2014 ef1a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and...

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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http://dx.doi.org/doi:10.1016/j.vetmic.2014.05.034

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

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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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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

2014 ef1a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and...

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