Accepted Manuscript
Title: EF1A interacting with nucleocapsid protein ofTransmissible Gastroenteritis Coronavirus and plays a role invirus replication
Author: Xin Zhang Hongyan Shi Jianfei Chen Da ShiChanglong Li Li Feng
PII: S0378-1135(14)00280-6DOI: http://dx.doi.org/doi:10.1016/j.vetmic.2014.05.034Reference: VETMIC 6640
To appear in: VETMIC
Received date: 25-4-2014Revised date: 29-5-2014Accepted date: 30-5-2014
Please cite this article as: Zhang, X., Shi, H., Chen, J., Shi, D., Li, C., Feng,L.,EF1A interacting with nucleocapsid protein of Transmissible GastroenteritisCoronavirus and plays a role in virus replication, Veterinary Microbiology (2014),http://dx.doi.org/10.1016/j.vetmic.2014.05.034
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EF1A interacting with nucleocapsid protein of Transmissible Gastroenteritis Coronavirus 1
and plays a role in virus replication 2
3
Xin Zhang1, Hongyan Shi1, Jianfei Chen1, Da Shi1, Changlong Li1, Li Feng1,* 4
1 Division of Swine Infectious Diseases, National Key Laboratory of Veterinary 5
Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of 6
Agricultural Sciences, Harbin 150001, China. 7
8
* To whom correspondence should be addressed: 9
Division of Swine Infectious Diseases, National Key Laboratory of Veterinary 10
Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of 11
Agricultural Sciences, No. 427 Maduan Street, Nangang District, Harbin 150001, China. 12
E-mail: [email protected]/[email protected] 13
Tel: +86-18946066048. 14
Fax:+86-451-51997164 15
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ABSTRACT 16
Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus 17
that causes diarrhea in pigs, which is correlated with high morbidity and mortality in 18
suckling piglets. Using the method of GST pull-down with the nucleocapsid (N), N 19
protein was found to interact with swine testes (ST) cells elongation factor 1-alpha 20
(EF1A), an essential component of the translational machinery with an important role in 21
cells. In vitro and in virus-infected cells interaction was then confirmed by 22
co-precipitation. Knockdown of EF1A impairs N protein proliferation and TGEV 23
replication in host cell. In was demonstrated that EF1A plays a role in TGEV replication. 24
The present study thus provides a protein-related information that should be useful for 25
underlying mechanism of coronavirus replication. 26
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Introduction 27
Coronaviruses (CoVs) includes four genera, alpha-, beta-, gamma-,and 28
deltacoronavirus, which have been clustered in the Coronavirinae subfamily (de Groot et 29
al., 2011; Reguera et al., 2012). Coronaviruses (CoVs) are pleomorphic, enveloped 30
viruses (Perlman and Netland, 2009). Transmissible gastroenteritis virus (TGEV) is a 31
representative CoV in the alphacoronavirus genus; severe acute respiratory 32
syndrome-related coronavirus (SARS-related CoV) is a representative of the 33
betacoronavirus genus; infectious bronchitis virus (IBV) is a representative of the 34
gammacoronavirus genus; and Bulbul-CoV is a representative of the deltacoronavirus 35
genus (de Groot et al., 2011). TGEV is positive RNA viruses, which is a large family of 36
enveloped virus (Masters, 2006). The infection of TGEV causes severe diarrhea in 37
suckling piglets (about 2 weeks old), which results in enormous economic loss in 38
swine-producing areas in the world (Kim and Chae, 2001; Sestak et al., 1996). TGEV 39
genome (28.5 kb) encodes the replicase gene (rep) at the 5′ end and encodes other viral 40
genes at the 3′ end (5′-S-3a-3b-E-M-N-7-3′)(Penzes et al., 2001). TGEV genome encodes 41
four structural proteins: spike (S), membrane (M), minor envelope (E), and nucleocapsid 42
(N). 43
CoVs N proteins are highly basic with a molecular mass ranging from 40 to 63 kDa, 44
depending on the species and strains. N protein binds to the RNA genome, forming a 45
helical nucleocapsid (Escors et al., 2001; Sturman et al., 1980). N protein has a structural 46
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role in coronavirus assembly (Risco et al., 1996) and is a growing evidence for a role in 47
RNA synthesis (Almazan et al., 2004; Baric et al., 1988; Stohlman et al., 1988). Some 48
reports have been studied the response of host cell to TGEV (Ding et al., 2012; Wei et al., 49
2012). Howerer, there is few report about the interaction of N protein with host cell. 50
Elongation factor 1-alpha (EF1Α) is a major translation factor involved in protein 51
synthesis in mammalian cells. EF1Α is an abundant G protein that delivers 52
aminoacyl-tRNA to the elongating ribosome (Carvalho et al., 1984b). EF1Α hydrolyzes 53
GTP, dissociates from the aminoacyl-tRNA, and leaves the ribosome (Moldave, 1985). 54
Except a major translation factor, EF1Α plays important multifunctional roles in 55
mammalian cells. EF1Α Interacts with newly synthesized polypeptides for quality 56
surveillance (Hotokezaka et al., 2002). In ubiquitin-dependent degradation, EF1Α 57
interacted with ubiquitinated proteins and is essential for ubiquitin-dependent degradation 58
(Chuang et al., 2005; Gonen et al., 1994). EF1Α undergoes several post-translational 59
modifications, mainly phosphorylation and methylation, and plays important role in 60
facilitating apoptosis (Lamberti et al., 2004). 61
Recently, some reports showed that EF1A interacted with viral proteins. The 62
interaction between EF1A and N protein of SARS-CoV was founded (Zhou et al., 2008). 63
There is no report about whether EF1A interacted with N protein of TGEV. In this study, 64
we demonstrate that EF1Α associates with N protein of TGEV and plays a role in virus 65
replication. This study will provide protein-related information for underlying mechanism 66
of coronavirus replication. 67
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Materials and methods 68
Cells and virus 69
Swine testes (ST) cells were obtained from ATCC. ST cells were grown in RPMI-1640 70
medium supplemented with 10% fetal calf serum under standard culture conditions (5% 71
CO2, 37°C). TGEV infectious strain H (Accession No. FJ755618) and TGEV attenuated 72
strain H (Accession No. EU074218) were propagated on an ST cell monolayer (Wang et 73
al., 2010). Pathogenicity of the TGEV infectious strain H is stronger than TGEV 74
attenuated strain H. However, the attenuated TGEV virus was better to adapt ST cells 75
than infectious TGEV. 76
Antibodies 77
Mouse monoclonal antibody (mAb) to glyceraldehyde-3-phosphate dehydrogenase 78
(GAPDH) (ab9484) and Rabbit polyclonal antibody (pAb) to EF1A (ab140632) were 79
purchased from Abcam. FITC-labeled goat anti-mouse IgG was purchased from 80
Kirkegaard and Perry Laboratories (KPL). TRITC-labeled goat anti-rabbit IgG was 81
purchased from Sigma mAb to N protein of TGEV was prepared in our lab. 82
Cell infection 83
ST cells were infected with TGEV infectious strain H or TGEV attenuated strain H at a 84
multiplicity of infection (MOI) of 1. After adsorption for 1 h, cells were washed and 85
incubated in fresh RPMI-1640 until required post inoculation (hpi). 86
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Construction of recombinant expression plasmid 87
N gene of TGEV was amplified with primers F-TGEV-N (5′- 88
CAGGATCCGCCAACCAGGGACAACGT-3′) and R-TGEV-N 89
5′-CACTCGAGGTTCGTTACCTCATCAATCA-3′) containing Bam HI and Xho I 90
enzyme sites. PCR products were subcloned into a prokaryotic expression pGEX-6p-1 91
vector (GE Healthcare). Recombinant expression plasmid was designated as 92
pGEX-TGEV-N and confirmed by DNA sequencing. 93
GST pull-down assay 94
GST-N protein was expressed was expressed in Escherichia coli BL21 (DE3) under 95
induction of 1 mM isopropyl-β-D-thiogalactopyranoside. GST-N fusion protein was 96
immobilized on beads at 4 °C for 2 h. The lysate of ST cells was prepared using 1 ml 97
RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% 98
Triton X-100) containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF; 1 99
mM). After centrifugation at 12,000×g for 15 min, cell lysate (500 μg) was incubated 100
with the GST-N protein preparation at 4 °C overnight. After washing four times with 101
buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% NP-40), the isolated pull-down 102
proteins were then analyzed by 12% PAGE analysis. Expressed GST protein was used as 103
a control. 104
Co-immunoprecipitation (Co-IP) assay 105
The lysate of ST cells infected with TGEV for 24 h was prepared with RIPA lysis 106
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buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate) 107
containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF) (1 mM). After 108
centrifugation at 12,000×g for 15 min, lysate supernatant was pretreated with protein A/G 109
plus-agarose (Beyotime) for 30 min at 4 °C to eliminate non-specific binding to the 110
agarose beads. The lysate supernatant (500 µg) was incubated with 1 µg of rabbit pAb to 111
EF1Α for overnight at 4 °C. Then, 20 μl resuspended Protein A/G PLUS-Agarose was 112
added to this mixture and incubated at 4 °C on a rocker platform for 2 h. After washing 113
four times with lysis buffer, the isolated immunoprecipitated proteins were then analyzed 114
by western blotting using mAb to N protein of TGEV and rabbit pAb to EF1A. The lysate 115
of TGEV mock-infected ST cells was used as a control. 116
Western blotting 117
Equivalent amounts of cell lysates were subjected to 12% PAGE and then transferred 118
to 0.22 µm nitrocellulose membranes (Hybond-C Extra, Amersham Biosciences). After 119
blotting, the membranes were incubated with rabbit pAb to EF1A for 1 h. After washing 120
three times with PBST, the membranes were inoculated with HRP-conjugated goat 121
anti-rabbit IgG (Sigma) at 37°C for 1 h and visualized using 122
3,3',5,5'-tetramethylbenzidine-stabilized substrate (TMB, Amresco). 123
Immunofluorescence assay 124
ST cells inoculated with TGEV were cultured for 24 h. The cells were washed twice 125
with PBS and fixed with paraformaldehyde (4%) for 30 min at 4 °C, and then allowed to 126
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air dry. After blotting with 5% skimmed milk powder, the fixed cells were incubated with 127
mAb to TGEV N protein (1:100) and rabbit pAb to EF1A (1:50) for 1 h at 37°C in a 128
humidified chamber. After washing three times with PBST, the fixed cells were incubated 129
with FITC-labeled goat anti-mouse IgG (1:100, KPL) and TRITC-labeled goat anti-rabbit 130
IgG (1:200, Sigma). The additional nuclear staining with 4',6-diamidino-2-phenylindole 131
(DAPI, Sigma) was performed as described previously (Jungmann et al., 2001). The 132
triple-stained cells were washed three times with PBST and subsequently examined under 133
a Leica TCS SP5 laser confocal microscopy. 134
Transfection of siRNA against EF1A 135
siRNA against EF1A (GenePharma) was used for transfection. The sequence of the 136
siRNA strands was as follows: 5'-GUGGUAUUACCAUUGACAUTT-3' (sense) and 137
5'-AUGUCAAUGGUAAUAACCACTT-3' (antisense). Transfection with siRNA was 138
performed with X-tremeGENE siRNA reagent (Roche) by following the manufacturer’s 139
instructions. ST cells were cultured overnight in six-well tissue culture plates. The siRNA 140
(20 nM) was complexed with X-tremeGENE siRNA reagent by incubating together at 141
room temperature for 30 min. After removing the cell culture supernatant, the complex 142
was added for incubation 36 h. 143
Virus titer assay 144
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ST cells were re-plated 1 day before infection in 96 well plates for the 50% infectious 145
dose (TCID50) assays. Treated samples and their paired controls were thawed as 146
described and immediately serially diluted. Cell cultures were then infected for 1 h. After 147
48 h of incubation, CPE was observed. TCID50 is calculated using the method of Reed 148
and Munch. Virus titer assay were performed three times for each condition and were 149
performed using the Student’s t-test. 150
Results 151
Expression and purification of TGEV N protein 152
Full-length TGEV N protein with a GST tag was expressed in E. coli BL21 (DE3) 153
using a T7 polymerase expression system. GST-N protein was successfully expressed and 154
purified in BL21 (DE3) in soluble fractions (Fig. 1). Western blot analysis for detection 155
of the GST tag confirmed expression of an ~70-kDa recombinant GST-N protein (Fig. 1). 156
Purified full-length recombinant GST-N protein was used in subsequent experiments. 157
EF1A interacting with N protein in vitro 158
The expressed GST-N protein immobilized on GST-agarose beads was used as a bait to 159
pull down cellular proteins of ST cells that form a complex with N protein. GST protein 160
was used as control to eliminate non-specifically binding proteins. Cellular proteins 161
immobilized on GST-agarose beads in GST pull-down assay were examined with specific 162
antibodies to EF1A (Fig. 2). From the GST pull-down results, we can see that the EF1A 163
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protein was found in GST-N protein immobilized beads but not in GST protein 164
immobilized beads. 165
Cellular EF1A interacts with N protein of TGEV in virus-infected cells 166
The immunoprecipitation assay was utilized to elucidate further whether TGEV N protein 167
interacted with cellular EF1A in TGEV-infected ST cells. From the immunoprecipitation 168
results (Fig. 3), we can see that the N protein of TGEV was precipitated by the antibody 169
to cellular EF1A in TGEV-infected ST cells but not in mock-infected ST cells. 170
Furthermore, the same results were obtained with TGEV infectious strain or with TGEV 171
attenuated strain (Fig. 3). These results demonstrated that the cellular EF1A interacted 172
with the N protein of TGEV. 173
Co-localization of EF1A with N protein in TGEV infected cells 174
The subcellular localization of EF1A was investigated in TGEV-infected ST cells using 175
indirect immunofluorescence confocal microscopy. The results indicated that the 176
subcellular localization of EF1A was distributed in the cytoplasm after TGEV infection 177
(Fig. 4). Furthermore, the red fluorescence of the TRITC-labeled goat anti-rabbit IgG 178
binding with cellular EF1A was covered with the green fluorescence of the FITC-labeled 179
goat anti-mouse IgG binding with N protein of TGEV. The evidence indicated that 180
cellular EF1A was co-localized with N protein of TGEV within the ST cells during 181
infection. 182
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Knockdown of EF1A impairs TGEV replication in host cell 183
To further investigate the role of EF1A in TGEV virus replication, EF1A protein of ST 184
cells was inhibited using siRNA. TGEV attenuated virus was used for siRNA analysis. 185
The transfected cells expressed lower protein levels of EF1A when compared with the 186
control siRNA-transfected cells (Fig. 5A and 5B). ST cells were infected with TGEV for 187
another 8 h or 24 h after transfection with siRNA at an MOI of 1. The viral RNA was 188
measured by quantative real-time RT-PCR and the N protein of TGEV was measured by 189
western blotting. TGEV infection was greatly reduced in the EF1A-knockdown cells, as 190
shown by a reduction in viral N protein expression (Fig. 5A and 5B). To demonstrate the 191
involvement of EF1A on TGEV replication, we quantified the amounts of cell-associated 192
virus and virus releasing in culture supernatant at 8 h and 24 h after inoculation. Virus 193
titer assay were performed three times for each condition and were performed using the 194
Student’s t-test. At 8 h inoculation, the specific numerical TCID50 of supernatant virus in 195
control siRNA group was 103.1/mL and the EF1A siRNA group was 102.3/mL. The 196
specific numerical TCID50 of cell-associated virus in control siRNA group was 103.9/mL 197
and the EF1A siRNA group was 103.2/mL. At 24 h inoculation, the specific numerical 198
TCID50 of supernatant virus in control siRNA group was 104.6/mL and the EF1A siRNA 199
group was 103.7/mL. The specific numerical TCID50 of cell-associated virus in control 200
siRNA group was 104.5/mL and the EF1A siRNA group was 103.6/mL. Fig. 5C shows that 201
knock-down of EF1A resulted in significant reduction of cell-associated virus, which 202
reflected viral replication. 203
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Discussion 204
N protein of CoVs facilitates template switching and is required for efficient 205
transcription (Schelle et al., 2005; Thiel et al., 2003; Zuniga et al., 2010). In addition, N 206
protein displays pleiotropic effect when expressed in host cells, such as induction of 207
apoptosis or cell-cycle arrest (He et al., 2003; Surjit et al., 2004). Some of the functional 208
outcomes that result from N gene expression in host cells are due to direct or indirect 209
interaction between N protein and cellular proteins. Studying the interaction of cellular 210
protein with N protein will provide new information for understanding the mechanism of 211
TGEV infection. 212
Results from previous study demonstrate that EF1A can anchor mRNA, suggesting that 213
EF1A is involved in sorting and regulating the expression of specific cellular mRNAs 214
(Bassell et al., 1994). EF1 complex is composed of four different subunits, alpha, beta, 215
gamma, and delta (2:1:1:1) in mammalian cells (Carvalho et al., 1984a). In TGEV 216
infected cells, EF1Α may interact with N protein of TGEV that bind to TGEV RNA in a 217
similar manner. Instead of an enzymatic activity, EF1Α may provide protein-RNA and 218
protein-protein interactions that promote the assembly of TGEV replication complexes. 219
In this study, the results demonstrate that EF1A can interact with N protein of TGEV. It is 220
possible that EF1A is involved in targeting TGEV N onto intracellular membranes that 221
provide a microenvironment for the efficient replication of the viral RNA. From the Fig.3, 222
we can see that the attenuated strain of TGEV N protein appears to be pulled down much 223
more with EF1A than the wild type N protein in. The reason maybe that attenuated TGEV 224
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virus was better to adapt ST cells than infectious TGEV (data not shown). We assumed 225
that the interaction of EF1A and N protein maybe play a role in cell culture adaptation. 226
The intrinsic characteristics of EF1A make it a suitable host protein for RNA virus 227
replication. Results in this study support a role for EF1A in the replication of CoVs. In 228
host cells, EF1A is found in high concentrations, approximately 1% of the total protein in 229
animal cells (Condeelis, 1995) and 5% in plant cells (Browning et al., 1990). For viral 230
replication, the abundance of EF1A would make it unnecessary to compete with cellular 231
processes. CoVs replicate in many hosts (Enjuanes et al., 2006). It is likely that host 232
factors selected for virus replication would be both structurally and functionally 233
conserved across different species. EF1A affords an excellent model system for the 234
further analysis of host protein and virus interactions. 235
EF1Α play an important role in some virus infection. Several viral proteins have been 236
observed to bind to EF1Α. The NS5A protein of bovine viral diarrhea virus (BVDV) 237
interacts with EF1Α, which may play a role in the replication of BVDV (Johnson et al., 238
2001). The nucleocapsid protein of SARS-CoV interacted with EF1A and inhibited cell 239
proliferation (Zhou et al., 2008). The RNA-dependent RNA polymerase of Turnip mosaic 240
virus (TuMv) interacts with EF1Α in virus-induced vesicles (Thivierge et al., 2008). RNA 241
polymerase of vesicular stomatitis virus (VSV) specifically associates with EF1Α (Das et 242
al., 1998). Gag polyprotein of Human immunodeficiency virus type 1 (HIV-1) interacts 243
with EF1Α requires tRNA, and EF1Α may contribute to tRNA incorporation into HIV-1 244
virions (Cimarelli and Luban, 1999). Studying the mechanism of EF1A and N protein of 245
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TGEV will help to understand the pathogenesis of CoVs. 246
Conclusions 247
In summary, EF1A interaction with N protein of TGEV was found. The eficiency of 248
TGEV replication depends on the presence of EF1A, which may facilitate virus 249
replication. EF1A may promote viral replication by interaction with N protein. The 250
present study thus provides information that should be useful for underlying mechanism 251
of coronavirus replication. 252
Acknowledgements 253
This work was supported by the National Natural Science Foundation of China (Grant 254
No.31172350 and No.31101823), Heilongjiang Provincial Natural Science Foundation 255
(Grant No.JC201118) and Research Team Program on Scientific and Technological 256
Innovation in Heilongjiang Provincial University (Grant No.2011TD001). 257
258
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Figure legends 259
Fig. 1. Expression and purification of TGEV GST-N protein. TGEV N protein was 260
expressed in E. coli, and lysates were resolved and purified by 12% PAGE. Proteins were 261
visualized by PhastGel Blue R staining (lanes 1-4) or N protein was detected by western 262
blotting with a GST mAb (lanes 5-6). Lane 1, protein molecular weight marker; lane 2, 263
uninduced culture of E. coli transformed with pGEX-6p-TGEV-N;lanes 4 and 5, 264
recombinant N protein purified by GST agarose; lanes 3 and 6, induced culture of E. coli 265
transformed with pGEX-6p-1. 266
Fig. 2. Cellular EF1A interacts with N protein of TGEV in vitro. EF1A binding to 267
GST-N agarose or to GST protein were resolved by Wb. PM, protein marker. GST-N and 268
GST proteins were visualized using mAb to GST; Cellular EF1A of ST cells was 269
visualized using pAb to EF1A. 270
Fig. 3. Cellular EF1A interacts with N protein of TGEV in virus-infected cells. N 271
protein of TGEV was precipitated by mAb to cellular EF1A in TGEV-infected ST cells 272
but not in mock-infected ST cells. T+ and T- represent the TGEV infected and uninfected 273
ST cells, respectively. T+1 represent the TGEV infectious H strain. T+2 represent the 274
TGEV attenuated H strain. 275
Fig. 4. Localization of cellular EF1A and N protein of TGEV. Cells were infected with 276
TGEV. Virus assembly sites were located using antibodies specific for the N protein 277
(green). EF1A was visualized using antibodies specific for EF1A (red). The nucleus was 278
stained with DAPI (blue). The triple-stained cells were observed by Leica TCS SP5 laser 279
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confocal microscopy. Bars, 10 μm. 280
Fig. 5. Gene silencing of EF1A reduced TGEV replication in ST cells. 281
EF1A-knockdown cells and negative control knockdown cells were adsorbed with TGEV 282
(MOI=1) at 37 °C for 1 h. The cells were washed and further incubated with TGEV. The 283
cell lysates were harvested for western blotting with antibodies against EF1A, TGEV N 284
protein and GAPDH, as indicated (A). The averaged densitometric intensity of EF1A and 285
N protein in immunoblot analysis, with GAPDH as a loading control (B). The culture 286
supernatants of cells and the virus-associated cells infected with TGEV for 8 h and 24 h 287
were collected for viral titration (C). The virus titers shown here are the averages and 288
standard deviations of three independent samples. * p<0.05. 289
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405
406
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Highlights 406 Interaction between TGEV N protein and EF1A is found in vitro. 407 Interaction between TGEV N protein and EF1A is found in vivo. 408 EF1A plays a role in TGEV replication. 409 Interaction between TGEV N protein and EF1A was firstly found in this study. 410 411
412
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Fig. 1
kDa
26
34
43
55
72 95
130 170
1 2 3 4 5 6
GST-N
Figure
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Fig. 2
72
kDa
26
34
43 55
95
34
PM GST GST-N
GST-N
EF1A
GST
55
43
Figure
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Fig. 3
kDa PM T− T+1
T+2
43
55
55 EF1A
N
Figure
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Fig. 4
T G
EV
−
EF1A
LPHA
N DAPI Merge
T G
EV
+
Figure
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Fig. 5
kDa EF1A
SiRNA
Control
SiRNA
55
43
EF1A
GAPDH
N
A
55
34
C
Lg T
CID
50
Supernatant
virus
Cell-associ
ated virus
8 hpi 24 hpi
EF
1A
siRN
A
Contro
l siRN
A
EF
1A
siRN
A
Contro
l siRN
A
EF
1A
siRN
A
Contro
l siRN
A
Contro
l siRN
A
Supernatant
virus
Cell-associ
ated virus
EF
1A
siRN
A
*
*
*
*
*
B EF1A SiRNA
Control SiRNA
Data 1
0
50
100
150Legend
Legend
Legend
Legend
Legend
Legend
EF1A N GAPDH
Inte
nsi
ty
Figure