2 Infectious Bursal Disease (IBD) is an acute, highly contagious and 23 immunosuppressive avian disease caused by IBD virus (IBDV). Although 24 IBDV-induced immuno-suppression has been well-established, the underlying exact 25 molecular mechanism for such induction is not very clear. We report here the 26 identification of IBDV VP4 as an interferon-suppressor by interacting with the 27 Glucocorticoid-induced leucine zipper (GILZ) in host cells. We found that VP4 28 suppressed the expression of type I interferon in HEK293T cells after Tumor Necrosis 29 Factor(TNF)-α treatment or Sendai virus (SeV) infection, and in DF-1 cells post Poly 30 I:C stimulation. In addition, the VP4-induced suppression of type I interferon could be 31 completely abolished by knockdown of GILZ by siRNA. Furthermore, knockdown of 32 GILZ significantly inhibited IBDV growth in host cells, and this inhibition could be 33 markedly mitigated by anti-interferon α/β antibodies in the cell cultures (p<0.001). 34 Thus, VP4-induced suppression of type I interferon is mediated by interacting with 35 GILZ, a protein that appears to inhibit cell responses to viral infection. 36 37 38 39 40 41 42 43 3 INTRODUCTION 44 Infectious bursal disease (IBD), also called Gumboro disease, is an acute, highly 45 contagious disease in young chickens that occurs across the world (33). Its causative 46 agent, IBDV, destroys its target cells, the B-lymphocyte precursors. The diseased 47 chickens suffer from a severe immunosuppression which leads to an increased 48 susceptibility to other pathogens (42). 49 IBDV is an Avibirnavirus belonging to Birnaviridae family, which is composed of 50 nonenveloped viruses containing two segments of double-stranded RNAs (A and B) 51 (1). Whereas the short RNA, segment B (2.8kb) encodes VP1, a RNA-dependent 52 RNA polymerase (RdRp)(30,45), segment A, the large molecule (3.17kb) contains 53 two partially overlapping open reading frames (ORFs) (21,42). The first ORF encodes 54 the nonstructural viral protein 5 (VP5) and the second one encodes for a 110-kDa 55 pVP2-VP4-VP3 precursor that can be cleaved by the proteolytic activity of VP4 to 56 form viral proteins VP2, VP3, and VP4 (1,18,20,21). VP2 and VP3 are the major 57 structural proteins, constituting 51% and 40% of the virion, respectively (13). VP4, a 58 viral protease, is able to cleave in trans and is responsible for the interdomain 59 proteolytic autoprocessing of the pVP2- VP4-VP3 polyprotein encoded by RNA 60 segment A into pVP2 precursor (48 kDa) as well as VP4 (28 kDa) and VP3 (32 kDa) 61 (4,42), and the pVP2 is further processed at its C-terminal domain by VP4 to generate 62 the mature capsid protein VP2 (41 kDa) and four small peptides (10). VP4 63 self-assembles and forms tubes with a diameter of about 25 nm (15). VP5, a highly 64 basic, cysteine-rich nonstructural (NS) protein(17 kDa) is not present in the virion, 65 4 and can only be detected in IBDV-infected cells (28). Several lines of evidence 66 suggest that it may play a role in the induction of apoptosis during IBDV infection 67 (25,28,52,53). 68 Viruses have refined various strategies to suppress host response against viral 69 dissemination (14). IBDV infection induces altered expressions of multiple genes that 70 are related to T- and B-cell activation and differentiation, and activation of genes 71 involved in Toll-like receptor (TLR)- and interferon (IFN)-mediated antiviral 72 responses (50). Recently, it has been reported that IFN alpha has strong antiviral 73 activity in IBDV-infected cells (29), suggesting that type I interferon of host cells may 74 play a critical role in combating IBDV. 75 Although VP4 has been known as a viral protease to cleave polyprotein 76 pVP2-VP4-VP3 (23,37), it may also be involved in viral pathogenesis (48). Thus, the 77 overall functions of VP4 remain to be elucidated. In this study, we found that VP4 78 acts as a major IBDV component responsible for suppressing type I interferon 79 expression via interaction with the Glucocorticoid-induced leucine zipper (GILZ) of 80 host cells. In support of a role of GILZ in cytokine response induced by VP4, 81 knockdown of GILZ by siRNA abolished VP4-induced suppression of type I 82 interferon expression, accompanied by inhibiting IBDV replication. 83 84 85 86 87 5 88 MATERIALS AND METHODS 89 Cells and virus 90 Both DF-1 (immortal chicken embryo fibroblast) and HEK293T cells were obtained 91 from ATCC. All cells were cultured in Dulbecco modified Eagle medium (DMEM) 92 (Invitrogen, USA) supplemented with 10 % fetal bovine serum (FBS) in 5% CO2 93 incubator. Lx, a cell culture-adapted IBDV strain, was kindly provided by Dr. Jue Liu 94 (Beijing Academy of Agriculture and Forestry, Beijing). 95 Reagents 96 Endo-toxin Free Plasmid Preparation Kits were purchased from Aidlab (Beijing, 97 China). All the restriction enzymes were purchased from NEB (USA). PCMV-Myc, 98 pDsRed-monomer-N1, pRK5-FLAG and pEGFP-N1 vectors were obtained from 99 Clontech (USA). Anti-c-Myc (sc-40), anti-GFP (sc-9996), anti-GILZ (sc-33780) and 100 anti-β-actin (sc-1616-R) antibodies were obtained from Santa Cruz Biotechnology 101 (USA). Anti-human IFN-α1 (ab11408) and anti-human IFN-β (ab6979) were 102 purchased from Abcam (UK). Poly (I: C) and anti-FLAG monoclonal antibody 103 (F1804) were purchased from Sigma. FITC-conjugated goat anti-mouse IgG, 104 TRITC-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse and 105 anti-rabbit IgG antibodies were purchased from DingGuo(China). OPTI-MEM I, 106 RNAiMAX and Lipofectamine LTX were purchased from Invitrogen (USA). 4’, 107 6-diamino-2-phenylindole (DAPI) was purchased from Beytime company 108 (China).Recombinant Human TNF-α was purchased from PeproTech company (USA). 109 6 Enhanced chemiluminescence (ECL) kit was purchased from Kangwei Biological 110 Company (China). 111 Constructs 112 IFN-α4, IFN-β and NF-κB promoter luciferase reporter plasmids (pGL3-IFN-α4, 113 pGL3-IFN-β, and pGL3-NF-κB) were kindly provided by Drs. Hongbin Shu (51,55). 114 pCMV-Myc, pDsRed-monomer-N1 and pEGFP-N1 vectors were purchased from 115 Clontech (USA). IBDV vp4 was cloned from IBDV strain Lx using the following 116 specific primers (Sense: 5′-AGGATAGCTGTGCCGGTGGTCTCCACAT-3′; 117 Anti-sense:5′-TTTGATGAACGTTGCCCAGTT-3′, GenBank Gen-ID: 6539893). 118 Human gilz was cloned from HEK293T cells using the specific primers 119 (Sense:5′-ATGGCCCAGTCCAAGCTCGA-3′;Anti-sense:5′-TTACACCGCAGAAC120 CACCA-3′) according to the sequence in GenBank (Gene ID: 62865623). Chicken 121 gilz was cloned from DF-1 cells using the specific primers 122 (Sense:5′-ATGAGCACCGGCGTGTACCA-3′;Anti-sense:5′-TTACACCGCAGAAC123 CACCA-3′ ) with reference to the sequence in GenBank (Gene ID: 124 NM_001077234).All the primers were synthesized by Sangon Company (China). 125 Immunofluorescence antibody assay (IFA) 126 HEK293T cells were infected with IBDV Lx at an MOI of 10 and incubated for 3 hr at 127 37°C. Uninfected HEK293T cells were used as negative controls. Three hours after 128 incubation, the medium was changed for fresh DMEM with 2% fetal bovine serum 129 (FBS), and further incubated for 24 hours. After incubation, the cells were fixed with 130 4% paraformaldehyde, permeabilized with 0.2%TritonX-100, blocked with 1% 131 7 bovine serum albumin, and incubated with chicken anti-IBDV antiserum, followed 132 with FITC-conjugated goat anti-chicken IgG antibodies and were visualized by a 133 fluorescence microscope. After infection with IBDV at an MOI of 10, HEK293T cells 134 were fixed with 4% paraformaldehyde, permeabilized with 0.2%TritonX-100, blocked 135 with 1% bovine serum albumin, and incubated with mouse anti-IBDV VP4 antiserum 136 and rabbit anti-GILZ antibodies, followed by FITC-conjugated goat anti-mouse IgG 137 (green) and TRITC-conjugated goat anti-rabbit IgG(red) and were visualized by a 138 fluorescence microscope. 139 RNA isolation and qRT-PCR analysis 140 Total RNA was prepared from HEK293T or DF-1 cells using Qiagen RNeasy kit per 141 manufacturer’s instruction. The total RNA was treated with DNase I, and 1μg of total 142 RNA was used for cDNA synthesis by reverse transcription using RT-PCR kit 143 (TaKaRa). The specific primers for human IFN-α1 144 (5′-CAATATCTACGATGGCCTCGC-3′, 5′-AGAGATGGCTGGAGCCTTCTG-3′), 145 IFN-β (5′-GATTCATCTAGCACTGGCTGG-3′, 146 5′-CTTCAGGTAATGCAGAATCC-3′), p50 (5′-GCGAGAGGAGCACAGATACC-3′, 147 5′-CTGATAGCCTGCTCCAGGTC-3′) and 148 GAPDH (5′-CAACTACATGGTTTACATGTTCC-3′, 149 5′-GGACTGTGGTCATGAGTCCT-3′) were designed with reference to previous 150 publications (22,31,40) and synthesized by Sangon Company (Shanghai, China). The 151 specific primers for Chicken IFN-α1(5′-CCAGCACCTCGAGCAAT-3′, 5′-GGCGCT 152 GTAATCGTTGTCT-3′), IFN-β (5′-GCCTCCAGCTCCTTCAGAATACG-3′, 5′- 153 8 CTGGATCTGGTTGAGGAGGCTGT-3′), p65 (5′-CCACAACACAATGCGCTC 154 TG-3′, 5′- AACTCAGCGGCGTCGATG-3′) and GAPDH 155 (5′-TGCCATCACAGCCACACAGAAG-3′,5′-ACTTTCCCCACAGCCTTAGCAG-3156 ′) (2,24,26). The analysis of real-time PCR was carried out by the Light Cycler 157 480(Roche, USA). The PCR was performed in a 20µl volume containing 1µl cDNA, 158 10µl 2×SYBR Green Premix Ex Taq(TaKaRa), 0.4µM of each gene-specific primer. 159 Thermal cycling parameters were as follows: 94°C for 2 min, 40 cycles of 94°C for 20 160 s, 55°C for 20 s, and 72°C for 20 s, and followed by one cycle of 95°C for 30 s ,60°C 161 for 30 s, and 95 for 30 s. The final step was to obtain a melt curve for the PCR 162 products to determine the specificity of the amplification. All samples were carried 163 out in triplicate on the same plate and GAPDH was utilized as the reference gene. 164 Expression levels of genes were calculated relative to the expression of the GAPDH 165 and expressed as an n-fold increase or decrease relative to the control samples. 166 Coimmunoprecipitation and Western Blot analysis 167 For immunoprecipitation, HEK293T or DF-1 cells (6×105) were seeded on 6-well 168 plates and cultured for 24 h before co-transfected with pCMV-Myc-GILZ and 169 pEGFP-VP4 or pRK5-flag-VP4 or empty vectors as controls by the standard calcium 170 phosphate precipitation. Twenty-four hours after transfection, cell lysates were 171 prepared using a non-denaturing lysis buffer (50mM Tris-HCl, PH8.0; 150mM NaCl; 172 1%NP-40; 5mM EDTA; 10% Glycerol; 10mM DTT; 1× complete cocktail protease 173 inhibitor). The cell lysates were incubated with 2 µg of anti-c-Myc or anti-FLAG 174 antibody at 4°C for 2 h, and then mixed with 20 µl of 50% slurry of protein A/G 175 9 plus-agarose and incubated for another 2 h. Beads were washed three times with the 176 lysis buffer and boiled with 2×SDS loading buffer for 10 min. The samples were 177 fractionated by electrophoresis on a 12% SDS-PAGE gels and resolved proteins were 178 transferred onto a PVDF membranes. After blocking with 5% skim milk, the 179 membranes were incubated with either anti-Myc or anti-FLAG or anti-GFP antibodies 180 followed by an appropriate HRP-conjugated secondary antibody. Blots were 181 developed using an enhanced chemiluminescence (ECL) kit. For endogenous 182 pull-down assay, HEK293T cells or DF-1 cells were transfected with pEGFP-VP4 or 183 pRK5-VP4 or with empty vector. Thirty-six hours after transfection, the cell lysates 184 were subjected to immunoprecipitation with anti-GFP antibody or anti-FLAG 185 antibody and immunoblotted with anti-GILZ or anti-GFP or anti-FLAG antibodies. 186 Confocal laser scanning microscopy assays 187 HEK293T cells (2×105) or DF-1 cells (1×105) were seeded on coverslips in 24-well 188 plates and were cultured overnight before transfection with pEGFP-VP4 and 189 pDsRed-GILZ. Twenty-four hours after transfection, the cells were fixed with 1% 190 paraformaldehyde and the nuclei were stained with DAPI. For endogenous protein 191 staining, For endogenous protein staining, IBDV or mock infected cells were fixed 192 with 1% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 15 min, 193 blocked with 1% bovine serum albumin, and then probed with mouse anti-IBDV VP4 194 antiserum and rabbit anti-GILZ antibodies, followed by FITC-conjugated goat 195 anti-mouse IgG (green) and TRITC-conjugated goat anti-rabbit IgG(red). After three 196 washes with PBS, the cells were stained for nuclei with DAPI. The samples were 197 10 analyzed with a laser confocal scanning microscope (Nikon C1 Standard Detector, 198 Japan). 199 Luciferase reporter gene assays 200 The HEK293T cells (2.0×105) were seeded on 24-well plates and transfected with 201 indicated reporter gene plasmids (pGL3-IFN-α4, pGL3-IFN-β, or pGL3-NF-κB) by 202 the standard calcium phosphate precipitation. To normalize for transfection efficiency, 203 we added 0.01 μg of pRL-TK Renilla luciferase reporter plasmid to each transfection. 204 Six hours after transfection, cells were mock-infected or infected with IBDV at an 205 MOI of 20. Twelve hours after infection, cells were treated with TNF-α at a final 206 concentration of 20ng/ml or medium as control. Twelve hours after TNF-α treatment, 207 luciferase reporter gene assays were performed. For the measurement of TNF-or 208 SeV-induced activation of type I interferon and NF-κB promoters, cells were 209 transfected with indicated reporter gene plasmids (pGL3-IFN-α4, pGL3-IFN-β, or 210 pGL3-NF-κB), eighteen hours after transfection, cells were treated with TNF-α at a 211 final concentration of 20ng/ml or infected with SeV at an MOI of 10 and medium or 212 mock-infection as controls. Twelve hours after TNF-α treatment or 24 hours after SeV 213 infection, luciferase reporter gene assays were performed with a dual-specific 214 luciferase assay kit (Promega, USA). Firefly luciferase activities were normalized on 215 the basis of Renilla luciferase activities. All reporter assays were repeated for at least 216 three times. Data shown were average values ± SD from one representative 217 experiment. 218 Nuclear protein extraction and EMSA 219 11 Crude nuclear proteins were extracted from HEK293T cells using a 220 nuclear-cytoplasmic extraction kit (Thermo Fisher, USA) with a protease inhibitor 221 mixture. 5′-Biotin-labeled NF-κB consensus double stranded oligonucleotides 222 (5′-AGTTGAGGGGACTTTCCCAGG-3′) were synthesized by AuGCT 223 Biotechnology (7). Detection of the NF-κB-oligonucleotide complex was performed 224 using a LightShift chemiluminescent EMSA kit (Thermo Fisher, USA) per the 225 manufacturer’s instruction. Briefly, nuclear protein (3–5µg) was incubated with 20 226 fmol of biotin-labeled oligonucleotides for 20 min at room temperature in a 20µl 227 reaction volume containing 10 mM HEPES-KOH (pH 7.9), 50 mM KCl, 2.5 mM 228 MgCl2, 1 mM DTT, 10% glycerol, 1µg of DNase-free BSA, and 2.5µg of 229 polydeoxyinosinic-deoxycytidylic acid. The resulting products were resolved by 230 electrophoresis on a 6% polyacrylamide gel using 0.5×Tris-borate EDTA (TBE) buffer. 231 NF-κB-oligonucleotide complex was electroblotted to a nylon membrane (Millipore). 232 After incubation in blocking buffer for 15 min at room temperature, the membrane 233 was incubated with streptavidin-HRP conjugate for 15 min at room temperature. 234 Color was developed using Light Shift chemiluminescence detection reagents 235 (Thermo Fisher, USA). 236 RNAi knockdown of GILZ 237 The siRNA was designed by Genechem Company (Shanghai, China) and used to 238 knockdown GILZ in HEK293T cells. The sequences of siRNA for targeting GILZ in 239 HEK293T cells included: RNAi#1 (sense, 5′-GUGAGAACACCCUGUUGAAtt-3′; 240 antisense, 5′-UUCAACAGGGUGUUCUCACtt-3′), RNAi#2 (sense, 5′-241 12 GAAGAAUCAUCUGAUGUAUtt-3′; antisense, 5′-242 AUACAUCAGAUGAUUCUUCtt-3′), RNAi#3 (sense, 5′-243 UCUGGUGAAGAAUCAUCUGtt-3′; antisense, 5′-244 CAGAUGAUUCUUCACCAGAtt-3′), negative control (sense, 5′-245 UUCUCCGAACGUGUCACGUtt-3′; antisense, 5′-246 ACGUGACACGUUCGGAGAAtt-3′). The sequences of siRNA for targeting GILZ 247 in DF-1 cells included: RNAi#1 (sense, 5′-GCGUGGUGGCCAUUGACAAtt-3′; 248 antisense, 5′-UUGUCAAUGGCCACCACGCtt-3′), RNAi#2 (sense, 5′-249 AGGAACUGUUGGAGAAGAAtt-3′; antisense, 5′-250 UUCUUCUCCAACAGUUCCUtt-3′), RNAi#3 (sense, 5′-251 GCCAGCGUGGUGGCCAUUGtt-3′; antisense, 5′-252 CAAUGGCCACCACGCUGGCtt-3′), negative control (sense, 5′-253 UUCUCCGAACGUGUCACGUtt-3′; antisense, 5′-254 ACGUGACACGUUCGGAGAAtt-3′).To transfect cells with the interference RNAs 255 against GILZ, we seeded HEK293T or DF-1 cells (4×105) cells on 6-well plates and 256 cultured for at least 20 h prior to transfection. The cells were transfected with siRNA 257 using RNAiMAX according to the manufacturer’s instructions (Invitrogen, USA). 258 Double transfection was performed at a 24-h interval. Forty-eight hours after the 259 second transfection, cells were harvested for further analysis. 260 Measurement of IBDV growth in DF-1 cells and HEK293T cells 261 Untreated DF-1 cells or HEK293T cells or cells receiving GILZ specific siRNAs, or 262 control siRNA were infected with IBDV at an MOI of 10, and cell cultures were 263 13 collected at different time points (12, 24, 48, 72 h) after infection. The cell culture 264 samples were freeze-thawed three times and centrifuged at 2,000× g for 10 min, and 265 the supernatants were saved at -80ºC till use. To neutralize interferon’s activity, 2µg of 266 anti-human IFN-α1 (ab11408, Abcam, UK) and anti-human IFN-β (ab6979, Abcam, 267 UK) were added to GILZ RNAi cell culture or RNAi controls 3 hours before IBDV 268 infection. Forty-eight hours after IBDV infection, the cell culture samples were 269 freeze-thawed three times and centrifuged at 2,000g for 10 min, and the supernatants 270 were saved at-80ºC till use. The viral contents in the supernatants were titrated using 271 TCID50 (50% tissue culture infective doses) in DF-1 cells. Briefly, the viral solution 272 was diluted by 10-fold in DMEM. A 100µL aliquot of each diluted sample was added 273 to the wells of 96-well plates, followed by addition of 100µLof DF-1 cells at a density 274 of 5 x105 cells /ml. Cells were cultured for 5 days at 37°C in 5% CO2. Tissue culture 275 wells with cytopathic effect (CPE) were determined as positive. The titer was 276 calculated based on a previously described method (35). 277 Statistical analysis 278 The significance of the differences between GILZ RNAi cells and controls in gene 279 expressions, promoter activities and viral growth was determined by the 280 Mann-Whitney and ANOVA accordingly. 281 282 RESULTS 283 Infection of HEK293T cells with IBDV inhibits TNF-induced expression of type I 284 interferon 285 14 IBDV is capable of replicating in multiple types of cells, including chicken B cells 286 (38), CEF and DF-1 cells (47), Vero and HEK293T cells (43). To determine if IBDV 287 Lx strain could replicate in HEK293T cells, we infected this cell line with the virus at 288 an MOI of 10 and examined the viral growth with immunofluorescence antibody 289 assay (IFA). Twenty-four hours after IBDV infection, a large number of 290 immunofluorescent cells could be detected after IFA staining using chicken anti-IBDV 291 antiserum (Fig. 1A-D). In addition, a kinetic of virus production was also examined in 292 IBDV-infected cells (Fig. 1E). These data indicate that IBDV Lx strain could replicate 293 in HEK293T cells. As Type I interferon plays a critical role in host response against 294 IBDV infection (29) and HEK293T cells do not produce detectable TLR3 activity 295 (11), we examined the expression of type I interferon in TNF-α-stimulated HEK293T 296 cells with or without IBDV infection using qRT-PCR asssay (49). Because TNF 297 induces activation of NF-κB (8,17), and NF-κB regulates type I IFN expressions(49), 298 we employed TNF-α as an inducer in our assay to examine the effect of IBDV 299 infection on TNF-induced type I interferon expression. We found that TNF-induced 300 expressions of IFN-α and IFN-β were significantly reduced in IBDV-infected cells as 301 compared to that of controls (p<0.01) (Fig. 1F-G), suggesting an inhibitory effect of 302 IBDV infection on type I interferon expression in host cells. Since the expression of 303 type I IFN is regulated by transcriptional regulator NF-κB (49), we next examined the 304 mRNA expressions of NF-κB in IBDV-infected cells stimulated with TNF-α. 305 Consistent with the above observation, infection of HEK293T cells with IBDV Lx 306 strain inhibited TNF-induced expression of NF-κB (Fig. 1H) (p<0.001), suggesting 307 15 that IBDV-induced suppression of type I IFN may be associated with transcriptional 308 regulator NF-κB. 309 To determine whether the inhibitory effect of IBDV on Type I IFN expression occurs 310 at or upstream of the transcriptional level, we employed luciferase reporter gene 311 assays to examine the cytokine gene promoter activities (6,49). HEK293T cells were 312 transiently transfected with type I interferon or NF-κB reporter gene plasmids, and 313 then cells were mock-infected or infected with IBDV at an MOI of 20. Twelve hours 314 after IBDV infection, cells were stimulated with TNF-α for 12 hr and their luciferase 315 activities were measured. All luciferase activities were normalized to a cotransfected 316 Renilla luciferase plasmid control. As shown in Fig. 1I-K, IBDV infection 317 significantly inhibited the reporter activities of IFN-α, IFN-β and NF-κB promoters 318 following stimulation with TNF-α (p<0.001). Thus, the inhibitory effect of IBDV on 319 the transactivation of these promoters suggests that IBDV suppress type I interferon 320 expression at or upstream of the transcriptional level, which may eventually lead to 321 suppressed immune response in host cells. 322 VP4 is mainly responsible for IBDV-induced suppression of type I interferon 323 expression. 324 Since IBDV infection suppressed type I interferon expression in host cells, we 325 proposed that one or more components of IBDV affect type I interferon expression by 326 engaging host proteins involved in regulating this cellular response. To test this 327 hypothesis, we cloned the vp1, vp2, vp3, vp4, and vp5 genes from the IBDV Lx strain 328 16 and made a GFP fusion for each of these proteins and expressed them in HEK293T 329 cells by transfection. All five protein fusions were expressed well in this cell line 330 when transfected with 5 μg of each individual plasmid (Fig. 2A). As SeV infection 331 causes typical type I interferon expression (16), we used this strategy to examine the 332 effect of each protein fusion on type I interferon expression. We found that transient 333 expression of GFP-VP4 significantly suppressed SeV-induced type I interferon 334 expression in host cells as compared to the GFP controls (p<0.05) (Fig. 2B&C). In 335 comparison, expressions of GFP-VP1, -VP2, -VP3 or –VP5 did not display marked 336 down-regulated expression of type I interferon in SeV-infected cells. In contrast, 337 GFP-VP3 and -VP5 enhanced SeV-induced type I interferon expression. Similar 338 results were also obtained from the examination of mRNA expression of NF-κB (Fig. 339 2D). As overexpression of GFP-VP4 does not affect cell viability (25), these results 340 suggest that VP4 is the major viral component responsible for IBDV-induced 341 suppression of type I interferon expression, leading to suppressed immune responses 342 in host cells. 343 VP4 suppresses TNF-induced activation of type I interferon and NF-κB 344 The fact that VP4 is the major IBDV component responsible for suppressing type I 345 interferon expression prompted us to investigate the role of VP4 in this suppression. 346 We examined the type I interferon response in pEGFP-vp4 transfected cells by 347 measuring TNF-α-induced activation of type I IFN and NF-κB promoters (49). 348 Consistent with above observation, transient expression of VP4 markedly inhibited 349 17 TNF-induced activation of type I IFN and NF-κB promoters as compared to that of 350 controls (Fig. 3A-C) (p<0.01). Similar results were also obtained from the 351 examination of reporter activities using SeV-infection (Fig. 3D-F). These results 352 indicate that IBDV VP4 suppresses type I interferon expressions at or upstream of the 353 transcriptional level in host cells. 354 To further determine the inhibitory effects of VP4 on NF-κB-mediated signaling, we 355 extracted nuclear proteins from pEGFP-vp4- or pEGFP-transfected cells with or 356 without TNF-α treatment, and performed EMSA to examine the impact of VP4 on 357 TNF-induced nuclear translocation of NF-κBp65, a master regulator of all 358 TLR-induced responses (5). As shown in Fig. 3G&H, the nuclear translocation of 359 NF-κBp65 remarkably increased in pEGFP-N (empty vector)- transfected cells after 360 TNF-α treatment. In contrast, the TNF-induced nuclear translocation of NF-κBp65 361 was markedly reduced in cells transfected with pEGFP-vp4, indicating that VP4 plays 362 an inhibitory role in NF-κB mediated cell response. 363 VP4 interacts with Glucocorticoid-induced leucine zipper (GILZ) 364 After identification of VP4 as a suppressor of antiviral response in host cells, we 365 furthered our investigation to study the mechanism of such suppression by searching 366 for its cellular targets. To this end, we used VP4 as a bait in the yeast two-hybrid 367 system to screen a cDNA library generated from the chicken bursa of Fabricius. The 368 positive clones were tested for β-galactosidase activity in two additional rounds of 369 selection (turning blue) (Figure 3A-C), and the plasmids from these clones were 370 18 rescued, sequenced, and BLASTed against NCBI. Among fifty-six positive clones, 371 twenty-five Glucocorticoid-induced leucine zipper (GILZ) clones were identified. In 372 addition, we found that during the course of IBDV infection, endogenous levels of 373 GILZ in DF-1 cells markedly increased (data not shown). This protein might be 374 relevant to VP4 function because it inhibits immune response (3,9). Thus, we 375 constructed a plasmid that allows the expression of Myc-GILZ for analyzing its 376 interaction with VP4 in HEK293T cells. When lysates of cells expressing both 377 EGFP-VP4 and Myc-GILZ were immunoprecipitated with Myc antibody, EGFP-VP4 378 was detected in the precipitate, indicating that VP4 interacted with ectopically 379 expressed GILZ in HEK293T cells (Fig. 4D). Similar results were obtained in 380 experiment using the DF-1 cells (Fig. 4E), indicating that the observed interaction 381 between these two proteins is not cell type specific. To further substantiate the binding 382 of VP4 to GILZ, we expressed VP4 in HEK293T or DF-1 cells and examined its 383 interaction with endogenous GILZ using a pull-down assay. The binding of GFP-VP4 384 or FLAG-VP4 with endogenous GILZ was readily detectable in cells expressing the 385 viral protein VP4 (Fig. 4F&G). These results demonstrate that VP4 interacts with 386 GILZ in host cells. 387 VP4 colocalizes with GILZ in host cells 388 To determine the subcelluar localization of VP4 and GILZ, we performed confocal 389 microscopy assay with HEK293T cells transfected to express DsRed-GILZ and 390 GFP-VP4. Transfection of HEK293T cells with pEGFP-vp4 or pDsRed-gilz indicated 391 19 that both VP4 and GILZ were located in cytoplasm (Fig. 5A&B). When cells were 392 transfected with both plasmids, we found the colocalization of VP4 with GILZ in the 393 transfected cells (Fig. 5C-E). To determine whether endogenous GILZ colocalizes 394 with VP4 in IBDV infected cells, we infected HEK293T or DF-1 cells and performed 395 an immunofluorescence antibody assay (IFA) to examine the interaction of VP4 with 396 endogenous GILZ. Consistent with the above observation, the endogenous GILZ was 397 also co-localized with VP4 in the cytoplasma of IBDV-infected cells (Fig. 5F-Q). 398 These results clearly demonstrate that VP4 interacts with GILZ in the cytoplasm of 399 host cells. 400 GILZ is required for VP4-induced suppression of type I interferon expression 401 The facts that VP4 inhibits type I interferon expression and it interacts with GILZ 402 suggest that GILZ might play a critical role in VP4-induced suppression of cytokine 403 response, and that knockdown of GILZ would therefore affect cell responses. To test 404 this hypothesis, we made three GILZ RNAi constructs, and found that one could 405 effectively lower the cellular level of GILZ without causing discernable changes in 406 cell morphology (Fig. 6A&B). We then transfected HEK293T cells receiving this 407 siRNA or control siRNA with pEGFP-vp4 plasmid and examined the activation of 408 type I interferon and NF-κB promoters in these cells after stimulation with TNF-α or 409 infection with SeV. As a result, knockdown of GILZ completely abolished 410 VP4-induced suppression of TNF-induced activation of type I interferon and NF-κB 411 promoters (Fig. 6C-E). Consistently, knockdown of GILZ also abolished 412 20 VP4-induced suppression of SeV-induced activation of type I interferon and NF-κB 413 promoters (Fig. 6F-H). Furthermore, we knocked down GILZ expression in DF-1 414 cells (immortal chicken embryo fibroblast) by siRNA (Fig. 7A&B), and examined the 415 expressions of type I interferon and NF-κB in these cells after stimulation with 416 Poly(I:C). Consistent with the above observation, VP4 suppressed Poly(I:C)-induced 417 expressions of type I interferon and NF-κB in DF-1 cells, and this suppression could 418 be abolished by knockdown of GILZ (Fig. 7C-E). These data clearly demonstrate that 419 GILZ is required for the VP4-induced suppression of type I interferon expression at or 420 upstream of the transcriptional level in host cells. 421 IBDV growth is inhibited by type I interferon or GILZ knockdown 422 To determine the role of GILZ in Type I interferon expression in IBDV-infected cells, 423 we knocked down GILZ expression in HEK293T cells by siRNA, infected these cells 424 with IBDV Lx strain at an MOI of 10, and examined type I interferon expression by 425 qRT-PCR assay. As expected, knockdown of GILZ markedly enhanced mRNA 426 expression of type I interferon in IBDV-infected cells (p<0.001) (Fig. 8A&B). These 427 results suggest that GILZ is involved in suppressing type I interferon expression in 428 IBDV-infected cells. 429 Since Type I interferon plays a critical role in host response to IBDV infection (29) 430 and GILZ suppresses type I interferon expression, we hypothesized that IBDV might 431 take advantage of GILZ to suppress host response for its own benefit, and that 432 knockdown of GILZ would therefore inhibit IBDV growth in host cells. To test this 433 21 hypothesis, we examined the viral replication in GILZ knockdown cells by measuring 434 viral loads in IBDV-infected DF-1 or HEK293T cell cultures at different time points 435 post infection. Consistent with its postulated role in suppression of antiviral response, 436 cells with lower GILZ levels markedly inhibited IBDV growth (p<0.01) (Fig.8C&D), 437 and this inhibition could be effectively blocked by specific antibodies against type I 438 IFN but not by its isotype-matched IgG controls (p<0.001) (Fig. 8E), suggesting that 439 GILZ suppresses immune response via inhibiting type I IFN expression, which might 440 be employed by IBDV as an important strategy to evade host antiviral response. 441 442 DISCUSSION 443 IBD is an acute, highly contagious viral disease causing damages in lymphoid organs 444 in birds, especially bursa of Fabricius (27). Importantly, the survivals of 445 IBDV-infected chickens suffer from immunosuppression with compromised humoral 446 and cellular immune responses (39,44), leading to susceptibility of chickens to other 447 diseases. Thus, IBD remains a threat to poultry industry worldwide. 448 Although IBDV-induced immunosuppression has been well-established (32,39,44), 449 the exact molecular mechanism for such induction is unclear. IBDV-induced 450 immunosuppression in host may involve multiple factors such as those involved in 451 pro-inflammatory response and apoptosis, cytokine regulation, and the cellular 452 immune response (24). It was reported that IBDV infection interferes with the 453 transcription of chicken type I and II interferon mRNA (34), suggesting that the 454 22 immunosuppressive effects of IBDV might be attributed, at least in part, to the 455 suppression of chicken interferon by IBDV infection. In addition, type I interferon 456 inhibits IBDV growth in chicken embryo fibroblast cultures (29), indicating that type 457 I interferon plays an important role in host anti-IBDV response. Our data show that 458 IBDV infection inhibits TNF-induced expressions of IFN-α, IFN-β and NF-κB at or 459 upstream of the transcriptional level in host cells, and transfection of HEK293T cells 460 with pEGFP-vp4 inhibits TNF-induced activation of interferon-α and-β promoters. 461 Using Sev-infection system (16,19), we also found that VP4 inhibited SeV-induced 462 activation of type I interferon and NF-κB promoters. As type I interferon expression is 463 regulated by transcriptional regulator NF-κB (49), VP-induced suppression of type I 464 interferon response might result from the inhibitory effect of VP4 on the activation of 465 NF-κB. Consistent with these observations, transfection with pEGFP-vp4 inhibits 466 TNF or SeV-induced activation of NF-κB reporter as demonstrated by luciferase 467 reporter gene assay and EMSA (Fig. 3). Thus, VP4-induced suppression of type I 468 interferon expression is not specific to IBDV-infected cells but a general signaling 469 inhibition for type I interferon expression in host cells. 470 Innate immunity is the first line of host defense against pathogenic infection. 471 TNF-induced NF-κB signaling is an essential portion of innate immune response in 472 host to viral infection (8,17). It was reported that proinflammatory cytokine TNF-α 473 had been detected in the tissues of IBDV infected chickens (54), suggesting that TNF 474 may induce inflammatory response in IBDV infected host. In the present study, we 475 first found that TNF-induced type I interferon expression in host cells was inhibited 476 23 by IBDV infection (Fig. 1F-K). Secondly, among the viral components, VP4 477 markedly suppressed the expression of type I interferon in host cells after 478 SeV-infection, and the suppressive effect of VP4 on the expression of I interferon and 479 NF-κB occurs at or upstream of the transcriptional level in host cells as demonstrated 480 by reporter gene assay and EMSA (Fig. 3). Thirdly, VP4 specifically interacts with 481 GILZ under all tested conditions (Fig. 4&5). Fourthly, the abolishment of 482 VP4-induced suppression of type I interferon could be achieved by the knockdown of 483 GILZ expression (Fig.6-7). Finally, knockdown of GILZ inhibited viral growth and 484 this inhibition could be effectively abolished by anti-type I IFN antibodies in the cell 485 culture (Fig. 8). Clearly, VP4 suppresses type I interferon response of host by 486 interacting with GILZ. As type I interferon is a critical anti-IBDV cytokine (29,34), 487 these results provide a strong evidence that VP4, via engagement with GILZ, 488 suppresses innate immune response. 489 GILZ, also known as TSC22 domain family protein 3, is a glucocorticoid-responsive 490 molecule (3,46). The GILZ protein consists of three major domains: the N-terminal, 491 LZ and C-terminal domains (3). A proline-rich region in the C-terminal domain of 492 GILZ is necessary for direct binding to the p65 subunit of NF-κB (12). Currently 493 three isoforms of the GILZ protein in human (GenBank ID: NM_198057.2 for the 494 longest one, NM_004089.3 for the medium, and NM_001015881.1 for the shortest), 495 four in mice (41) and one in chicken (GenBank ID: DQ917420.1) have been 496 identified. The similarity of chicken GILZ with human ranges from 55.5% to 80%, 497 while with mice it ranges from 30% to 90%. Interestingly, the amino acid sequence of 498 24 the C-terminal domain of chicken GILZ is identical to that of the three isoforms of 499 human GILZ, indicating that this portion (containing NF-κB binding site) is highly 500 conservative. GILZ plays an important role in the regulation of immune response, 501 such as inhibiting inflammation (3,36) and preventing T cell response (9). It has been 502 reported that GILZ inhibits activities NF-κB (12). Our data show that VP4 markedly 503 suppressed TNF-α,SeV or Poly(I:C)-induced activation of NF-κB (Fig. 3) and this 504 suppression can be abolished by the knockdown of GILZ expression (Fig. 6-7), 505 suggesting that VP4, by interacting with GILZ, inhibits NF-κB signaling, thus leading 506 to the immunosuppression of host. In this regard, it can be proposed that knockdown 507 of GILZ may enhance antiviral response in IBDV infected cells. The fact that cells 508 with lower GILZ levels markedly inhibited IBDV growth and this inhibition could be 509 effectively inhibited by specific antibodies against type I interferon provides a strong 510 evidence in support of this hypothesis (Fig. 8). 511 Of note, the mechanism underlying the immunosuppression induced by pathogenic 512 infection may vary. Suppression of cytokine expression in host cells in such a case 513 might only be one of the tricks exploited by the pathogens to evade immune response. 514 As such, several questions are raised. For example, how VP4 activates GILZ, by 515 phosphorylation, acetylation, enzymatic cleavage or something else? Similarly, what 516 molecular features of VP4 that interacts with GILZ, leading to suppressed cytokine 517 response? And is GILZ taken advantage of by any other pathogens to suppress 518 immune response in host cells? 519 25 In summary, our results reveal that IBDV VP4 interacts with Glucocorticoid-induced 520 leucine zipper (GILZ) to suppress innate immune response in host cells. The 521 observations that knockdown of GILZ abolished VP4-induced suppression of type I 522 interferon expression and reduced IBDV growth in host cells suggest that GILZ plays 523 a critical role in IBDV-induced immunosuppression. These findings have provided 524 insights for further studies of the molecular mechanism of IBDV infection. 525 526 ACKNOWLEDGMENTS 527 We thank Dr. Zhao-Qing Luo for critical reading of the manuscript. This work was 528 supported by grants from the National Natural Science Foundation of China (# 529 31272543 and 31072117) and Earmarked Fund for Modern Agro-industry Technology 530 Research System (#NYCYTX-41). 531 532 REFERENCES 533 534 1. Azad, A. A., S. A. Barrett, and K. J. Fahey. 1985. 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HEK293T cells were 727 mock-infected (A-B) or infected with IBDV Lx at an MOI of 10 (C-D) and incubated 728 for 3 hr at 37°C. Twenty-four hours after IBDV infection, cells were subjected to IFA 729 staining using chicken anti-IBDV antiserum and followed with FITC-conjugated 730 rabbit anti-chicken IgG antibodies, and were visualized under a fluorescence 731 microscope. (E) Kinetics of IBDV replication in HEK293T cells. HEK293T cells 732 were infected with IBDV at an MOI of 10. At different time points (12, 24, 48 and 72 733 h) post IBDV infection, the viral titers in the cell cultures were determined by TCID50 734 using 96-well plates. (F-H) Effects of IBDV infection on TNF-induced mRNA 735 expressions of IFN-α, IFN-β and NF-κB. HEK293T cells (2×105) were 736 mock-infected or infected with IBDV at an MOI of 20. Twelve hours after IBDV 737 infection, cells were treated with TNF-α at a final concentration of 20ng/ml. Twelve 738 hours after TNF treatment, mRNA expressions of IFN-α, IFN-β and NF-κB were 739 measured by qRT-PCR using specific primers. The relative level of gene expression is 740 calculated as follows: gene expression of IBDV-infected cells or TNF-treated (mock 741 or IBDV-infected cells) / gene expression of mock-infected controls. (I-K) Effects of 742 IBDV infection on TNF-induced activation of IFN-α, IFN-β and NF-κB promoters. 743 The HEK293T cells (2.0×105) were seeded on 24-well plates and transfected with 744 indicated reporter gene plasmids. Six hours after transfection, cells were 745 32 mock-infected or infected with IBDV at an MOI of 20. Twelve hours after IBDV 746 infection, cells were treated with TNF-α at a final concentration of 20ng/ml. Twelve 747 hours after TNF-α treatment, luciferase reporter gene assays were performed. The data 748 are normalized as in (F-H). Results are representative of three independent 749 experiments. Results are representative of three independent experiments. Data are 750 represented as mean +/- SD, n=3. *** stands for p<0.001 and ** for p<0.01. 751 FIG 2 The VP4 protein is mainly responsible for IBDV-induced suppression of type I 752 interferon expression in HEK293T cells 753 (A) Expression of GFP-VP1, -VP2, -VP3, -VP4, or –VP5 fusion proteins in 754 HEK293T cells. HEK293T cells were transfected with 5 μg of pEGFP-N1, 755 pEGFP-VP1, pEGFP-VP2, pEGFP-VP3, pEGFP-VP4, or pEGFP-VP5 plasmid. 756 Twenty-four hours after transfection, cell lysates were prepared and examined with 757 Western Blot (WB) using anti-GFP antibodies. (B-D) Effects of viral components on 758 mRNA expressions of the type I IFN and NF-κB in host cells after SeV-infection. 759 HEK293T cells were non-transfected or transfected with 5 μg of pEGFP-N1, 760 pEGFP-VP1, pEGFP-VP2, pEGFP-VP3, pEGFP-VP4, or pEGFP-VP5 as described 761 for panel A. Six hours after transfection, cells were mock-infected or infected with 762 SeV at an MOI of 10. Twenty-four hours after SeV infection, mRNA expressions of 763 IFN-α, IFN-β and NF-κB were measured by quantitative RT-PCR using specific 764 primers. The relative level of mRNA expression is calculated as follows: gene 765 expression of plasmid transfected cells or SeV-infected cells / gene expression of 766 mock-infected non-transfected controls. Results are representative of three 767 33 independent experiments. Data are represented as mean +/- SD, n=3. ** stands for 768 p<0.01 and * for p<0.05. 769 FIG 3 Expression of VP4 inhibits TNF-α or Sendai virus (SeV)- induced activation of 770 IFN-α, IFN-β and NF-κB promoters. 771 (A-C) Effects of VP4 on TNF-induced activation of IFN-α, IFN-β and NF-κB 772 promoters. HEK293T cells (2.0×105) were transfected with pEGFP-vp4 or empty 773 vector (pEGFP-N1) as control together with the indicated reporter plasmids. Eighteen 774 hours after transfection, cells were treated with 20ng/ml of TNF-α or medium as 775 controls for 12 hr before reporter activities were examined with a dual-specific 776 luciferase assay kit. (D-F) Effects of VP4 on SeV-induced activation of IFN-α, IFN-β 777 and NF-κB promoters. HEK293T cells (2.0×105) were transfected with pEGFP-vp4 778 or empty vector as controls together with the indicated reporter plasmids. Eighteen 779 hours after transfection, cells were mock-infected or infected with SeV at an MOI of 780 10 for 24 hr before reporter activities were examined with a dual-specific luciferase 781 assay kit. Results are representative of three independent experiments. Data are 782 represented as mean +/- SD, n=3. *** stands for p<0.001, ** for p<0.01 and * for 783 p<0.05. (G) Impacts of VP4 on TNF-induced nuclear translocation of NF-κB p65. 784 HEK293T cells (5.0×105) were transfected with pEGFP-vp4 or empty vector as 785 controls. Twenty hours after transfection, cells were treated with 100ng/ml of TNF-α 786 or medium as controls for 45 min before crude nuclear proteins were extracted. The 787 nuclear proteins were then subjected to EMSA for determining the nuclear 788 translocation of NF-κB p65. The detection of the NF-κB-oligonucleotide complex 789 34 was performed using a Light Shift chemiluminescenct EMSA kit. (H) The density of 790 relative nuclear translocated NF-κB p65 bands in (G) was quantitated by densitometry 791 and normalized to that of vector control. Results are representative of three 792 independent experiments. 793 FIG 4 The interaction of IBDV VP4 with host cellular protein GILZ 794 (A-C) Yeast two-hybrid screening of IBDV VP4 binding proteins. Yeast colonies 795 co-transformed with pGBKT7-VP4 and pGADT7-GILZ (A) or co-transformed with 796 pGBKT7-Lam and pGADT7-T antigen as negative control (B) or co-transformed with 797 pGBKT7-p53 and pGADT7-T antigen as positive control (C) were incubated at 30°C 798 and checked periodically for the positive (turning blue). (D-E) The interaction of VP4 799 with exogenous GILZ. HEK293T (D) or DF-1 (E) cells were transfected with 2.5 μg 800 of indicated plasmids. Twenty-four hours after transfection, cell lysates were prepared 801 and immunoprecipitated (IP) with anti-Myc(D) or anti-FLAG (E) antibodies. VP4 and 802 GILZ in the immune complex were immunoblotted with anti-GFP or anti-Myc or 803 anti-FLAG antibodies. (F-G) The interaction of VP4 with endogenous GILZ. 804 HEK293T (F) or DF-1 (G) cells were transfected with 5 μg of pEGFP-vp4 (F), 805 pRK5-vp4(G), pRK5-vp5 (G) or empty vectors as controls, and immunoprecipitation 806 assays were performed with anti-GFP or anti-FLAG antibodies. GILZ in the immune 807 complex was examined with Western Blot using anti-GILZ antibody. Data are 808 representative of three experiments with similar results. 809 FIG 5 Colocalization of VP4 with GILZ in the cell. 810 35 (A-B) Expression of exogenous VP4 (A) or GILZ (B) in HEK293T cells. HEK293T 811 cells (2×105) were seeded on 24-well plates with coverslips in the wells and cultured 812 overnight. Cells were transfected with pEGFP-vp4 (A) or pDsRed-gilz (B). 813 Twenty-four hours after transfection, cells were fixed and observed with a laser 814 confocal scanning microscope. (C-E) Colocalization of VP4 with exogenous GILZ. 815 HEK293T cells (2×105) were seeded on 24-well plates with coverslips in the wells 816 and cultured overnight. Cells were transfected with both pEGFP-vp4 and pDsRed-gilz. 817 Twenty-four hours after transfection, cells were fixed and observed with a laser 818 confocal scanning microscope. (F-Q) Colocalization of IBDV VP4 with endogenous 819 GILZ in IBDV-infected cells. HEK293T (F-K) or DF-1 (L-Q) cells were 820 mock-infected (F-H and L-N) or infected with IBDV Lx (I-K and O-Q) at an MOI of 821 10 and incubated for 3 hr at 37°C. Twenty-four hours after IBDV infection, cells were 822 fixed and probed with mouse anti-VP4 antibodies and rabbit anti-GILZ antibodies, 823 followed by the FITC-conjugated goat anti-mouse antibody (green) and 824 TRITC-conjugated goat anti-rabbit antibody (red). Nuclei were counterstained with 825 DAPI (blue) and were visualized under a fluorescence microscope. 826 FIG 6 GILZ mediates the inhibitory effect of VP4 on TNF-α or Sendai virus 827 (SeV)-induced activation of IFN-α, IFN-β and NF-κB promoters 828 (A-B) Effects of GILZ RNAi on the expression of endogenous GILZ. HEK293T 829 cells (2.0×105) were transfected with siRNA (#1-3) or controls as described in 830 Materials and Methods. Forty-eight hours after the second transfection, cell lysates 831 were prepared and examined by Western Blot with anti-GILZ antibody. Endogenous 832 36 β-actin expressions were used as internal controls. The band density of GILZ in GILZ 833 RNAi treated cells (A) was quantitated by densitometry, and the relative levels of 834 GILZ (B) were calculated as follows: the band density of GILZ / the band density of 835 β-actin. (C-H) Knockdown of GILZ abolished the inhibitory effect of VP4 on TNF-α 836 or SeV-induced activation of IFN-α, IFN-β and NF-κB promoters. HEK293T cells 837 (2.0×105) were transfected with the GILZ RNAi#1 construct or RNAi control. 838 Twenty-four hours after the first transfection, cells were co-transfected with RNAi 839 construct together with of pEGFP-vp4 and indicated reporter plasmids. Eighteen 840 hours after transfection, cells were treated with 20ng/ml of TNF-α (C-E) or infected 841 with SeV (F-H) at an MOI of 10. Medium or mock-infected cells were used as 842 controls. Twelve hours after TNF-α treatment or 24 hours after SeV infection, cells 843 were collected and the reporter activities of IFN-α, IFN-β and NF-κB were 844 determined by luciferase reporter gene assays using a dual-specific luciferase assay kit. 845 Data are presented as mean +/- SD, n=3. *** stands for p<0.001, and ** for p<0.01. 846 FIG 7 GILZ mediates the inhibitory effect of VP4 on Poly(I:C)-induced expressions 847 of IFN-α, IFN-β and NF-κB in DF-1 cells. 848 (A-B) Effects of GILZ RNAi on the expression of endogenous GILZ in DF-1 cells. 849 DF-1 cells (2.0×105) were transfected with siRNA constructs (#1-3) or controls. 850 Double transfection was performed at a 24-h interval. Forty-eight hours after the 851 second transfection, cell lysates were prepared and examined by Western Blot with 852 anti-GILZ antibody. Endogenous β-actin expressions were used as internal controls 853 (A). The band density of GILZ in GILZ RNAi treated cells (A) was quantitated by 854 37 densitometry, and the relative levels of GILZ (B) were calculated as follows: the band 855 density of GILZ / the band density of β-actin. (C-E) Knockdown of GILZ abolished 856 the inhibitory effect of VP4 on Poly(I:C)-induced expressions of IFN-α, IFN-β and 857 NF-κB. DF-1 cells (2.0×105) were co-transfected with RNAi constructs together with 858 of pEGFP-vp4 expression plasmids or pEGFP-N1 (vector) controls. Eighteen hours 859 after transfection, cells were treated with 0.8 μg of Poly (I:C). Twenty-four hours after 860 Poly (I:C) treatment, mRNA expressions of IFN-α, IFN-β and NF-κB were measured 861 by qRT-PCR using specific primers. The expressions level of mRNA were calculated 862 in relation to the expression level of GAPDH .Results are representative of three 863 independent experiments. Data are represented as mean +/- SD, n=3. *** stands for 864 p<0.001 and ** for p<0.01. 865 FIG 8 Knock down of GILZ inhibits IBDV growth by type I interferon 866 (A-B) GILZ is required for IBDV-induced suppression of Type I interferon 867 expressions in host cells after TNF-α treatment. HEK293T cells were treated with 868 GILZ-RNAi or RNAi-ctrl constructs or medium only as untreated control, and were 869 mock-infected or infected with IBDV at an MOI of 10. Twelve hours after IBDV 870 infection, cells were treated with TNF-α at a final concentration of 20ng/ml. Twelve 871 hours after TNF-α treatment, mRNA expressions of IFN-α and IFN-β were measured 872 by qRT-PCR using specific primers. The expression levels of mRNA were calculated 873 in relation to that of GAPDH .Results are representative of three independent 874 experiments. Data are represented as mean +/- SD, n=3. *** stands for p<0.001. (C-D) 875 Knockdown of GILZ inhibits IBDV growth. DF-1 (C) or HEK293T (D) cells were 876 38 treated with GILZ-RNAi or control-RNAi constructs or medium only as controls, and 877 were infected with IBDV at an MOI of 10. At different time points (12, 24, 48 and 72 878 h) post IBDV infection, the viral titers in the cell cultures were determined by TCID50 879 using 96-well plates. The significance of the differences between GILZ-RNAi and 880 controls was performed by ANOVA (p<0.01). (E) Type I interferon mediates the 881 inhibitory effect of GILZ-RNAi on IBDV growth. Anti-human IFN-α1 (4×104 882 neutralizing units/ml) and anti-human IFN-β (5×104 neutralizing units/ml) were added 883 to GILZ knockdown cells or RNAi controls 3 hours ahead of IBDV infection. 884 Forty-eight hours after IBDV infection, the culture samples were freeze-thawed three 885 times and centrifuged at 2,000g for 10 min. The viral titers were titrated using TCID50 886 (50% tissue culture infective doses) in DF-1 cells. Results are representative of three 887 independent experiments. Data are represented as mean +/- SD, n=3. *** stands for 888 p<0.001. 889