantly, R-RNA was no more abundant 6 h after electroporation in C20b 342

cells than in MA104 cells (and even less so), contrary to what might have been 343

expected if NSP3 protected rotavirus mRNAs from degradation. 344

Figure 3C and D illustrate the luciferase activities measured in each condition; 345

these activities were normalized to the amounts of reporter mRNA present at T0 to 346

take into account the slight, although statistically insignificant, differences in the 347

amount of transfected RNAs (figure 3B). The luciferase activity driven by each 348

reporter was enhanced in C20b cells (compared with the parental MA104 cells), 349

albeit to different degrees. The translation of R-RNA was stimulated by 125-fold, 350

whereas the translation of pA-RNA and N-RNA were stimulated by only 2.2 and 2.6-351

fold, respectively. Thus, if we consider that the C20b cells translated mRNAs twice as 352

well as MA104 cells, then R-RNA is still translated >60-fold better in cells expressing 353

NSP3 than in cells that lack NSP3 expression. This stimulation is even more 354

pronounced than the stimulation of translation of the same R-RNA in RF- or RRV-355

infected cells versus mock-infected cells (60-fold versus 29- or 46-fold; figure 2C) 356

despite the much lower NSP3 expression in C20b cells (figure 3A). 357


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Importantly, the higher (2.2 or 2.6) translation activity of the N- or pA-type 358

RNAs in C20b cells was not related to a higher steady-state RNA level in these cells 359

(figure 3B) and more likely reflected an overall increase in translation. Because the 360

amounts of reporter RNAs remaining in MA104 and C20b cells were quite similar, 361

using the amount of RNA measured 6 h after electroporation to normalize reporter 362

expression (figure 3E and F) did not modify the above conclusions. 363

These experiments showed that even a low amount of NSP3 is sufficient to 364

strongly stimulate the translation of mRNAs ending with GACC. They also reveal that 365

the strong stimulation of R-RNA translation by NSP3 cannot be attributed to NSP3-366

dependent RNA protection. Furthermore, it appeared that NSP3 per se does not 367

inhibit but rather induces the translation of both poly(A) and non-poly(A) mRNAs, 368

suggesting that NSP3 alone can substitute for the PABP-poly(A) complex in 369

stimulating translation initiation. 370

371

Differences in cellular poly(A) mRNA expression between RF and RRV are not 372

genetically linked to NSP3. 373

The above results showed that the extent of pA-RNA translation inhibition 374

differs between the RRV and RF rotavirus strains and that R-RNA translation 375

stimulation but not pA-RNA translation inhibition can be obtained by expressing 376

NSP3 alone. 377

To establish whether the RF and RRV rotavirus strains also differed in their 378

capacity to inhibit cellular poly(A) mRNA expression, an MA104 cell line constitutively 379

expressing a destabilized renilla luciferase (25) was established. The use of a 380

destabilized luciferase increases the sensitivity and dynamic range of the luciferase 381

reporter assay (32). Luciferase activities were then measured at several time points 382


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after cells were infected with the RF or RRV viruses. The results are reported in 383

figure 4. It can be noted that a slight increase in luciferase activity was observed 1-2 384

hours after infection probably due to a transient inhibition of the proteasome 385

consequential to the activation of the unfolded protein response triggered by rotavirus 386

infection (19, 33). RF required 3.5 h and RRV required 6 h to inhibit 50% of the 387

cellular protein synthesis, and whereas RF reduced expression to 10% of the initial 388

value after 8 h of infection, RRV reduced expression to just below 50% (figure 4) 389

during the same period. To determine if these "slow" or "fast" phenotypes were linked 390

to the genetic origin of NSP3, reassortants were generated between the RRV and RF 391

viruses. Two monoreassortants were selected and plaque purified twice. The first 392

monoreassortant (RF/07RRV) bears the RRV NSP3 gene (gene 7) but retains the 393

remaining ten RF genes. Conversely, the second selected monoreassortant 394

(RRV/07RF) bears an RF gene 7 but retains the other ten RRV genes. Interestingly, 395

the different kinetics of protein synthesis inhibition imposed by the two 396

monoreassortants (figure 4) were independent of NSP3 origin, as the RF/07RRV 397

reassortant displayed a "fast" phenotype quite similar to that of RF, whereas the 398

RRV/07RF reassortant displayed a slower phenotype than that of parental RRV. This 399

experiment indicated that the different capacities of the RF and RRV viruses to inhibit 400

cellular poly(A) mRNA translation is not linked only to NSP3. 401

402

RRV and RF NSP3 stimulate R-RNA translation similarly. 403

Transfection of R-RNA into cells infected with RF or RRV showed that R-RNA 404

translation enhancement was higher with the former virus (figure 2C and 2E). We 405

therefore tested whether such a difference could be attributed to strain-specific 406

NSP3s. To this end, NSP3 from the RF (NSP3-RF) or the RRV (NSP3-RRV) strain 407


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was expressed at similar levels in BSRT7 cells (figure 5). These cells were then 408

electroporated with three different types of rotavirus-like reporter mRNAs: R-RNA 409

ending with the canonical GACC sequences or Nc-RNAs ending with either a 410

GAACC or a GGCC sequence. The GAACC sequence corresponds to the non-411

canonical 3' end found on gene 5 of the SA11 rotavirus strain, with GGCC being 412

found at the 3' end of genes 5 and 7 of the RRV and SA11 strains, respectively (34). 413

The luciferase activities measured are reported in figure 5 and show that NSP3 of 414

either origin stimulated R-RNA translation equally well (7- to 8-fold) compared with 415

translation in the absence of NSP3. Thus, the different translation stimulation of R-416

RNA observed with the RRV and RF strains (figure 2C, E), was not due to the 417

different efficiencies of their cognate NSP3s. Note that a slight (x2) but significant 418

enhancement of Nc-RNA GGCC translation was observed in cells expressing NSP3-419

RRV versus cells lacking NSP3 (but not versus cells expressing NSP3-RF), 420

suggesting that NSP3-RRV could exhibit a recognition specificity for the 3' end of 421

RNA that differs slightly from that of NSP3-RF. Similar results have been obtained 422

using NSP3 from the SA11 strain (20). 423

424

NSP3 binding to eIF4G stabilizes the eIF4E-eIF4G interaction. 425

We next wished to gain insights into the molecular mechanism underlying the 426

stimulation of poly(A) and non-poly(A) mRNA translation by NSP3. Because both 427

poly(A) and non-poly(A) mRNAs are devoid of the 3' GACC sequence required for 428

specifically binding to the N-terminal RNA-binding domain of NSP3, we anticipated 429

that the RNA-binding domain of NSP3 is not involved. Given that both poly(A) and 430

non-poly(A) mRNAs are capped, we instead tested the possibility that NSP3 equally 431

stimulated their translation through its interaction with eIF4G and through subsequent 432


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changes in eIF4G affinity for the cap-binding protein eIF4E. To this end, three 433

deletion fragments of RF-NSP3 that lack the RNA-binding domain were created and 434

fused to a eGFP tag (figure 6A) as follows: one C-terminal fragment (cNSP3) 435

containing both RoXaN and the eIF4G-binding domains, one shorter fragment 436

lacking the eIF4G-binding domain (cΔ4G), and another shorter fragment lacking the 437

RoXaN-binding domain (cΔRX). The ability of these different fragments to interact 438

with eIF4G was first verified in transiently transfected HeLa cells. As expected, co-439

immunoprecipitation (IP) experiments using anti-GFP antibodies confirmed that 440

NSP3 fragments containing the eIF4G-binding domain (cNSP3 and cΔRX) indeed 441

interacted with eIF4G, whereas the NSP3 fragment devoid of the eIF4G-binding 442

domain (cΔ4G) did not (figure 6B). Next, co-IP experiments performed using anti-443

eIF4GI antibodies revealed that whereas PABP was evicted from eIF4GI (as 444

expected), interactions between the cNSP3 or cΔRX fragments and eIF4GI were 445

associated with a slight but reproducible increase in the amount of co-446

immunoprecipitated eIF4E compared with non-transfected cells (figure 6C, left). 447

Similarly, a slight but reproducible increase in the amount of eIF4GI that was co-448

immunoprecipitated using anti-eIF4E antibodies was observed with cells expressing 449

either of the two NSP3 fragments containing the eIF4G-binding domain (figure 6C, 450

right). In contrast, changes in the eIF4E-eIF4GI interaction were not significant when 451

anti-eIF4GI or anti-eIF4E co-IPs were performed with cells expressing the cΔ4G 452

NSP3 fragment devoid of the eIF4G-binding site (figure 6B, left and right). 453

These data indicate that despite the eviction of PABP from eIF4G (and the 454

consequent disruption of the mRNA closed-loop conformation), the interaction of 455

NSP3 with eIF4G somehow enhances the affinity of eIF4G for eIF4E. This effect 456

required neither the RNA-binding domain nor the RoXaN-interacting site of NSP3. 457


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This result may at least partially explain how NSP3 stimulates the translation of both 458

poly(A) and non-poly(A) mRNAs independently of its binding to any mRNA 3' end. 459

460

461

DISCUSSION 462

In this work, we have shown that, in living cells, the rotavirus protein NSP3 463

apart from other viral proteins is able to enhance the translation of a rotavirus-like 464

mRNA but has no effect on RNA stability. These results are in agreement with results 465

we previously obtained using in vivo and in vitro translation assays (15), as well as 466

with those of Chizikoff and Patton, who used infected cells to show an enhancement 467

of translation when the reporter mRNA ends with a GACC sequence (14). 468

The role of NSP3 in stimulating rotavirus mRNA translation had been 469

questioned (18). Through the siRNA-mediated down-regulation of NSP3 in infected 470

cells, Montero et al. detected via pulse labeling only a transient decrease in de novo 471

viral proteins synthesis early after infection. In these experiments, however, NSP3 472

down-regulation was only partial, and it is likely that the remaining NSP3 was 473

sufficient to ensure viral protein synthesis, albeit at a slower rate. Indeed our data 474

support this idea, as we show that even a very low amount of NSP3 is capable of 475

enhancing rotavirus-like mRNA translation a hundred fold. Thus, NSP3 is likely most 476

important, though at low levels, at the onset of infection, when competition with 477

cellular mRNAs for access to the cell translation machinery is fiercest. 478

We also showed that the translation capability of R-RNA slightly differs 479

depending on the strain used (RF or RRV), a difference that cannot be attributed to 480

the two different strain-specific NSP3s. This result indicates that other viral-481

dependent mechanisms are required to modify R-RNA translation efficiency in 482


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rotavirus-infected cells. The competition between viral mRNAs and R-RNA for NSP3 483

is likely one of these mechanisms. 484

The interaction of NSP3 with eIF4G, which results in the eviction of PABP from 485

translation initiation complexes and its subsequent nuclear re-localization, has been 486

hypothesized to cause the inhibition of host poly(A) mRNA translation following 487

rotavirus infection (9, 13, 15). The reduction of cellular poly(A) mRNA translation 488

induced by the addition of recombinant truncated NSP3 to a rabbit reticulocyte lysate 489

in vitro translation (RRL) assay (6) and the reduction of vaccinia-virus gene 490

expression with recombinant vaccinia virus expressing full-length or truncated NSP3 491

(17), substantiated this hypothesis. Here, we did observe a reduction in the 492

translation of pA-RNA introduced into rotavirus-infected cells via electroporation, with 493

the level of translation varying from one viral strain to another and being noticeable in 494

RRV-infected cells only after normalizing to the amount of pA-RNA remaining in the 495

cell. Similar reductions in translation were not observed by Rubio et al. (19) using the 496

RRV rotavirus strain and RNA lipofection even after normalizing to the amount of 497

RNA; it is possible that RNA lipofection distorted RNA quantification, as lipofection 498

has been shown to interfere with RNA half-life, thus precluding its use in monitoring 499

the expression of reporter RNA (35). The same difference in the capacities of the RF 500

and RRV strains to abate cellular gene expression was also observed in cells 501

constitutively expressing a reporter gene (i.e., expressed as a polyadenylated mRNA 502

synthesized in the nucleus and translated in the cytoplasm); the extent of reduction of 503

cellular mRNAs expression was higher and quicker in RF- than in RRV-infected cells. 504

However, the use of monoreassortant strains showed that this variation was not 505

entirely linked to the NSP3 genetic background. Cellular gene expression involves 506

many steps that lie upstream of translation, including transcription, splicing and 507


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nuclear export. Our results suggest that the RF and RRV strains may differentially 508

impact one or more of these steps and that the inhibition of translation by NSP3 is 509

probably not the sole factor involved in shutting down cellular protein synthesis. 510

Another explanation is that the numerous viral mRNA copies present during infection 511

(19, 36) simply titrate away translation initiation factors and thus render them 512

inaccessible to host poly(A) mRNAs. Thus, the inhibition of cellular polyadenylated 513

mRNA translation during rotavirus infection would more likely result from the high 514

level of viral mRNA translation facilitated by NSP3 rather than by the direct exclusion 515

of cellular mRNAs from translation initiation complexes. 516

Whereas pA-RNA translation was reduced in RF-infected cells, it unexpectedly 517

increased 2.6-fold in cells expressing only NSP3, and similarly a 2.2-fold increase 518

was observed with N-RNA. Thus, in the absence of viral mRNA, NSP3 seems to non-519

specifically increase RNA translation. One possibility that could explain such an 520

observation is that the eviction of PABP from eIF4G by NSP3 abolishes the 521

competition between polyadenylated and non-polyadenylated RNA and renders all 522

RNAs equally competent for translation regardless of their 3' ends. A similar 523

phenomenon was observed when short poly(A) RNAs were added to an in vitro 524

translation assay using ribosome-depleted RRL (37) or when non-polyadenylated 525

mRNA degradation was abolished in yeast (38). However, our data showing that 526

NSP3 promotes the eIF4E-eIF4G interaction provides a more direct explanation for 527

this unexpected observation and actually suggests that through its interaction with 528

eIF4G, NSP3, independently of its binding to any 3' end, can substitute for the PABP-529

poly(A) complex in facilitating the initiation of capped mRNA translation. Indeed, the 530

binding of PABP to eIF4G has been shown to similarly enhance eIF4E affinity for the 531

cap structure (39, 40), itself further increased by binding of eIF4E to eIF4G (41). Yet, 532


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it remains to be determined whether stabilization of eIF4E-eIF4G interaction in 533

NSP3-expressing cells is directly due to NSP3-binding to eIF4G or whether a more 534

indirect effect such as liberation of eIF4E form its sequestering proteins (i.e. the 535

eIF4E-binding proteins 1 and 2) is involved. 536

The results described here, in addition to the study using NSP3 that lacks 537

RNA or eIF4G binding (20), are totally consistent with the notion that NSP3 behaves 538

as a PABP surrogate for rotavirus mRNA translation through its simultaneous 539

interaction with eIF4G and the viral mRNA GACC 3' end. However, our results 540

indicate that NSP3 per se cannot explain the inhibition of host poly(A) mRNA 541

translation observed in rotavirus-infected cells. Instead, NSP3 separated from other 542

viral proteins appears to also substitute for PABP in cellular mRNA translation, given 543

that this protein slightly enhances the translation of both poly(A) and non poly(A) 544

mRNAs, likely through its interaction with eIF4G and the resulting stabilization of the 545

eIF4E-eIF4G complex. 546

547

Acknowledgements 548

This work was supported by a grant from Agence Nationale de la Recherche ANR-549

09-MIEN-04 "Trans-Inf-Rot" to DP and SP. MG was supported by a PhD thesis 550

fellowship from Ministère de l'Education Nationale, de l'Enseignement Supérieur et 551

de la Recherche. ES was supported by a post-doc grant from ANR. P.V., A.C. and 552

D.P. are staff members from the Institut National de la Recherche Agronomique 553

(INRA). M.D. is a staff member from University of Evry Val d'Essones. SP is staff 554

member from INSERM. Thanks to Cécile Laroche for technical assistance. 555


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Plasmid pcDNA3.1purodsRluc-4xJCV was kindly provided by Dr Sullivan, The 556

University of Texas at Austin (USA). eIF4G1 antiserum was kindly provided by Dr 557

Sonenberg, McGill University (Canada). 558

559

FIGURES LEGENDS 560

561

Figure 1. Schematic representation of reporter mRNAs and quantification of 562

GAPDH RNA in rotavirus-infected cells. 563

Panel A; the schematic structures of rotavirus-like mRNA (R-RNA), non-rota and non-564

poly(A) RNA (N-RNA), polyadenylated RNA (pA-RNA) and non-canonical rotavirus-565

like mRNAs (Nc-RNA) are indicated with their 5' and 3' ends. The Renilla luciferase 566

ORF (Rluc) is boxed and the rotavirus GACC 3' motif and the poly(A) tail appear in 567

bold letters. All mRNAs were 5' capped (m7G). 568

Panel B; GAPDH RNA quantification in mock- and rotavirus-infected cells. Total 569

mRNA was purified from mock- or rotavirus (strain RF or RRV)-infected cells at the 570

indicated times (in hours), and RT-qPCR was performed with GAPDH primers. Data 571

shown represent the mean Ct values (+/- SEM) obtained from three independent 572

experiments performed in triplicate. No significant differences were observed, as 573

determined by a two-tailed Student t test. 574

575

Figure 2. Quantification and translation of reporter mRNAs introduced into 576

mock- and rotavirus-infected cells. 577

Mock- or rotavirus (strain RF or RRV)-infected cells were electroporated with the R-, 578

N-, and pA-RNA reporters and were recovered 6 h after electroporation (8.5 h after 579

infection). The amount of reporter RNA was quantified by RT-qPCR (A), and Rluc 580


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activities were measured (B,C). In panel A, the cellular mRNA encoding GAPDH was 581

used as an internal control, and the amount of reporter RNA relative to GAPDH RNA 582

is presented. In panels B and C, the Rluc activity measured in mock-infected cells 583

electroporated with R-RNA was set to one, but no correction was performed for RNA 584

abundance. In panels D and E, Rluc activities were normalized to the relative amount 585

of reporter RNA, as measured by RT-qPCR (A), with the ratio (Rluc/RNA) obtained 586

with mock-infected cells electroporated with R-RNA being set to one. Numbers 587

indicate the fold increase/decrease. The means +/- SEM for three independent 588

experiments performed in triplicate are shown. Asterisks indicate significant 589

differences (*; p<0.05) as determined by a two-tailed Student t test. 590

591

Figure 3: Quantification and translation of reporter mRNAs in MA104 and C20b 592

cells. 593

Panel A; Expression of NSP3 in C20b cells. Cell lysates were prepared using 594

identical numbers of C20b and MA104 cells infected with the RF rotavirus strain at an 595

MOI of 10 for the indicated times (hpi; hours post infection). Viral (NSP3) and cellular 596

(GAPDH) proteins were detected by western blot and quantitated using a LICOR 597

Odyssey Fc fluoroimager. The amounts of NSP3 relative to GAPDH (NSP3/GAPDH) 598

are indicated at the bottom of the figure. The ratio obtained for C20b cells was set to 599

one. 600

Panels B-F; MA104 or C20b cells were electroporated with the R-, N-, and pA-RNA 601

reporters, total RNA was recovered immediately (T0) or 6 h (T6) after electroporation, 602

and the amount of electroporated reporter RNA was quantified by RT-qPCR. Rluc 603

activities were measured at T6. The amount of reporter RNA relative to GAPDH RNA 604

is presented (panel B). Rluc activities were normalized to the relative amounts of 605


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reporter RNAs present at T0 (panels C and D) or at T6 (panels E and F). The ratio 606

(Rluc/RNA) obtained with mock-infected cells electroporated with R-RNA was set to 607

one. The means +/- SEM for three independent experiments performed in triplicate 608

are shown. Numbers indicate the increase/decrease rate between points. Asterisks 609

indicate significant differences (*; p<0.05 or **p; <0.01) as determined by a two-tailed 610

Student t test. 611

612

Figure 4: Kinetics of cellular gene expression after infection with parental (RF 613

and RRV) and monoreassortant (RRV/RF07 and RF/RRV07) strains. 614

615

Cells constitutively expressing dsRenilla luciferase were mock infected or 616

infected with the indicated parental or reassortant viruses at an MOI of 10 and 617

recovered at the indicated times after infection. Rluc activities were measured, and 618

the level of Rluc measured 20 minutes after infection was set to 100. The means +/- 619

SEM for three independent experiments are shown. 620

621

Figure 5: Expression of R- and Nc-RNAs in cells expressing NSP3 of RF or RRV 622

origin. 623

Panel A: NSP3-RF and NSP3-RRV expression in BSRT7 cells. Lysates from BSRT7 624

cells transfected for 24 h with expression vectors encoding NSP3 from the RF 625

(NSP3-RF) or RRV (NSP3-RRV) strains or killed NSP3-RF (NSP3 -), were analyzed 626

by western blotting with an anti-NSP3 rabbit polyclonal antibody and a mouse 627

monoclonal antibody against the cellular protein GAPDH (used as a loading control). 628

The ratio of NSP3-to GAPDH-fluorescence (NSP3/GAPDH) is indicated at the bottom 629


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of the figure. MW: molecular weight marker in Kd. The three lanes are from the same 630

membrane and experiment. 631

Panel B: Cells expressing RF-, or RRV-, or killed NSP3 for 24 h were electroporated 632

with R-RNA or Nc-RNAs ending with a GGCC or GAACC and with a standard EMCV-633

Fluc RNA. Fluc and Rluc activities were measured 6 h after electroporation. The 634

means +/- SEM of the Renilla-to-Firefly luciferase ratio (Rluc/Fluc) of three 635

independent experiments are presented. The Rluc/Fluc ratio obtained with R-RNA 636

electroporated into cells expressing NSP3-RF was set to 100. Numbers indicate the 637

increase/decrease rate between points. Asterisks indicate significant differences (*; 638

p<0.05 or **p; <0.01) as determined by two-tailed Student t tests. 639

640

Figure 6: NSP3 binding to eIF4G stabilizes the eIF4E-eIF4G interaction 641

Panel A; Schematic representation of NSP3 fragments. Different fragments of NSP3 642

(RF strain) containing the RoXaN and eIF4G-binding sites (cNSP3) or devoid of 643

either the eIF4G- or RoXaN-binding domain (cΔ4G or cΔRX) were fused to GFP. 644

RNA refers to the RNA-binding domain of NSP3, and the numbers refer to the NSP3 645

amino acid sequence. 646

Panel B. HeLa cells were transiently transfected with the different GFP-tagged NSP3 647

fragments, and their relative expression and interaction with eIF4GI (direct 648

interaction), eIF4E (indirect interaction through eIF4G) and PABP (no interaction) 649

were visualized by western blotting using the indicated antibodies either directly 650

(input) or after immunoprecipitation with anti-GFP antibodies (IP anti-GFP). NT: non-651

transfected; -Ab: IP without anti-GFP antibodies; *: non-specific signal. The results 652

are representative of three separate experiments. 653


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Panel C. eIF4E-eIF4GI interaction in HeLa cells transiently transfected with the 654

different GFP-tagged NSP3 fragments was visualized by western blotting after IP 655

using either anti-eIF4GI (left) or anti-eIF4E (right) antibodies. Co-immunoprecipitated 656

PABP was also visualized. Input: direct western-blotting; NT: non-transfected; -Ab: IP 657

without any antibodies. The results are representative of three separate experiments. 658

659

660

661

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39. Haghighat A, Sonenberg N. 1997. eIF4G dramatically enhances the binding 773 of eIF4E to the mRNA 5'-cap structure. J. Biol. Chem. 272:21677-21680. 774

40. Wei CC, Balasta ML, Ren J, Goss DJ. 1998. Wheat germ poly(A) binding 775 protein enhances the binding affinity of eukaryotic initiation factor 4F and 776 (iso)4F for cap analogues. Biochemistry 37:1910-1916. 777

41. von Der Haar T, Ball PD, McCarthy JE. 2000. Stabilization of eukaryotic 778 initiation factor 4E binding to the mRNA 5'-Cap by domains of eIF4G. J. Biol. 779 Chem. 275:30551-30555. 780

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figure 1

B

A 5’ m7GGC... ...UUAUGUGACC 3' R-RNA: R. Luc.

5’ m7GGC... ...UUAUGUGGCC 3' Nc-RNA: R. Luc.

5’ m7GGC... ...UUAUGUGAACC 3' Nc-RNA: R. Luc.

5’ m7GGC... ...UUAUGUG 3' N-RNA: R. Luc.

5’ m7GGC... pA-RNA: ...UUAUGUGACC(A) 3'

65 R. Luc.

Mock

3 6 9

RRV RF

12

14

16

13

15

0 9

Ct

3 6 9


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A

B C

D E

Mock RRV RF

R-RNA N-RNA

Mock RRV RF

0

20

40

60

80

100

R.

lucif

era

se

fo

ld i

nc

rea

se

x46 *

x6 *

x4 *

x29 *

x15 *

x5 *

0

10

20

30

40

50

60

Mock RRV RF

RN

A (

rela

tiv

e a

mo

un

t, A

.U.)

0

10

20

30

40

50

60

Mock RRV RF Mock RRV RF

R-RNA N-RNA pA-RNA

x2 *

x2.5 * x3 *

x3.4 *

x3 *

pA-RNA

Mock RRV RF

0

100

200

300 R

. lu

cif

era

se f

old

in

cre

as

e

/6 *

/5 *

pA-RNA

Mock RRV RF

0

100

200

300

Rlu

c

RN

A

400

500

700

800

600

/19 *

/4 *

figure 2

50

60

0

10

20

30

40 x10 *

x2 *

x2 *

Rlu

c

RN

A

x34 * x8

Mock RRV RF

R-RNA

Mock RRV RF

N-RNA


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0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

MA104 MA104 C20b C20b

R N pA R N pA R N pA R N pA

T0 T6

RN

A (

rela

tiv

e a

mo

un

t, A

.U.)

**

*

*

0

5

10

15

20

25

30

35

40

C D

E

B

A

MA104 C20b

R N

R.

luc

ife

ras

e r

ela

tiv

e f

old

in

cre

ase

0

R N

20

40

80

60

100

120

140

160

180

** x2.2

** x125

MA104 C20b

pA pA

0

400

800

1200

600

200

1000

* x2.6

MA104 C20b

R N

50

R.

lucif

era

se r

ela

tive

fo

ld i

nc

rea

se

0 R N

100

250

300

350

400

450

150

200

** x2.4

** x350

0

500

1000

1500

2500

2000

* x3.4

MA104 C20b

pA pA

F

figure 3

infected MA104 (hpi)

C20b mock .5 1 2 4 6

NSP3-

GAPDH-

NSP3 GAPDH

1 - - - .2 3.2 6.8


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figure 4

time post-infection (hours)

RF

RRV

m7RRV

m7RF

MOCK

50

0

100

150

200

0 1 2 4 6 8

% R

luc

ex

pre

ssio

n

RF/07RRV

RRV/07RF

RF

RRV

Mock


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figure 5

A B

NSP3

RF RRV -

- NSP3

- GAPDH

MW

NSP3

GAPDH 100 103 0

120-

80-

46-

31-

24-

19-

rela

tive

Rlu

c/F

luc

20

40

60

80

100

0

x2.7 ** x4.5 **

x6.9-7.8 **

x6.5-7.4 **

x4** x4.7**

NSP3-RF

GACC

GGCC

GAACC

R-RNA Nc-RNA

NSP3-RRV

GACC

GGCC

GAACC

R-RNA Nc-RNA

NSP3-

GACC

GGCC

GAACC

R-RNA Nc-RNA


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C

B A

figure 6

input IP a-GFP

eIF4GI

PABP

GFP

eIF4E

-Ab

GF

P-c

NS

P3

GF

P-c

D4

G

GF

P-c

DR

X

NT

GF

P-c

NS

P3

GF

P-c

D4

G

GF

P-c

DR

X

NT

NT

*

1 149 313 206 240

NSP3

RNA

RoXaN

eIF4G

GFP-cD4G GFP

GFP-cDRX GFP

GFP-cNSP3 GFP

input IP a-eIF4GI -Ab

GF

P-c

NS

P3

GF

P-c

D4

G

GF

P-c

DR

X

NT

GF

P-c

NS

P3

GF

P-c

D4

G

GF

P-c

DR

X

NT

NT

IP a-eIF4E -Ab

GF

P-c

NS

P3

GF

P-c

D4

G

GF

P-c

DR

X

NT

NT

eIF4GI

PABP

GFP

eIF4E


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Documents

Rotavirus NSP3 is a translational surrogate of the PABP-poly(A