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• W2022211158 abstract "Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process. Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process. Infectious bursal disease (IBD), 3The abbreviations used are: IBDinfectious bursal diseaseIBDVinfectious bursal disease virusCEFchicken embryo fibroblastTCID5050% tissue culture infective doseTRITCtetramethylrhodamine isothiocyanate. also called Gumboro disease, is an acute, highly contagious disease in young chickens that occurs across the world (1.Pitcovski J. Gutter B. Gallili G. Goldway M. Perelman B. Gross G. Krispel S. Barbakov M. Michael A. Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens.Vaccine. 2003; 21: 4736-4743Crossref PubMed Scopus (87) Google Scholar). Its causative agent, IBD virus (IBDV), destroys its target cells, the B lymphocyte precursors (2.Liu M. Vakharia V.N. VP1 protein of infectious bursal disease virus modulates the virulence in vivo.Virology. 2004; 330: 62-73Crossref PubMed Scopus (68) Google Scholar, 3.Sharma J.M. Kim I.J. Rautenschlein S. Yeh H.Y. Infectious bursal disease virus of chickens: pathogenesis and immunosuppression.Dev. Comp. Immunol. 2000; 24: 223-235Crossref PubMed Scopus (299) Google Scholar4.Yao K. Vakharia V.N. Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis.Virology. 2001; 285: 50-58Crossref PubMed Scopus (80) Google Scholar). IBDV infection may cause mortality in naïve chickens and very high mortality in chickens with low levels of neutralizing antibodies or no mortality at all but a high degree of immunosuppression (5.Müller H. Islam M.R. Raue R. Research on infectious bursal disease: the past, the present and the future.Vet. Microbiol. 2003; 97: 153-165Crossref PubMed Scopus (280) Google Scholar). The survival chickens suffer from a severe immunosuppression that leads to an increased susceptibility to other pathogens (6.Stricker R.L. Behrens S.E. Mundt E. Nuclear factor NF45 interacts with viral proteins of infectious bursal disease virus and inhibits viral replication.J. Virol. 2010; 84: 10592-10605Crossref PubMed Scopus (34) Google Scholar). infectious bursal disease infectious bursal disease virus chicken embryo fibroblast 50% tissue culture infective dose tetramethylrhodamine isothiocyanate. IBDV belongs to the genus Avibirnavirus of the family Birnaviridae and has two segments of double-stranded genomic RNAs (A and B) (7.Azad A.A. Barrett S.A. Fahey K.J. The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus.Virology. 1985; 143: 35-44Crossref PubMed Scopus (169) Google Scholar). Segment B encodes VP1 (97 kDa), a RNA-dependent RNA polymerase (8.Pan J. Lin L. Tao Y.J. Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site.Virology. 2009; 395: 87-96Crossref PubMed Scopus (24) Google Scholar), affecting viral replication and virulence (2.Liu M. Vakharia V.N. VP1 protein of infectious bursal disease virus modulates the virulence in vivo.Virology. 2004; 330: 62-73Crossref PubMed Scopus (68) Google Scholar, 9.Escaffre O. Le Nouën C. Amelot M. Ambroggio X. Ogden K.M. Guionie O. Toquin D. Müller H. Islam M.R. Eterradossi N. Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus.J. Virol. 2013; 87: 2767-2780Crossref PubMed Scopus (69) Google Scholar, 10.Wang Y. Sun H. Shen P. Zhang X. Xia X. Effective inhibition of infectious bursal disease virus replication by recombinant avian adeno-associated virus-delivered microRNAs.J. Gen. Virol. 2009; 90: 1417-1422Crossref PubMed Scopus (20) Google Scholar). Segment A contains two partially overlapping ORFs. The first ORF encodes nonstructural viral protein 5 (VP5), and the second one encodes a pVP2-VP4-VP3 precursor (110 kDa) that can be cleaved by the proteolytic activity of VP4 to form viral proteins VP2, VP3, and VP4 (7.Azad A.A. Barrett S.A. Fahey K.J. The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus.Virology. 1985; 143: 35-44Crossref PubMed Scopus (169) Google Scholar, 11.Casañas A. Navarro A. Ferrer-Orta C. González D. Rodríguez J.F. Verdaguer N. Structural insights into the multifunctional protein VP3 of birnaviruses.Structure. 2008; 16: 29-37Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12.Kibenge F.S. McKenna P.K. Dybing J.K. Genome cloning and analysis of the large RNA segment (segment A) of a naturally avirulent serotype 2 infectious bursal disease virus.Virology. 1991; 184: 437-440Crossref PubMed Scopus (47) Google Scholar). VP2, a major structural protein (13.Dobos P. Hill B.J. Hallett R. Kells D.T. Becht H. Teninges D. Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes.J. Virol. 1979; 32: 593-605Crossref PubMed Google Scholar), is involved in antigenicity, cell tropism, pathogenic phenotype, and apoptosis (14.Brandt M. Yao K. Liu M. Heckert R.A. Vakharia V.N. Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus.J. Virol. 2001; 75: 11974-11982Crossref PubMed Scopus (184) Google Scholar). VP3 also participates in the formation of viral particles and is involved in serotype specificity (15.Mahardika G.N. Becht H. Mapping of cross-reacting and serotype-specific epitopes on the VP3 structural protein of the infectious bursal disease virus (IBDV).Arch. Virol. 1995; 140: 765-774Crossref PubMed Scopus (17) Google Scholar), viral assembly (11.Casañas A. Navarro A. Ferrer-Orta C. González D. Rodríguez J.F. Verdaguer N. Structural insights into the multifunctional protein VP3 of birnaviruses.Structure. 2008; 16: 29-37Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16.Luque D. Saugar I. Rodríguez J.F. Verdaguer N. Garriga D. Martín C.S. Velázquez-Muriel J.A. Trus B.L. Carrascosa J.L. Castón J.R. Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch.J. Virol. 2007; 81: 6869-6878Crossref PubMed Scopus (44) Google Scholar, 17.Maraver A. Oña A. Abaitua F. González D. Clemente R. Ruiz-Díaz J.A. Castón J.R. Pazos F. Rodriguez J.F. The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus, plays a critical role in capsid assembly.J. Virol. 2003; 77: 6438-6449Crossref PubMed Scopus (63) Google Scholar18.Delgui L.R. Rodríguez J.F. Colombo M.I. The endosomal pathway and the Golgi complex are involved in the infectious bursal disease virus life cycle.J. Virol. 2013; 87: 8993-9007Crossref PubMed Scopus (28) Google Scholar), and apoptotic regulation (19.Busnadiego I. Maestre A.M. Rodríguez D. Rodríguez J.F. The infectious bursal disease virus RNA-binding VP3 polypeptide inhibits PKR-mediated apoptosis.PloS ONE. 2012; 7: e46768Crossref PubMed Scopus (28) Google Scholar). VP4, a viral protease, is able to cleave in trans and is responsible for the interdomain proteolytic autoprocessing of the pVP2-VP4-VP3 polyprotein into the pVP2 precursor (48 kDa) and VP4 (28 kDa) as well as VP3 (32 kDa) (6.Stricker R.L. Behrens S.E. Mundt E. Nuclear factor NF45 interacts with viral proteins of infectious bursal disease virus and inhibits viral replication.J. Virol. 2010; 84: 10592-10605Crossref PubMed Scopus (34) Google Scholar, 20.Birghan C. Mundt E. Gorbalenya A.E. A non-canonical lon proteinase lacking the ATPase domain employs the Ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus.EMBO J. 2000; 19: 114-123Crossref PubMed Scopus (167) Google Scholar). pVP2 is further processed at its C-terminal domain by VP4 to generate the mature capsid protein VP2 (41 kDa) and four small peptides (21.Da Costa B. Chevalier C. Henry C. Huet J.C. Petit S. Lepault J. Boot H. Delmas B. The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2.J. Virol. 2002; 76: 2393-2402Crossref PubMed Scopus (108) Google Scholar). A recent report indicates that VP4 is responsible for IBDV-induced immune suppression (22.Li Z. Wang Y. Li X. Li X. Cao H. Zheng S.J. Critical roles of glucocorticoid-induced leucine zipper in infectious bursal disease virus (IBDV)-induced suppression of type I Interferon expression and enhancement of IBDV growth in host cells via interaction with VP4.J. Virol. 2013; 87: 1221-1231Crossref PubMed Scopus (64) Google Scholar). The nonstructural viral protein VP5 only exists in IBDV-infected cells and plays different roles in IBDV-induced apoptosis during IBDV infection. VP5 inhibits apoptosis early during infection (23.Liu M. Vakharia V.N. Nonstructural protein of infectious bursal disease virus inhibits apoptosis at the early stage of virus infection.J. Virol. 2006; 80: 3369-3377Crossref PubMed Scopus (58) Google Scholar, 24.Wei L. Hou L. Zhu S. Wang J. Zhou J. Liu J. Infectious bursal disease virus activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by interaction of VP5 protein with the p85α subunit of PI3K.Virology. 2011; 417: 211-220Crossref PubMed Scopus (42) Google Scholar), whereas it induces apoptosis at a later stage of infection (4.Yao K. Vakharia V.N. Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis.Virology. 2001; 285: 50-58Crossref PubMed Scopus (80) Google Scholar, 25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar, 26.Mundt E. Köllner B. Kretzschmar D. VP5 of infectious bursal disease virus is not essential for viral replication in cell culture.J. Virol. 1997; 71: 5647-5651Crossref PubMed Google Scholar). In a previous study, we found that VP5 induces apoptosis in DF-1 cells via interaction with voltage-dependent anion channel 2 (VDAC2) (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar). However, the molecular mechanism underlying such an induction remains elusive. In this study, we expanded our investigation to search for the interacting proteins for VDAC2 by yeast two-hybrid screening, immunoprecipitation, and confocal microscopy assays. We found that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they can form a complex. Importantly, overexpression of RACK1 suppressed IBDV-induced apoptosis. Furthermore, knockdown of RACK1 by siRNA markedly induced the activation of caspases 9 and 3 and suppressed IBDV growth. Both HEK293T and DF-1 (immortal chicken embryo fibroblast) cells were obtained from the ATCC. All cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS in a 5% CO2 incubator. Primary chicken embryo fibroblast (CEF) cells were prepared from 10-day-old specific pathogen-free chicken embryos. Lx, a cell culture-adapted IBDV strain, was provided by Dr. Jue Liu (Beijing Academy of Agriculture and Forestry, Beijing, China). All restriction enzymes were purchased from New England Biolabs. The pRK5-FLAG, pDsRed-monomer-N1, pCMV-Myc, pEGFP-C1, and pEGFP-N1 vectors were obtained from Clontech. Anti-c-Myc (catalog no. sc-40), anti-GFP (catalog no. sc-9996), anti-RACK1 (catalog no. sc-17754), and anti-β-actin (catalog no. sc-1616-R) monoclonal antibodies were obtained from Santa Cruz Biotechnology. Rabbit anti-VDAC2 polyclonal antibodies (catalog no. ab47104) were purchased from Abcam. Anti-VP5 monoclonal antibody (catalog no. EU0208) was purchased from CAEU Biological Co. (Beijing, China). Rabbit anti-GFP antibodies (catalog no. 2956S) were purchased from Cell Signaling Technology. Anti-FLAG (catalog no. F1804) antibody, propidium iodide, Annexin V-phycoerythrin (Annexin V-PE) and 7-amino-actinomycin D were purchased from Sigma. Opti-MEM I, RNAiMAX, and Lipofectamine LTX were purchased from Invitrogen. Gallus RACK1 was cloned from DF-1 cells using the specific primers 5′-ATGACGGAGCAGATGACC-3′ (sense) and 5′-TCATCTGGTTCCAATGGT-3′ (antisense) according to the published sequence in GenBank (accession No. AY393848.1). pRK5-FLAG-rack1, pCMV-Myc-rack1, pDsRed-rack1, and pEGFP-rack1 were constructed by standard molecular biology techniques. All primers were obtained from a commercial source (Sangon, Shanghai, China). pRK5-FLAG-vdac2, pEGFP-vdac2, pRK5-FLAG-vp5, pEGFP-vp5, and truncated VP5 expression plasmids were kept in our laboratory. Yeast two-hybrid screening was performed according to the protocol of the manufacturer (Matchmaker Two-Hybrid System 3). Briefly, the pGBKT7-vdac2 plasmid expressing the fusion protein GAL4-BD-vdac2 was used as bait, and the bursa of Fabricius cDNA expression library fusion to the GAL4-activation domain in the pGADT7 plasmid was used as prey. Positive clones were selected on S.D./Ade/His/Leu/Trp medium and tested for β-galactosidase activity. The coimmunoprecipitation approach used to analyze protein interaction has been described previously (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar). Briefly, HEK293T cells or DF-1 cells were cotransfected with the indicated plasmids or empty vectors as controls. Twenty-four hours after transfection, cell lysates were subjected to immunoprecipitation with anti-Myc (or anti-FLAG) antibody at 4 °C for 3 h and then mixed with 20 μl of a 50% slurry of protein A/G plus agarose and incubated for another 3 h. Beads were washed three times with lysis buffer and boiled with 2× SDS loading buffer for 10 min. The samples were fractionated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane. After blocking with 5% skimmed milk, the membranes were incubated with the indicated antibodies. Blots were developed using an ECL kit. For the endogenous pulldown assay, DF-1 cells were transfected with pEGFP-VP5 or infected with IBDV. Twenty-four hours after transfection or 36 h after infection, the cell lysates were subjected to immunoprecipitation with anti-RACK1 antibody and immunoblotted with anti-VDAC2, anti-RACK1, anti-VP5, or anti-GFP antibodies. HEK293T cells transfected with pDsRed-rack1 and pEGFP-vdac2 (or pEGFP-vp5) were fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI. For endogenous protein staining, mock- or IBDV-infected DF-1 cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, blocked with bovine serum albumin, and then probed with the indicated antibodies. The cells were stained for nuclei with DAPI. The samples were analyzed with a laser confocal scanning microscope (Nikon C1 standard detector). DF-1 or CEF cells were transfected with pEGFP-rack1 or pEGFP-N1 plasmids by Lipofectamine LTX before infection with IBDV at an m.o.i. of 1. Twenty-four hours after infection, cells were harvested and stained with propidium iodide (1 μg/ml) or PE-conjugated Annexin V in a binding buffer for 15 min. GFP-positive cells were gated for apoptosis analysis. Fluorescence-activated cell sorter data were analyzed with CellQuest software (BD Biosciences). DF-1 cells receiving RACK1-specific siRNA or control siRNA were harvested and washed twice with PBS, resuspended in a binding buffer, and incubated with PE-conjugated Annexin V and 7-amino-actinomycin D for 15 min at room temperature. Samples were subjected to flow cytometry analysis as described above. siRNAs designed by Genechem Co. (Shanghai, China) were used to knock down RACK1 in DF-1 cells. The sequences of siRNA for targeting RACK1 in DF-1 cells included RNAi#1 (sense, 5′-CCAUCAUCAUGUGAAGCUtt-3′; antisense, 5′-AGCUUCACAUGAUGAUGGtt-3′), RNAi#2 (sense, 5′-CCCGAGAUGAGACCAACUAtt-3′; antisense, 5′-UAGUUGGUCUCAUCUCGGGtt-3′), RNAi#3 (sense, 5′-UCAAACUCUGGAACACUUU tt-3′; antisense, 5′-AAAGUGUUCCAGAGUUUGA tt-3′), and a negative siRNA control (sense, 5′-UUCUCCGAACGUGUCACGUtt-3′; antisense, 5′-ACGUGACACGUUCGGAGAAtt-3′). DF-1 cells were transfected with siRNA using RNAiMAX reagent according to the instructions of the manufacturer (Invitrogen). Triple transfections were performed at a 24-h interval. Twenty-four hours after the third transfection, cells were harvested for further analysis. DF-1 cells receiving RACK1-specific siRNA or control siRNA were lysed in chilled cell lysis buffer, the cell lysates were centrifuged, and the supernatants were collected to measure caspase 3 and 9 activities according to the instructions of the manufacturer (BioVision). The results of all experiments are reported as mean ± S.D. DF-1 cells receiving RACK1-specific siRNA or control siRNA or transfected with pEGFP-rack1 or pEGFP-N1 as a control were infected with IBDV at an m.o.i. of 10, and cell cultures were collected at different time points (12, 24, 48, and 72 h) after infection. The viral titers in the supernatants or cell cultures were titrated using 50% tissue culture infective doses (TCID50) as described previously (27.Reed L M.H. A simple method of estimating fifty percent endpoints.Am. J. Epidemiol. 1938; 27: 493-497Crossref Scopus (16192) Google Scholar). Briefly, the viral solution was serially diluted 10-fold in DMEM. A 100-μl aliquot of each diluted sample was added to the wells of 96-well plates, followed by addition of 100 μl of DF-1 cells at a density of 5 × 105 cells/ml. Cells were cultured for 5 days at 37 °C in 5% CO2. Tissue culture wells with a cytopathic effect were considered to be positive. The significance of the differences between RACK1 RNAi cells and controls in caspase 9 and 3 activation, between RACK1 RNAi cells and controls in viral growth or apoptosis, and between pEGFP-rack1-transfected cells and controls in viral growth or apoptosis was determined by Mann-Whitney test or analysis of variance, respectively. Our previous report indicated that VP5 induced apoptosis in DF-1 cells via interaction with VDAC2, which affected the viral release (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar). To elucidate the mechanism of such an induction, we set out to search for the cellular targets of VDAC2. We used VDAC2 as bait in a yeast two-hybrid system to screen a cDNA library generated from the chicken bursa of Fabricius. Among the proteins that potentially interacted with VDAC2, RACK1 proteins were identified twice. The interaction between VDAC2 and RACK1 was further verified with a colony lift filter assay (Fig. 1). Because RACK1 is related to apoptosis (28.Wu Y. Wang Y. Sun Y. Zhang L. Wang D. Ren F. Chang D. Chang Z. Jia B. RACK1 promotes Bax oligomerization and dissociates the interaction of Bax and Bcl-XL.Cell Signal. 2010; 22: 1495-1501Crossref PubMed Scopus (15) Google Scholar, 29.Mamidipudi V. Cartwright C.A. A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways.Oncogene. 2009; 28: 4421-4433Crossref PubMed Scopus (57) Google Scholar30.Choi D.S. Young H. McMahon T. Wang D. Messing R.O. The mouse RACK1 gene is regulated by nuclear factor-κ B and contributes to cell survival.Mol. Pharmacol. 2003; 64: 1541-1548Crossref PubMed Scopus (42) Google Scholar), we proposed that RACK1 might be involved in VDAC2-mediated apoptosis. Therefore, we constructed a plasmid that allowed the expression of Myc-RACK1 for analyzing its interaction with VDAC2 in HEK293T cells. When lysates of cells expressing both Myc-RACK1 and GFP-VDAC2 were immunoprecipitated with Myc antibody, GFP-VDAC2 was detected in the precipitate, indicating that RACK1 interacted with exogenous VDAC2 in HEK293T cells (Fig. 2A). Similar results were obtained in an experiment using DF-1 cells (Fig. 2B), indicating that the interaction observed between these two proteins is not cell type-specific. Considering the binding capacity of RACK1 as a signaling hub (31.Ron D. Adams D.R. Baillie G.S. Long A. O'Connor R. Kiely P.A. RACK1 to the future: a historical perspective.Cell Commun. Signal. 2013; 11: 53Crossref PubMed Scopus (27) Google Scholar), we expanded our investigation to study whether RACK1 could interact with IBDV VP5. Lysates of cells expressing Myc-RACK1 and FLAG-VP5 were immunoprecipitated with FLAG antibody, and Myc-RACK1 was detected in the precipitate, indicating that RACK1 interacted with ectopically expressed VP5 in HEK293T cells (Fig. 2C). Similar results were obtained in an experiment using DF-1 cells (Fig. 2D). To further substantiate the binding of RACK1 to VDAC2 or IBDV VP5, we expressed RACK1 or VDAC2 in DF-1 cells and examined their interaction with endogenous partners using a pulldown assay. The binding of FLAG-RACK1 with endogenous VDAC2 was readily detectable in cells expressing RACK1 protein (Fig. 3A), and the binding of FLAG-VDAC2 with endogenous RACK1 was also detectable (Fig. 3B). These results demonstrate that VDAC2 interacts with RACK1 in host cells. When lysates of DF-1 cells expressing GFP-VP5 were immunoprecipitated with RACK1 antibody, GFP-VP5 was detected in the precipitate, indicating that VP5 interacted with endogenous RACK1 in host cells (Fig. 3C). These data suggest that RACK1 interacts with both VDAC2 and VP5 proteins. Because RACK1 interacted with both VDAC2 and VP5 and was considered to play a role in complex assembly, we expanded our investigation to study whether RACK1, VDAC2, and IBDV VP5 form a complex. Lysates of HEK293T cells expressing FLAG-RACK1, GFP-VP5, and GFP-VDAC2 were immunoprecipitated with FLAG antibody, and both GFP-VP5 and GFP-VDAC2 were detected in the precipitate (Fig. 4A), indicating that ectopically expressed VP5, RACK1, and VDAC2 formed a complex. To further examine the complex of VDAC2 and RACK1 to VP5, we expressed VP5 in DF-1 cells and examined the complex using a pulldown assay. Lysates of DF-1 cells expressing GFP-VP5 were immunoprecipitated with RACK1 antibody, and both VP5 and VDAC2 were detected in the precipitate (Fig. 4B), indicating that VP5, endogenous RACK1, and VDAC2 formed a complex in host cells. To determine whether the IBDV VP5, VDAC2, and RACK1 could form a complex under physiological conditions, we infected DF-1 cells and performed a pulldown assay using anti-RACK1 antibodies. As a result, both VP5 and VDAC2 could be detected in the precipitate of the lysates from IBDV-infected cells but not from mock-infected controls (Fig. 4C). These data clearly show that VDAC2, RACK1, and IBDV VP5 formed a complex in host cells. To determine the region of VP5 responsible for interacting with RACK1, we constructed a series of VP5 truncated mutants fused to the GFP tag (Fig. 5A). These VP5 derivatives were coexpressed individually with FLAG-RACK1 in HEK293T cells, and lysates of cells were immunoprecipitated with FLAG antibody. These VP5 derivatives were detected with anti-GFP antibody. Our results indicated that, with the exception of mutants (Δ3 and Δ4) lacking residues 100–145, the VP5 mutant with residues 100–145 only (Δ2 mutant) retained the ability to interact with RACK1 (Fig. 5B), suggesting that the region with 100–145 of VP5 is important for its interaction with RACK1. Because we have established previously that a domain that spans residues 1–50 of VP5 interacts with VDAC2 (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar), and because our data from this study show that a domain with residues 100∼145 of VP5 interacts with RACK1 and that VDAC2 binds to RACK1, it is very likely that VP5, RACK1, and VDAC2 form a complex in cells (Fig. 5C). To determine the subcellular localization of VDAC2 and RACK1, we performed confocal microscopy assays with HEK293T cells transfected to express DsRed-RACK1 and GFP-VDAC2. As shown in Fig. 6, A and B, both RACK1 and VDAC2 were located in the cytoplasm of HEK293T cells transfected with pEGFP-vdac2 or pDsRed-rack1. When cells were transfected with both plasmids, we found that the fluorescence of exogenous VDAC2 and RACK1 merged in the cytoplasm (Fig. 6, C–E), suggesting that VDAC2 and RACK1 were colocalized in cells. To consolidate these findings, we examined the colocalization of endogenous VDAC2 with RACK1 in IBDV-infected DF-1 cells. We infected DF-1 cells with IBDV at an m.o.i. of 10 and performed an immunofluorescent antibody assay using anti-VDAC2 and anti-RACK1 antibodies. In mock-infected cells, both VDAC2 and RACK1 were diffused cytoplasmically (Fig. 6, F–H). However, when cells were infected with IBDV, endogenous VDAC2 and RACK1 changed their localization patterns from diffused to granular structures where both of them were colocalized (Fig. 6, I–K), indicating the interaction of VDAC2 with RACK1 in IBDV-infected cells. Because we have shown previously that VP5 colocalized with VDAC2 in the mitochondrion of host cells (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar) and because VP5-VDAC2-RACK1 formed a complex, as demonstrated by immunoprecipitation assays, we performed a confocal microscopy assay to examine the colocalization of VP5 with RACK1 in cells. As shown in Fig. 6, L–N, the colocalization of VP5 with exogenous RACK1 was found primarily in the cytoplasm of cells transfected with both pDsRed-RACK1 and pEGFP-VP5. When cells were transfected with pEGFP-VP5 only, endogenous RACK1 colocalized with VP5 (Fig. 6, O–Q). In addition, transfection of DF-1 cells with pDsRed-RACK1 indicated that RACK1 was also located in the mitochondria of DF-1 cells (Fig. 6, R–T). All these data strongly suggested that VP5, VADC2, and RACK1 formed a complex in the cytoplasm of cells and that they were all located in the mitochondria. Because IBDV VP5 protein was mainly responsible for IBDV-induced apoptosis via interaction with VDAC2 (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar) and because RACK1 was associated physically with both VP5 and VDAC2, RACK1 was assumed to affect IBDV-induced apoptosis. To test this hypothesis, DF-1 cells were transfected with pEGFP-rack1 or pEGFP-N1 as a control, followed by mock infection or infection with IBDV. As shown in Fig. 7A, exogenous RACK1 was expressed very well in both mock- and IBDV-infected cells. Interestingly, we found that overexpression of RACK1 markedly suppressed IBDV-induced apoptosis in DF-1 cells (Fig. 7, B and C). Similar results were obtained using primary cell culture (CEFs) (Fig. 7, D and E). These data suggest that RACK1 may play an antiapoptotic role in IBDV-induced apoptosis in host cells. Because IBDV-induced apoptosis is associated with viral growth (25.Li Z. Wang Y. Xue Y. Li X. Cao H. Zheng S.J. Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.J. Virol. 2012; 86: 1328-1338Crossref PubMed Scopus (68) Google Scholar), we examined the viral growth in pEGFP-rack1-transfected DF-1 cells and CEFs at different time points after IBDV infection. As shown in Fig. 8A, overexpression of RACK1 enhanced IBDV growth in the cell culture compared with that of controls (p < 0.05), wh" @default.
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• W2022211158 title "The Association of Receptor of Activated Protein Kinase C 1(RACK1) with Infectious Bursal Disease Virus Viral Protein VP5 and Voltage-dependent Anion Channel 2 (VDAC2) Inhibits Apoptosis and Enhances Viral Replication" @default.
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