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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23
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
45
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Rotavirus NSP3 enhances translation 3
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
60
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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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Rotavirus NSP3 enhances translation 9
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
235
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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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Rotavirus NSP3 enhances translation 13
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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Rotavirus NSP3 enhances translation 14
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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Rotavirus NSP3 enhances translation 22
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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Rotavirus NSP3 enhances translation 25
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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Rotavirus NSP3 enhances translation 26
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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Rotavirus NSP3 enhances translation 27
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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Rotavirus NSP3 enhances translation 28
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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36. Johnson MA, McCrae MA. 1989. Molecular biology of rotaviruses. VIII. 765 Quantitative analysis of regulation of gene expression during virus replication. 766 J. Virol. 63:2048-2055. 767
37. Borman AM, Michel YM, Malnou CE, Kean KM. 2002. Free poly(A) 768 stimulates capped mRNA translation in vitro through the eIF4G-poly(A)-769 binding protein interaction. J. Biol. Chem. 277:36818-36824. 770
38. Searfoss AM, Wickner RB. 2000. 3' poly(A) is dispensable for translation. 771 Proc. Natl. Acad. Sci. U. S. A. 97:9133-9137. 772
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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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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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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