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The association of ribosomal protein L18 (RPL18) with infectious bursal disease virus viral protein VP3 enhances viral replication
Accepted Manuscript Title: The Association of Ribosomal Protein L18 (RPL18) with Infectious Bursal Disease Virus Viral Protein VP3 Enhances Viral Replication Authors: Bin Wang, Xueyan Duan, Mengjiao Fu, Yanan Liu, Yongqiang Wang, Xiaoqi Li, Hong Cao, Shijun J. Zheng PII: DOI: Reference:
S0168-1702(17)30244-7 https://doi.org/10.1016/j.virusres.2017.12.009 VIRUS 97314
To appear in:
Virus Research
Received date: Revised date: Accepted date:
23-3-2017 14-12-2017 18-12-2017
Please cite this article as: Wang, Bin, Duan, Xueyan, Fu, Mengjiao, Liu, Yanan, Wang, Yongqiang, Li, Xiaoqi, Cao, Hong, Zheng, Shijun J., The Association of Ribosomal Protein L18 (RPL18) with Infectious Bursal Disease Virus Viral Protein VP3 Enhances Viral Replication.Virus Research https://doi.org/10.1016/j.virusres.2017.12.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The Association of Ribosomal Protein L18 (RPL18) with Infectious Bursal Disease Virus Viral Protein VP3 Enhances Viral Replication
Bin Wanga,b,c, Xueyan Duan a,b,c, Mengjiao Fua,b,c, Yanan Liua,b,c, Yongqiang Wanga,b,c,
a
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Xiaoqi Lia,b,c, Hong Caoa,b,c, and Shijun J. Zhenga,b,c* State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing
b
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100193, China
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, China
College of Veterinary Medicine, China Agricultural University, Beijing 100193,
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c
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Agricultural University, Beijing 100193, China
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*
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China
Corresponding author at: College of Veterinary Medicine, China Agricultural
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University, 2 Yuan-Ming-Yuan West Road, Beijing 100193, P.R.China Tel: 86-(10)-
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6273-4681. Fax: 86-(10)-6273-4681.
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Email: [email protected] Highlights
IBDV VP3 interacted with chRPL18 in host cells.
chPKR and chRPL18 could be pulled down by anti-VP3 mcAb in IBDV-infected cells
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Knockdown of chRPL18 by RNAi promoted Type I interferon expression
Knockdown of chRPL18 inhibited IBDV replication.
Abstract
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Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). IBDV VP3 is a multifunctional protein playing a key role in virus assembly and pathogenesis. To investigate the role of VP3 in pathogenesis, we transfected DF-1 cells with pRK5-FLAG-vp3 and found that VP3
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enhanced type I interferon expression and suppressed IBDV replication. Furthermore we found that VP3 interacted with chicken Ribosomal Protein L18 (chRPL18) in host
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cells and knockdown of chRPL18 by RNAi significantly promoted Type I interferon
expression and inhibited IBDV replication. Moreover, our data show that chicken
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double-stranded RNA-activated protein kinase (chPKR) interacted with both VP3 and
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chRPL18. Thus chRPL18 in association with VP3 and chPKR affects viral replication.
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Keywords: chRPL18; IBDV VP3; chPKR; IBDV replication 1. Introduction
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Infectious bursal disease virus (IBDV) causes an acute, highly contagious, and
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immunosuppressive disease in young chickens that causes great losses to the poultry industry across the world (Pitcovski et al., 2003). IBDV attacks the immune system,
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which causes severe damage of the B-lymphocyte precursors, resulting in a high mortality in the young chickens (Muller et al., 2003). The surviving chickens suffer
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from a severe immunosuppression, leading to an increased susceptibility to other pathogens (Stricker et al., 2010). IBDV, belonging to the genus Avibirnavirus of the family Birnaviridae, is a nonenveloped, double-stranded RNA virus with an icosahedral nucleocapsid (Tacken 2
et al., 2003). The virus genome consists of two segments of double-stranded RNA, segment A (3.2 kb) and B (2.8kb) (Azad et al., 1985; Tacken et al., 2004). Segment B, the short RNA, encodes VP1 (90kDa) (Morgan et al., 1988), an RNA-dependent RNA polymerase (Shwed et al., 2002; Tacken et al., 2004). Segment A, the large one,
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contains two partially overlapping open reading frames (ORFs) (Kibenge et al., 1991). The small ORF encodes the nonstructural viral protein VP5 (17kDa), involved in
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apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2) in IBDVinfected cells (Li et al., 2012). The large one encodes an approximate 110 kDa
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polyprotein precursor that can be self-cleaved by viral protease VP4 to form viral
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proteins pVP2 (41 kDa), VP3 (32 kDa) and VP4 (24 kDa) (Azad et al., 1985; Hudson
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et al., 1986; Jagadish et al., 1988; Kibenge et al., 1991). pVP2 is further processed into
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VP2 and several small peptides from the C terminal by VP4 (Sanchez and Rodriguez,
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1999), the puromycin aminopeptidase (Irigoyen et al., 2012) and autoproteolytically between residues Ala441-Phe442 (Irigoyen et al., 2009). Apart from acting as a viral
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protease, VP4 is also considered to be an interferon suppressor by interacting with
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GILZ in host cells (Li et al., 2013). VP2 and VP3 are the major structural proteins, constituting 51% and 40% of the mature viral particle, respectively (Dobos et al., 1979). VP3, acting as a scaffold protein (Maraver et al., 2003), recruits genome dsRNA and
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VP1 to form a ribonucleoprotein (RNP) complex and serves as a transcriptional activator (Casanas et al., 2008; Lombardo et al., 1999a), playing a critical role in virus assembly (Lombardo et al., 1999b; Maraver et al., 2003). It was reported that VP3 shows a higher affinity than MDA5 at binding IBDV genomic dsRNA to evade innate 3
immunity (Ye et al., 2014). Besides, VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing PKR-mediated apoptosis (Busnadiego et al., 2012). Although IBDV VP3 plays a critical role in viral assembly, the exact role of VP3 in the
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pathogenesis of IBDV infection is not clear. In this study, we found that VP3 enhanced
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type I interferon expression and suppressed IBDV replication. Furthermore，we found that VP3 interact with chRPL18, and knockdown of chRPL18 markedly suppressed
IBDV replication by enhancing type I interferon expression. Moreover, our data show
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that VP3, chRPL18 and chPKR form a complex affecting viral replication.
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2. Materials and Methods
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2.1. Cells and virus.
Both HEK-293T cells and DF-1 (immortal chicken embryo fibroblast) cells were
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obtained from the ATCC. All cells were cultured in Dulbecco modified Eagle medium
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(DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 incubator. Lx, a cell culture-adapted IBDV strain, was kindly provided by Dr. Jue
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Liu (Beijing Academy of Agriculture and Forestry Science, Beijing, China).
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2.2. Reagents.
Endotoxin-free plasmid preparation kits were purchased from ZYMO RESEARCH (USA). All restriction enzymes were purchased from Takara (Japan). RNase A was purchased from Aidlab (Beijing, China). Anti-c-Myc (sc-40), anti-green fluorescent protein (anti-GFP; sc-9996), and anti-β-actin (sc-1616-R) antibodies were obtained 4
from Santa Cruz Biotechnology (USA). Poly(I:C), Anti-RPL18 polyclonal antibody (SAB1100328) and anti-FLAG monoclonal antibody (F1804) were purchased from Sigma (USA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, tetramethyl rhodamine isocyanate (TRITC)-conjugated goat anti-rabbit IgG, and
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horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG antibodies were purchased from Ding-Guo (China). Anti-VP3 monoclonal antibody
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(EACU-c10) and anti-chPKR polyclonal antibody (EACU-2015) were purchased from
EACU (Beijing, China). jetPRIME® transfection reagent was purchased from Polyplus-
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transfection SA (France). OPTI-MEM I, RNAiMAX were purchased from Invitrogen
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(USA). 4’, 6-Diamino-2-phenylindole (DAPI) was purchased from Beytime (China).
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Coomassie brilliant blue R-250 was purchased from Abgene (USA). Fast mutagenesis
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2.3. Constructs.
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system was purchased from Transgene (Beijing, China).
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IBDV vp3 gene was cloned from IBDV strain Lx using the following specific primers: sense primer 5’-CGT TTC CCT CAC AAT CCA CGC GA-3’ and antisense primer 5’-
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CTC AAG GTC CTC ATC AGA GAC GGT-3’ (GenBank accession no. 126032566). Chicken rpl18 gene was cloned from DF-1 cells using the specific primers 5’- CGG
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CGC CGG GAG CCC AAAA-3’ (sense) and 5’-CCC CCG CGC CCG CTC GAA CTT-3’ (antisense) according to the sequence in GenBank (accession no. AY389963.1). Chicken pkr gene was cloned from DF-1 cells using the specific primers 5’- ATG GAC CGA GAG TGC ATG GC-3’ (sense) and 5’-TTA GTG ACT GTA AGC TTT ATG CGA G-3’ (antisense) with reference to the sequence in GenBank (accession 5
no.NM_204487). vp3 mutant containing four amino acid substitutions (K99D, R102D,K105D and K106D) was constructed using Fast mutagenesis system kit per the manufacturer’s instructions, using the following three pairs specific primers 5’- GAA GCA CAG AGG GAA GAC GACACA CGGA-3’ (K99D, sense) and 5’-GTC TTC
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CCT CTG TGC TTC CTC TGG TGTG-3’ (K99D, antisense), and 5’- GAA GCA CAG AGG GAA GAC GAC ACA GAC ATC TCA GACG-3’ (R102D,sense) and 5’-GTC
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TGT GTC GTC TT CCC TCT GTG CTTC CTC TGG TGTG-3’ (R102D, antisense), and 5’- GAC ACA CGG ATC TCA GAC GAC ATG GAG ACCA-3’ (K105D and
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K106D, sense) and 5’-GTC GTC TGA GAT CCG TGT GTC TTT TTC CCTC-3’
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(K105D and K106D, antisense). pCMV-Myc, pRK5-FLAG, pEGFP-N1 and pDsRed-
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monomer-N1 vectors were obtained from Clontech (USA). All the primers were
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2.4. Pull-down assays.
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synthesized by Sangon Company (China).
DF-1 cells were seeded on 100 mm cell culture dishes and cultured for 24 h before
Thirty-six hours after transfection, cell lysates were prepared using a
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reagent.
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transfection with pRK5-FLAG-vp3 or pRK5-FLAG using jetPRIME® transfection
nondenaturing lysis buffer (50nM Tris-HCl, pH 8.0; 150 nM NaCl; 1% TritonX-100,
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5nM EDTA, 10% glycerol, 10nM dithiothreitol, 1×complete cocktail protease inhibitor). The cell lysates were collected and centrifuged at 6000 rpm for 30 min, and the supernatants were incubated with 10μg of anti-FLAG antibody and 50 μl of a 50% slurry of protein A/G plus agarose at 4°C for 6 h. Beads were washed six times with the lysis buffer and boiled with 2×SDS loading buffer for 10 min. Samples were subjected 6
to 12% SDS-PAGE gel electrophoresis and stained with coomassie brilliant blue R250. After separation of proteins on SDS-PAGE gel, bands of interest were sliced and analyzed by MS. 2.5. Coimmunoprecipitation and Western Blot analysis.
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For immunoprecipitation, HEK-293T cells or DF-1 cells (4 ×105) were seeded on sixwell plates and cultured for 24 h before being cotransfected with pCMVMyc-chrpl18
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and pRK5-FLAG-vp3 or empty vectors as controls using jetPRIME® transfection reagent. Twenty-four hours after transfection, cell lysates were prepared using a
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nondenaturing lysis buffer (50mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM
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EDTA, 10% glycerol, 10mM dithiothreitol, 1×complete cocktail protease inhibitor).
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The cell lysates were incubated with 1μg of anti-FLAG antibody, 20μl of a 50% slurry of protein A/G plus agarose, and RNase A (10µg/ml) at 4°C overnight. Efficiency of
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RNase A treatment was detected by loading of 20 µl of protein samples incubated with
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or without RNase A on 1% agarose gel stained by ethidium bromide. Beads were washed three times with the lysis buffer and boiled with 2×SDS loading buffer for 10
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min. The samples were subjected to 12% SDS-PAGE gels electrophoresis, and resolved proteins were transferred onto polyvinylidene difluoride（PVDF）membranes. After
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blocking with 5% skimmed milk, the membranes were incubated with either anti-Myc or anti-FLAG antibodies, followed by an appropriate horseradish peroxidaseconjugated secondary antibody. Blots were developed using an enhanced chemiluminescence (ECL) kit. For endogenous chRPL18 or chPKR pull-down assay, HEK293T cells or DF-1 cells were infected with IBDV at an MOI of 1. Twenty-four 7
hours after infection, cell lysates were prepared using a nondenaturing lysis buffer. The cell lysates were subjected to immunoprecipitation with anti-VP3 antibody and immunoblotted with anti-RPL18, anti-VP3 or anti-chPKR antibodies. 2.6. Laser Confocal microscopy assays.
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DF-1 cells (1×105) were seeded on coverslips in 24-well plates and were cultured
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overnight before infection with IBDV. Twenty-four hours after infection, IBDV- or mock-infected cells were fixed with 1% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 15 min, blocked with 1% bovine serum albumin, and then
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probed with mouse anti-IBDV VP3 and/or rabbit anti-RPL18 antibodies, followed by
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FITC-conjugated goat anti-mouse IgG (green) or TRITC-conjugated goat anti-rabbit
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IgG (red) antibodies. After three washes with PBS, the cells were stained with DAPI
Nikon, Japan).
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for nuclei. The samples were observed with a laser confocal microscope (OLYMPUS;
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2.7. Knockdown of chRPL18 by RNA interference (RNAi). The siRNAs were designed by Genepharma Company (Shanghai, China) and used to
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knock down chRPL18 expressions in DF-1 cells. The siRNAs for targeting chRPL18 in DF-1 cells were as follows: RNAi#1 (sense, 5’- UCG UCA AGC UGU ACC GUU
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UTT-3’; antisense, 5’-AAA CGG UAC AGC UUG ACG ATT-3’), RNAi#2 (sense, 5’UCU UCA UGA GCC GAA CCA ATT -3’; antisense, 5’-UUG GUU CGG CUC AUG AAG ATT-3’), RNAi#3 (sense, 5’- CCU UCG ACC AAU UGG CCA UTT-3’; antisense, 5’- AUG GCC AAU UGG UCG AAG GTT-3’), and negative controls (sense, 5’-UUC UCC GAA CGU GUC ACG UTT-3’; antisense, 5’-ACG UGA CAC 8
GUU CGG AGA ATT-3’). To transfect cells with the interference RNAs against chRPL18, we seeded DF-1 cells (2×105) cells on 12-well plates and cultured them for at least 20 h prior to transfection. The cells were transfected with siRNA using RNAiMAX according to the manufacturer’s instructions (Invitrogen). Double
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transfections were performed at a 24h interval. Twenty-four hours after the second
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transfection, cells were harvested for further analysis. 2.8. Measurement of IBDV growth in DF-1 cells.
DF-1 cells receiving chRPL18-specific siRNAs or control siRNA were infected with
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IBDV at an MOI of 1, and the cell cultures (including cell and supernatant) or
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supernatants of cell cultures were collected at different time points (12, 24, 48, 72 h)
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after infection, respectively. The culture samples were freeze-thawed three times and centrifuged at 2,000×g for 10 min. The viral contents in the cellular lysates and
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supernatants of cell cultures were titrated using 50% tissue culture infective doses (TCID50) in DF-1 cells, respectively. Briefly, the viral solution was diluted by 10-fold
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in DMEM. A 100μl aliquot of each diluted sample was added to the wells of 96-well
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plates, followed by addition with 100μl of DF-1 cells at a density of 5×105 cells/ml. Cells were cultured for 5-7 days at 37°C in 5% CO2. Tissue culture wells with a
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cytopathic effect (CPE) were determined to be positive. The titer was calculated on the basis of a previously described method (Reed LJ, 1938). 2.9. RNA extraction and quantitative real-time PCR (qRT-PCR) analysis. Total RNA was prepared from DF-1 cells using EASYspin Plus kit (aidlab Biotechnology, China) following the manufacturer’s instructions. 0.5μg of total RNA 9
was used for cDNA synthesis by reverse transcription using an RT-PCR kit (TaKaRa). Quantitative RT-PCR analysis was performed using Tli RnaseH Plus (Takara) on LightCycler 480II (Roche, USA). Specific primers for chicken IFN-α (chIFN-α) (5’CCA GCA CCT CGA GCA AT-3’ and 5’-GGC GCT GTA ATC GTT GTC T-3’),
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chicken IFN-β (chIFN-β) (5’-GCC TCC AGC TCC TTC AGA ATA CG-3’and 5’-CTG GAT CTG GTT GAG GAG GCT GT-3’), chicken IRF3 (chIF3) (5’-GCT CTC TGA
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CTC TTT CAA CCT CTT-3’ and 5’-AAT GCT GCT CTT TTC TCC TCT G-3’),
chicken p65(chp65) (5′-CCA CAA CAC AAT GCG CTC TG-3′ and 5′- AAC TCA
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GCG GCG TCG ATG-3′), and chicken GAPDH (5’-TGC CAT CAC AGC CAC ACA
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GAA G-3’ and 5’-ACT TTC CCC ACA GCC TTA GCA G-3’) were designed with
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reference to previous publications (Abdul-Careem et al., 2008; Li et al., 2007; Liu et
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al., 2010). GAPDH gene was utilized as the reference gene. All quantitative real-time
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PCR experiments were performed in triplicate. The qRT-PCR was performed in a 20μl volume containing 1μl of cDNA, 10μl of 2×SYBR green Premix Ex Taq (TaKaRa),
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and a 0.4μM of each gene-specific primers. Thermal cycling parameters were as
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follows: 94°C for 2 min; 40 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s; and 1 cycle of 95°C for 30 s, 60°C for 30 s, and 95°C for 30 s. The final step was to obtain a melt curve for the PCR products to determine the specificity of the
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amplification.
2.10. Statistical analysis. The significance of the differences between chRPL18 RNAi cells and controls in gene expressions and viral growth, and between pCMV-Myc-chpkr-transfected cells and 10
controls in viral growth was determined by the Mann-Whitney test or analysis of variance (ANOVA) accordingly. 3. RESULTS 3.1. IBDV VP3 enhanced expressions of type I interferon, and inhibited IBDV
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replication.
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As a major structural protein, IBDV VP3 plays a key role in virus assembly and pathogenesis (Ye et al., 2014; Maraver et al., 2003). To examine whether VP3 affects
type I interferon expression, we transfected DF-1 cells with pRK5-FLAG-vp3 plasmid
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or control plasmid. We found that transient expression of VP3-FLAG significantly
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enhanced expression of type I interferon, chIRF3, and NF-κB (Fig1A-D). As type I
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interferon plays a critical role in the host response against IBDV infection (Ragland et al., 2002; Li et al., 2013; O'Neill et al., 2010), we infected pRK5-FLAG-vp3 transfected
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DF-1 cells with IBDV, and examined the viral growth in pRK5-FLAG-vp3 transfected
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DF-1 cells in 24h after IBDV infection. As shown in Fig.1E&F, expression of VP3 inhibited IBDV replication in cell cultures and the supernatants compared to that of
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controls. Taken together, these results suggest that IBDV VP3 enhanced expressions of type I interferon and inhibited IBDV replication.
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3.2. Screening and identification of IBDV VP3-interacting cellular proteins in DF1 cells. Pathogen-host interaction is the basis for the pathogenesis of IBDV infection (Li et al., 2012; Li et al., 2013; Qin and Zheng, 2017; Lin et al., 2015). To investigate the host cellular proteins that might be targeted by IBDV VP3, we transfected DF-1 cells with 11
pRK5-FLAG-vp3 and performed a pull-down assay using anti-FLAG McAb. As a result, nine extra protein bands were observed in the FLAG-VP3 lane on SDS-PAGE compared to that of FLAG controls (Fig.2A), indicating that IBDV VP3 interacted with some cellular proteins in DF-1 cells. To determine the amino acid sequences of VP3-
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interacting proteins, the arrow-pointed protein bands in Fig. 2A were cut-down and subjected to Mass Spectrometry analysis. As shown in Fig.2B, among nine proteins
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potentially interacting with VP3, chRPL18 might be relevant to IBDV replication in host cells because RPL18 was required during DENV replication cycle, facilitated
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translational transactivation of CaMV, and prevented PKR activation by dsRNA
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(Cervantes-Salazar et al., 2015; Leh et al., 2000; Kumar et al., 1999b). Thus, we
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selected chRPL18 for further investigation. The sequence of RPL18 protein identified
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in band F by Mass Spectrometry analysis is shown in red (Fig.2C).
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3.3. VP3 interacts with chRPL18.
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To verify the interaction of chRPL18 with VP3, we constructed a plasmid that allows the expression of Myc-chRPL18 for analyzing its interaction with VP3 in HEK293T
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cells by immunoprecipitation assays. Since both chRPL18 and VP3 possess RNAbinding domains (Casanas et al., 2008; Woestenenk et al., 2002), RNase A was added
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into cell lysates before immunoprecipitation assays. When the lysates of cells expressing both FLAG-VP3 and Myc-chRPL18 were immunoprecipitated with antiFLAG antibody, Myc-chRPL18 was detected in the precipitate, indicating that VP3 interacted with ectopically expressed chRPL18 in mammalian cells (Fig. 3A). Similar results were obtained in an experiment using the DF-1 cells (Fig. 3B), indicating that 12
the interaction observed between these two proteins is not cell type specific. To further confirm the interaction, we transfected DF-1 cells with pRK5-FLAG-vp3 plasmids and performed an immunoprecipitation assay with anti-FLAG McAb. The binding of FLAG-VP3 with endogenous chRPL18 was readily detectable in cells expressing the
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viral protein VP3 (Fig. 3C). In addition, the interaction of endogenous chRPL18 with viral VP3 was also confirmed by pull-down assay in IBDV-infected DF-1 cells (Fig.
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3D) .These results clearly demonstrate that VP3 interacts with chRPL18 in host cells.
3.4. A domain that spans residues 87 to 172 of VP3 is involved in interacting with
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chRPL18.
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It was reported that VP3 prevents VP2-induced protein synthesis arrest and PCD
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dependent upon the presence of function Patch1 dsRNA-bing domain, and the substitution of positively charged K and R resides for negatively charged D residues
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(K99D, R102D, K105D, and K106D) within the Patch1 region results in loss of ability
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to binding dsRNA (Busnadiego et al., 2012). To determine the region of VP3 responsible for interacting with RPL18 and examine whether the interaction of VP3
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with RPL18 was related to its ability of binding dsRNA, we constructed VP3 mutant with replacement of residues (K99D, R102D, K105D, and K106D) and truncated VP3
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fused with GFP (Fig. 4A). These VP3 derivatives were individually expressed in HEK293T cells, and their ability to interact with RPL18 was examined by immunoprecipitation (Fig. 4B). Our results indicated that RPL18 interacted with WT, △1, △2 and VP3 mutant, indicating that the interaction of VP3 with RPL18 is not dependent on the presence of dsRNA and that the portion of VP3 from amino acids 87 13
aa to 172 aa is responsible for binding to RPL18. 3.5. VP3 colocalized with chRPL18 in IBDV infected cells. To determine the subcelluar localization of IBDV VP3 and chRPL18, we performed a confocal microscopy assay, in which we infected DF-1 cells with IBDV and examined
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the interaction of VP3 with endogenous chRPL18 using mouse anti-IBDV VP3 and/or rabbit anti-RPL18 antibodies. Under this condition, both chRPL18 and VP3 were
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primarily found in the cytoplasm (Fig.5D&E). The results show that the IBDV VP3
colocalized with endogenous chRPL18 in the cytoplasm of IBDV-infected cells (Fig.
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5D-I), indicating the interaction of VP3 with chRPL18 in the cytoplasm of host cells.
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3.6. Knockdown of chRPL18 inhibits IBDV growth in DF-1 cells.
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To examine the role of chRPL18 in IBDV replication, we made three chRPL18 RNAi constructs and found that one could effectively lower the cellular level of chRPL18
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compared with that of siRNA negative controls (Fig. 6A&B). We then knocked down
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the expression of chRPL18 in DF-1 cells and examined the viral replication in these cells by measuring viral titers in the culture of IBDV-infected cells at different time
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points post infection. As a result, virus titres in cells with chRPL18 knockdown were one log less compared to control cells in both supernatant and cell cultures (Fig.
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6C&D). These results suggest that knockdown of chRPL18 inhibits viral propagation. 3.7. Knockdown of chRPL18 enhances type I interferon expression in IBDV infected cells As type I interferon plays a critical role in the host response against IBDV infection (Li et al., 2013; O'Neill et al., 2010; Ragland et al., 2002). We hypothesized that inhibition 14
of IBDV replication by knockdown of chRPL18 might be due to the promotion of type I interferon expression. To test this hypothesis, we examined the expressions of type I interferon, chIRF3 and NF-κB in these cells receiving chRPL18 siRNA or control siRNA after infection with IBDV. As a result, knockdown of chRPL18 by RNAi
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markedly enhanced type I interferon in IBDV infected DF-1 cells (Fig7A&B). The similar results were obtained from the examination of chIRF3 (Fig.7C), and NF- κB
3.8. VP3, chRPL18, and chPKR form a complex.
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expression in host cells in response to IBDV infection.
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(Fig.7D) in those cells. These data suggest that chRPL18 inhibit type I interferon
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The fact that RPL18 interacts with PKR in mammalian cells prompted us to investigate
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the possibility that chRPL18 might interact with chPKR in chicken cell lines (Kumar et al., 1999a). Thus, we constructed a plasmid that allows the expression of Myc-chPKR
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for the analysis of its interaction with chRPL18 in DF-1 cells. When lysates of cells
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expressing both EGFP-chRPL18 and Myc-chPKR were immunoprecipitated with Myc antibody, EGFP-chRPL18 was readily detectable in the precipitate, indicating that
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chRPL18 interacted with ectopically expressed chPKR in HEK293T cells (Fig. 8A). Since chRPL18 interact with VP3 and chPKR, we examined the possible interaction of
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VP3 with chPKR. Lysates of cells expressing Myc-chPKR and FLAG-VP3 were immunoprecipitated with FLAG antibody, and Myc-chPKR was detected in the precipitate, indicating that chPKR interacted with ectopically expressed VP3 in DF-1 cells (Fig. 8B). Since chRPL18 interacts with VP3 and chPKR, we expanded our investigation to determine whether chPKR, chRPL18, and VP3 could form a complex. 15
Lysates of HEK293T cells expressing Myc-chPKR, FLAG-VP3, and Myc-chRPL18 were immunoprecipitated with FLAG antibody, and both Myc-chPKR and MycchRPL18 were detected in the precipitate (Fig. 8C), indicating that ectopically expressed VP3, chPKR, and chRPL18 formed a complex.
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To further determine whether the IBDV VP3, chRPL18, and chPKR could form a
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complex under a physiological condition, we infected DF-1 cells with IBDV and performed a pull-down assay using anti-VP3 monoclonal antibody. As a result, both
chRPL18 and chPKR could be detected in the precipitate of the lysates from IBDV-
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infected cells but not from mock infected controls (Fig. 8D). These data clearly show
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that chRPL18, chPKR, and IBDV VP3 formed a complex in host cells.
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4. Discussion
IBDV infection causes severe damages in the bursa of Fabricius (Becht and Muller,
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1991)，which result in immunosuppression of IBDV-infected chickens (Zachar et al.,
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2016)，leading to susceptibility of the chickens to other diseases (Muller et al., 2003). Although IBDV VP3 plays a critical role in viral infection (Chevalier et al., 2004;
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Maraver et al., 2003; Ye et al., 2014), the exact role of VP3 in host cells is not very clear. In this study, we found that IBDV VP3-induced expressions of type I interferon. Furthermore, we found that VP3 interacted with chRPL18, and that knockdown of chRPL18 by RNAi suppressed IBDV replication and enhanced type I interferon
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expression, suggesting that the interaction of VP3 with chRPL18 induced the expressions of type I interferon and suppression of viral replication. The ribosomal proteins, acting as critical components of ribosome function, were mainly thought to participate in protein synthesis and translational regulation (Graifer
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et al., 2014; Jiang et al., 2015; Merrick, 1992). Mounting evidence suggest that
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ribosomal proteins are involved in interaction between host cells and viruses (Cheng et
al., 2011; Ganaie et al., 2014; Sharma et al., 2015). It was reported that efficient MMTV (mouse mammary tumor virus) particle assembly in the nucleoli of infected cells is
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dependent upon the interaction of Gag and ribosomal protein L9 (Beyer et al., 2013).
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In addition, ribosomal protein S6 was identified as an indispensable host factor for HCV
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propagation, and knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation (Huang et al., 2012). Besides, interaction of
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Hantavirus Nucleocapsid Protein with Ribosomal Protein S19 facilitates ribosome
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loading on capped mRNAs during N-mediated translation initiation (Haque and Mir, 2010). Our data show that IBDV VP3 specifically interacted with chRPL18 under all
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tested conditions, and knockdown of chRPL18 by RNAi significantly suppressed IBDV replication in host cells.
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RPL18 is a component of ribosomal 60S subunit, playing a remarkable role in ribosomal assembly (Stelter et al., 2015). Besides, RPL18 has functions outside of the ribosome (Cervantes-Salazar et al., 2015; Leh et al., 2000). It was reported that RPL18 participates in translational transactivation of cauliflower mosaic virus by interacting with P6 (Leh et al., 2000). A recent study shows that Dengue virus NS1 protein interacts 17
with the ribosomal protein RPL18 and this interaction is required during DENV replication cycle (Cervantes-Salazar et al., 2015). Our data show that knockdown of chRPL18 significantly inhibited IBDV growth, suggesting that chRPL18, by interacting with VP3, promoted IBDV replication Furthermore, knockdown of
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chRPL18 markedly enhanced IFN expression, which accounts for the inhibition of viral
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growth in chRPL18 knockdown cells.
PKR, also known as eukaryotic translation initiation factor 2-alpha kinase 2, is a 551 amino acid protein (Kuhen et al., 1996). It is established that PKR plays a major role in
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the antiviral immunity (Balachandran et al., 2000; Iordanov et al., 2001; Khabar et al.,
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2000). . Our data show that VP3, chPKR, and chRPL18 formed a complex. It was
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reported that ribosomes are inhibitory for PKR activity since they compete with dsRNA for binding to PKR, inhibit the activation of the protein kinase by dsRNA, and prevent
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the phosphorylation of the PKR substrate eIF2α (Raine et al., 1998). However, what
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prevents PKR activation while it bound to ribosomes remains elusive. It was found that RPL18 competed with dsRNA for binding to PKR and inhibited both PKR
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autophosphorylation and PKR-mediated phosphorylation of eIF-2a, and mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding
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and RPL18 interaction, suggesting the molecular mechanism that RPL18 inactivate PKR (Kumar et al., 1999b). It was reported that IBDV VP3 inhibited PKR phosphorylation and mediated programmed cell death (Busnadiego et al., 2012). Our data show that RPL18, VP3 and PKR formed complex and that knockdown of chRPL18 enhanced type I interferon expression and inhibited viral replication in cells. It was 18
possible that chRPL18 facilitated IBDV replication via antagonizing chPKR. More efforts will be required to elucidate the molecular mechanisms underlying the signal transduction initiated by VP3 via chRPL18, and chPKR. In summary, our results revealed that IBDV VP3 interacted with chRPL18 in host cells.
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Knockdown of chRPL18 significantly promoted Type I interferon expression and
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inhibited IBDV replication. Furthermore, our data show that chPKR interacted with
VP3 and chRPL18 respectively, suggesting that the formation of VP3, chRPL18 and chPKR complex affects viral replication and host response to IBDV infection. These
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underlying the pathogenesis of IBDV infection.
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findings have provided insights for further studies of the molecular mechanism
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Acknowledgements
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We thank Dr. Jue Liu for his kind assistance. This work was supported by grants from the National Natural Science Foundation of China (#31430085) and Earmarked Fund
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Figure Legends
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Fig.1. Overexpression of IBDV VP3 enhanced expressions of type I interferon, and
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inhibited IBDV replication. DF-1 cells were transfected with pRK5-FLAG-vp3 or control plasmids. Twenty-four hours after transfection, cells were harvested for
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quantifying the expressions of chIFN-α (A),chIFN-β (B), chIRF3 (C) , and NF-κB (D)
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by qRT-PCR analysis, or infected with IBDV at an MOI of 1. Twenty-four hours after infection, the viral titers in the cell cultures (E) or supernatants (F) were determined by
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TCID50 analysis using 96-well plates. (Data were normalized to normal cells). Results are representative of three independent experiments. Data were presented as mean±SD; ***, p<0.001; **, <0.01; and *, p<0.05.
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Fig.2. Screening and identification of IBDV VP3-interacting cellular proteins in DF-1 cell. (A) DF-1 cells transfected with pRK5-FLAG or pRK5-FLAG-vp3 were lysed and incubated with protein A/G-anti-FLAG McAb complex or protein A/G-antibody control complex for pull-down assays. The pull-down pellets were examined by 12%
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SDS-PAGE and subjected to coomassie blue R-250 staining. (B) Analysis of the arrowpointed protein bands in (A) by Mass Spectrometry. (C) Alignment of the chRPL18
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sequences (GenBank: AAS49582.1) with its matched peptides as shown in red
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identified by Mass Spectrometry analysis.
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Fig.3. Interaction of IBDV VP3 with chRPL18 in cells. (A&B) Interaction of VP3 with ectopically expressed RPL18. HEK293T cells (A) and DF-1 (B) were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated (IP) with anti-FLAG antibody and
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immunoblotted with anti-FLAG or anti-Myc antibodies. (C) Interaction of VP3 with endogenous chRPL18. DF-1 cells were transfected with pRK5-FLAG-vp3 or empty
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vector as controls. Twenty-four hours after transfection, cell lysates were prepared and
immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG or
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anti-chRPL18 antibodies. (D) The interaction of IBDV VP3 with endogenous chRPL18
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in IBDV-infection cells. DF-1 cells were infected with IBDV at an MOI of 1. Twenty-
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four hours after infection, cell lysates were prepared and immunoprecipiated with anti-
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VP3 antibody and immunoblotted with anti-VP3 or anti-chRPL18 antibodies.
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FIG 4. The portion of VP3 from amino acids 87 aa to 172 aa is responsible for binding to RPL18. (A) Schematics represent the genes encoding the full-length VP3, VP3 mutant ( △3 with K99D, R102D, K105D and K106D), and truncated VP3 molecules ( △1 and △2). The numbers indicate the amino acid positions in the molecule. (B)
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RPL18 interacted with different truncated VP3 proteins. HEK293T cells were transfected with Myc-RPL18 and different truncated GFP-VP3 molecules (△1 and △
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2) or empty vectors or VP3 mutant. Thirty-six hours after transfection, cell lysates were prepared and immunoprecipitated with anti-Myc monoclonal antibody. The pellets
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were examined by Western Blot using anti-GFP monoclonal antibody.
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Fig.5. Colocalization of VP3 with chRPL18 in DF-1 cells. DF-1 cells were mock infected or infected with IBDV at an MOI of 1. Twenty-four hours after infection, cells were fixed and probed with mouse anti-VP3 and rabbit anti-RPL18 antibodies, followed by the TRITC-conjugated goat anti-mouse antibody (red) and FITC-
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conjugated goat anti-rabbit antibody (green). Nuclei were counterstained with DAPI
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(blue). The cell samples were observed with a laser confocal scanning microscope.
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Fig.6. Knockdown of chRPL18 suppressed the replication of IBDV. (A and B) Effects of chRPL18 RNAi on the expression of endogenous chRPL18. (A) DF-1 cells were transfected with siRNA (RNAi#1 to RNA i#3) or controls as described in Materials and Methods. Twenty-four hours after the second transfection, cell lysates were prepared
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and examined by Western Blot with anti-RPL18 antibody (A). Endogenous β-actin expression was used as an internal control. The band density of chRPL18 in (A) was
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quantitated by densitometry, and the relative levels of chRPL18 were calculated as
follows: band density of chRPL18 / band density of β-actin (B). (C&D) DF-1 cells
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receiving chRPL18 RNAi, control RNAi or Mock transfection were infected with
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IBDV at an MOI of 1. At different time points (12, 24, 48 and 72h) after IBDV
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infection, the viral titers in the cell cultures (C) or supernatants (D) were determined by
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TCID50 analysis using 96-well plates. The significance of the difference between
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chRPL18 RNAi and control RNAi treatment was performed by ANOVA. The graph
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shows the average of viral titers in DF-1 cells from three individual experiments.
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Fig.7. Knockdown of chRPL18 enhanced expressions of chIFN-α, chIFN-β, chIRF3, and NF-κB in IBDV infected DF-1 cells. DF-1 cells were transfected with siRNA against chRPL18 or control siRNA. Twenty-four hours after the second siRNA transfection, cells were infected with IBDV at an MOI of 1. Twenty-four hours after
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IBDV infection, cells were harvested for quantifying the expressions of chIFN-α (A),chIFN-β (B), chIRF3 (C) , and NF-κB (D) by qRT-PCR analysis (Data were
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normalized to control RNAi). Results are representative of three independent
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experiments. Data were presented as mean±SD; ***, p<0.001; **, <0.01; and *, p<0.05.
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Fig.8. VP3, chRPL18, and chPKR formed a complex. (A and B) Interaction of MycchPKR with exogenous chRPL18 (A) or VP3 (B). (A) HEK293T cells (1×106) were transfected with 4μg of pCMV-Myc-chpkr and/or pEGFP-chrpl18 or empty vector plasmids. Twenty-four hours after transfection, cell lysates were prepared,
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immunoprecipitated (IP) with anti-Myc antibody and immunoblotted with anti-Myc or anti-GFP antibodies. (B) DF-1 cells (1×106) were transfected with 4μg of pCMV-Myc-
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chpkr and/or pRK5-FLAG-vp3 or empty vector plasmids. Twenty-four hours after
transfection, cell lysates were prepared, immunoprecipitated (IP) with anti-FLAG
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antibody, and immunoblotted with anti-Myc or anti-FLAG antibodies. (C) VP3,
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exogenous chRPL18 and chPKR form a complex. HEK293T cells (1×107) were
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transfected with 4μg of pRK5-FLAG-vp3, and/or pCMV-Myc-chpkr and pEGFP-
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chrpl18 or empty vector plasmids.. Twenty-four hours after transfection, cell lysates
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were prepared, immunoprecipiated with anti-FLAG antibody and immunoblotted with anti-Myc or anti-FLAG antibodies. (D) VP3, chRPL18, and chPKR form a complex in
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IBDV infected cells. DF-1 cells were infected with IBDV at an MOI of 1. Twenty-four
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hours post infection, cell lysates were prepared, immunoprecipiated with anti-VP3 antibody and immunoblotted with anti-VP3, anti-chRPL18, or anti-chPKR antibodies.
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(E) The illustration of complex formed by IBDV VP3, chRPL18 and chPKR.
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S0168-1702(17)30244-7 https://doi.org/10.1016/j.virusres.2017.12.009 VIRUS 97314
To appear in:
Virus Research
Received date: Revised date: Accepted date:
23-3-2017 14-12-2017 18-12-2017
Please cite this article as: Wang, Bin, Duan, Xueyan, Fu, Mengjiao, Liu, Yanan, Wang, Yongqiang, Li, Xiaoqi, Cao, Hong, Zheng, Shijun J., The Association of Ribosomal Protein L18 (RPL18) with Infectious Bursal Disease Virus Viral Protein VP3 Enhances Viral Replication.Virus Research https://doi.org/10.1016/j.virusres.2017.12.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The Association of Ribosomal Protein L18 (RPL18) with Infectious Bursal Disease Virus Viral Protein VP3 Enhances Viral Replication
Bin Wanga,b,c, Xueyan Duan a,b,c, Mengjiao Fua,b,c, Yanan Liua,b,c, Yongqiang Wanga,b,c,
a
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Xiaoqi Lia,b,c, Hong Caoa,b,c, and Shijun J. Zhenga,b,c* State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing
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100193, China
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, China
College of Veterinary Medicine, China Agricultural University, Beijing 100193,
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c
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Agricultural University, Beijing 100193, China
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*
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China
Corresponding author at: College of Veterinary Medicine, China Agricultural
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University, 2 Yuan-Ming-Yuan West Road, Beijing 100193, P.R.China Tel: 86-(10)-
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6273-4681. Fax: 86-(10)-6273-4681.
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Email: [email protected] Highlights
IBDV VP3 interacted with chRPL18 in host cells.
chPKR and chRPL18 could be pulled down by anti-VP3 mcAb in IBDV-infected cells
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Knockdown of chRPL18 by RNAi promoted Type I interferon expression
Knockdown of chRPL18 inhibited IBDV replication.
Abstract
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Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). IBDV VP3 is a multifunctional protein playing a key role in virus assembly and pathogenesis. To investigate the role of VP3 in pathogenesis, we transfected DF-1 cells with pRK5-FLAG-vp3 and found that VP3
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enhanced type I interferon expression and suppressed IBDV replication. Furthermore we found that VP3 interacted with chicken Ribosomal Protein L18 (chRPL18) in host
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cells and knockdown of chRPL18 by RNAi significantly promoted Type I interferon
expression and inhibited IBDV replication. Moreover, our data show that chicken
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double-stranded RNA-activated protein kinase (chPKR) interacted with both VP3 and
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chRPL18. Thus chRPL18 in association with VP3 and chPKR affects viral replication.
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Keywords: chRPL18; IBDV VP3; chPKR; IBDV replication 1. Introduction
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Infectious bursal disease virus (IBDV) causes an acute, highly contagious, and
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immunosuppressive disease in young chickens that causes great losses to the poultry industry across the world (Pitcovski et al., 2003). IBDV attacks the immune system,
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which causes severe damage of the B-lymphocyte precursors, resulting in a high mortality in the young chickens (Muller et al., 2003). The surviving chickens suffer
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from a severe immunosuppression, leading to an increased susceptibility to other pathogens (Stricker et al., 2010). IBDV, belonging to the genus Avibirnavirus of the family Birnaviridae, is a nonenveloped, double-stranded RNA virus with an icosahedral nucleocapsid (Tacken 2
et al., 2003). The virus genome consists of two segments of double-stranded RNA, segment A (3.2 kb) and B (2.8kb) (Azad et al., 1985; Tacken et al., 2004). Segment B, the short RNA, encodes VP1 (90kDa) (Morgan et al., 1988), an RNA-dependent RNA polymerase (Shwed et al., 2002; Tacken et al., 2004). Segment A, the large one,
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contains two partially overlapping open reading frames (ORFs) (Kibenge et al., 1991). The small ORF encodes the nonstructural viral protein VP5 (17kDa), involved in
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apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2) in IBDVinfected cells (Li et al., 2012). The large one encodes an approximate 110 kDa
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polyprotein precursor that can be self-cleaved by viral protease VP4 to form viral
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proteins pVP2 (41 kDa), VP3 (32 kDa) and VP4 (24 kDa) (Azad et al., 1985; Hudson
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et al., 1986; Jagadish et al., 1988; Kibenge et al., 1991). pVP2 is further processed into
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VP2 and several small peptides from the C terminal by VP4 (Sanchez and Rodriguez,
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1999), the puromycin aminopeptidase (Irigoyen et al., 2012) and autoproteolytically between residues Ala441-Phe442 (Irigoyen et al., 2009). Apart from acting as a viral
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protease, VP4 is also considered to be an interferon suppressor by interacting with
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GILZ in host cells (Li et al., 2013). VP2 and VP3 are the major structural proteins, constituting 51% and 40% of the mature viral particle, respectively (Dobos et al., 1979). VP3, acting as a scaffold protein (Maraver et al., 2003), recruits genome dsRNA and
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VP1 to form a ribonucleoprotein (RNP) complex and serves as a transcriptional activator (Casanas et al., 2008; Lombardo et al., 1999a), playing a critical role in virus assembly (Lombardo et al., 1999b; Maraver et al., 2003). It was reported that VP3 shows a higher affinity than MDA5 at binding IBDV genomic dsRNA to evade innate 3
immunity (Ye et al., 2014). Besides, VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing PKR-mediated apoptosis (Busnadiego et al., 2012). Although IBDV VP3 plays a critical role in viral assembly, the exact role of VP3 in the
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pathogenesis of IBDV infection is not clear. In this study, we found that VP3 enhanced
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type I interferon expression and suppressed IBDV replication. Furthermore，we found that VP3 interact with chRPL18, and knockdown of chRPL18 markedly suppressed
IBDV replication by enhancing type I interferon expression. Moreover, our data show
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that VP3, chRPL18 and chPKR form a complex affecting viral replication.
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2. Materials and Methods
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2.1. Cells and virus.
Both HEK-293T cells and DF-1 (immortal chicken embryo fibroblast) cells were
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obtained from the ATCC. All cells were cultured in Dulbecco modified Eagle medium
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(DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 incubator. Lx, a cell culture-adapted IBDV strain, was kindly provided by Dr. Jue
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Liu (Beijing Academy of Agriculture and Forestry Science, Beijing, China).
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2.2. Reagents.
Endotoxin-free plasmid preparation kits were purchased from ZYMO RESEARCH (USA). All restriction enzymes were purchased from Takara (Japan). RNase A was purchased from Aidlab (Beijing, China). Anti-c-Myc (sc-40), anti-green fluorescent protein (anti-GFP; sc-9996), and anti-β-actin (sc-1616-R) antibodies were obtained 4
from Santa Cruz Biotechnology (USA). Poly(I:C), Anti-RPL18 polyclonal antibody (SAB1100328) and anti-FLAG monoclonal antibody (F1804) were purchased from Sigma (USA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, tetramethyl rhodamine isocyanate (TRITC)-conjugated goat anti-rabbit IgG, and
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horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG antibodies were purchased from Ding-Guo (China). Anti-VP3 monoclonal antibody
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(EACU-c10) and anti-chPKR polyclonal antibody (EACU-2015) were purchased from
EACU (Beijing, China). jetPRIME® transfection reagent was purchased from Polyplus-
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transfection SA (France). OPTI-MEM I, RNAiMAX were purchased from Invitrogen
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(USA). 4’, 6-Diamino-2-phenylindole (DAPI) was purchased from Beytime (China).
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Coomassie brilliant blue R-250 was purchased from Abgene (USA). Fast mutagenesis
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2.3. Constructs.
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system was purchased from Transgene (Beijing, China).
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IBDV vp3 gene was cloned from IBDV strain Lx using the following specific primers: sense primer 5’-CGT TTC CCT CAC AAT CCA CGC GA-3’ and antisense primer 5’-
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CTC AAG GTC CTC ATC AGA GAC GGT-3’ (GenBank accession no. 126032566). Chicken rpl18 gene was cloned from DF-1 cells using the specific primers 5’- CGG
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CGC CGG GAG CCC AAAA-3’ (sense) and 5’-CCC CCG CGC CCG CTC GAA CTT-3’ (antisense) according to the sequence in GenBank (accession no. AY389963.1). Chicken pkr gene was cloned from DF-1 cells using the specific primers 5’- ATG GAC CGA GAG TGC ATG GC-3’ (sense) and 5’-TTA GTG ACT GTA AGC TTT ATG CGA G-3’ (antisense) with reference to the sequence in GenBank (accession 5
no.NM_204487). vp3 mutant containing four amino acid substitutions (K99D, R102D,K105D and K106D) was constructed using Fast mutagenesis system kit per the manufacturer’s instructions, using the following three pairs specific primers 5’- GAA GCA CAG AGG GAA GAC GACACA CGGA-3’ (K99D, sense) and 5’-GTC TTC
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CCT CTG TGC TTC CTC TGG TGTG-3’ (K99D, antisense), and 5’- GAA GCA CAG AGG GAA GAC GAC ACA GAC ATC TCA GACG-3’ (R102D,sense) and 5’-GTC
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TGT GTC GTC TT CCC TCT GTG CTTC CTC TGG TGTG-3’ (R102D, antisense), and 5’- GAC ACA CGG ATC TCA GAC GAC ATG GAG ACCA-3’ (K105D and
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K106D, sense) and 5’-GTC GTC TGA GAT CCG TGT GTC TTT TTC CCTC-3’
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(K105D and K106D, antisense). pCMV-Myc, pRK5-FLAG, pEGFP-N1 and pDsRed-
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monomer-N1 vectors were obtained from Clontech (USA). All the primers were
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2.4. Pull-down assays.
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synthesized by Sangon Company (China).
DF-1 cells were seeded on 100 mm cell culture dishes and cultured for 24 h before
Thirty-six hours after transfection, cell lysates were prepared using a
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reagent.
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transfection with pRK5-FLAG-vp3 or pRK5-FLAG using jetPRIME® transfection
nondenaturing lysis buffer (50nM Tris-HCl, pH 8.0; 150 nM NaCl; 1% TritonX-100,
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5nM EDTA, 10% glycerol, 10nM dithiothreitol, 1×complete cocktail protease inhibitor). The cell lysates were collected and centrifuged at 6000 rpm for 30 min, and the supernatants were incubated with 10μg of anti-FLAG antibody and 50 μl of a 50% slurry of protein A/G plus agarose at 4°C for 6 h. Beads were washed six times with the lysis buffer and boiled with 2×SDS loading buffer for 10 min. Samples were subjected 6
to 12% SDS-PAGE gel electrophoresis and stained with coomassie brilliant blue R250. After separation of proteins on SDS-PAGE gel, bands of interest were sliced and analyzed by MS. 2.5. Coimmunoprecipitation and Western Blot analysis.
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For immunoprecipitation, HEK-293T cells or DF-1 cells (4 ×105) were seeded on sixwell plates and cultured for 24 h before being cotransfected with pCMVMyc-chrpl18
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and pRK5-FLAG-vp3 or empty vectors as controls using jetPRIME® transfection reagent. Twenty-four hours after transfection, cell lysates were prepared using a
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nondenaturing lysis buffer (50mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM
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EDTA, 10% glycerol, 10mM dithiothreitol, 1×complete cocktail protease inhibitor).
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The cell lysates were incubated with 1μg of anti-FLAG antibody, 20μl of a 50% slurry of protein A/G plus agarose, and RNase A (10µg/ml) at 4°C overnight. Efficiency of
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RNase A treatment was detected by loading of 20 µl of protein samples incubated with
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or without RNase A on 1% agarose gel stained by ethidium bromide. Beads were washed three times with the lysis buffer and boiled with 2×SDS loading buffer for 10
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min. The samples were subjected to 12% SDS-PAGE gels electrophoresis, and resolved proteins were transferred onto polyvinylidene difluoride（PVDF）membranes. After
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blocking with 5% skimmed milk, the membranes were incubated with either anti-Myc or anti-FLAG antibodies, followed by an appropriate horseradish peroxidaseconjugated secondary antibody. Blots were developed using an enhanced chemiluminescence (ECL) kit. For endogenous chRPL18 or chPKR pull-down assay, HEK293T cells or DF-1 cells were infected with IBDV at an MOI of 1. Twenty-four 7
hours after infection, cell lysates were prepared using a nondenaturing lysis buffer. The cell lysates were subjected to immunoprecipitation with anti-VP3 antibody and immunoblotted with anti-RPL18, anti-VP3 or anti-chPKR antibodies. 2.6. Laser Confocal microscopy assays.
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DF-1 cells (1×105) were seeded on coverslips in 24-well plates and were cultured
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overnight before infection with IBDV. Twenty-four hours after infection, IBDV- or mock-infected cells were fixed with 1% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 15 min, blocked with 1% bovine serum albumin, and then
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probed with mouse anti-IBDV VP3 and/or rabbit anti-RPL18 antibodies, followed by
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FITC-conjugated goat anti-mouse IgG (green) or TRITC-conjugated goat anti-rabbit
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IgG (red) antibodies. After three washes with PBS, the cells were stained with DAPI
Nikon, Japan).
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for nuclei. The samples were observed with a laser confocal microscope (OLYMPUS;
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2.7. Knockdown of chRPL18 by RNA interference (RNAi). The siRNAs were designed by Genepharma Company (Shanghai, China) and used to
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knock down chRPL18 expressions in DF-1 cells. The siRNAs for targeting chRPL18 in DF-1 cells were as follows: RNAi#1 (sense, 5’- UCG UCA AGC UGU ACC GUU
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UTT-3’; antisense, 5’-AAA CGG UAC AGC UUG ACG ATT-3’), RNAi#2 (sense, 5’UCU UCA UGA GCC GAA CCA ATT -3’; antisense, 5’-UUG GUU CGG CUC AUG AAG ATT-3’), RNAi#3 (sense, 5’- CCU UCG ACC AAU UGG CCA UTT-3’; antisense, 5’- AUG GCC AAU UGG UCG AAG GTT-3’), and negative controls (sense, 5’-UUC UCC GAA CGU GUC ACG UTT-3’; antisense, 5’-ACG UGA CAC 8
GUU CGG AGA ATT-3’). To transfect cells with the interference RNAs against chRPL18, we seeded DF-1 cells (2×105) cells on 12-well plates and cultured them for at least 20 h prior to transfection. The cells were transfected with siRNA using RNAiMAX according to the manufacturer’s instructions (Invitrogen). Double
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transfections were performed at a 24h interval. Twenty-four hours after the second
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transfection, cells were harvested for further analysis. 2.8. Measurement of IBDV growth in DF-1 cells.
DF-1 cells receiving chRPL18-specific siRNAs or control siRNA were infected with
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IBDV at an MOI of 1, and the cell cultures (including cell and supernatant) or
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supernatants of cell cultures were collected at different time points (12, 24, 48, 72 h)
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after infection, respectively. The culture samples were freeze-thawed three times and centrifuged at 2,000×g for 10 min. The viral contents in the cellular lysates and
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supernatants of cell cultures were titrated using 50% tissue culture infective doses (TCID50) in DF-1 cells, respectively. Briefly, the viral solution was diluted by 10-fold
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in DMEM. A 100μl aliquot of each diluted sample was added to the wells of 96-well
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plates, followed by addition with 100μl of DF-1 cells at a density of 5×105 cells/ml. Cells were cultured for 5-7 days at 37°C in 5% CO2. Tissue culture wells with a
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cytopathic effect (CPE) were determined to be positive. The titer was calculated on the basis of a previously described method (Reed LJ, 1938). 2.9. RNA extraction and quantitative real-time PCR (qRT-PCR) analysis. Total RNA was prepared from DF-1 cells using EASYspin Plus kit (aidlab Biotechnology, China) following the manufacturer’s instructions. 0.5μg of total RNA 9
was used for cDNA synthesis by reverse transcription using an RT-PCR kit (TaKaRa). Quantitative RT-PCR analysis was performed using Tli RnaseH Plus (Takara) on LightCycler 480II (Roche, USA). Specific primers for chicken IFN-α (chIFN-α) (5’CCA GCA CCT CGA GCA AT-3’ and 5’-GGC GCT GTA ATC GTT GTC T-3’),
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chicken IFN-β (chIFN-β) (5’-GCC TCC AGC TCC TTC AGA ATA CG-3’and 5’-CTG GAT CTG GTT GAG GAG GCT GT-3’), chicken IRF3 (chIF3) (5’-GCT CTC TGA
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CTC TTT CAA CCT CTT-3’ and 5’-AAT GCT GCT CTT TTC TCC TCT G-3’),
chicken p65(chp65) (5′-CCA CAA CAC AAT GCG CTC TG-3′ and 5′- AAC TCA
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GCG GCG TCG ATG-3′), and chicken GAPDH (5’-TGC CAT CAC AGC CAC ACA
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GAA G-3’ and 5’-ACT TTC CCC ACA GCC TTA GCA G-3’) were designed with
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reference to previous publications (Abdul-Careem et al., 2008; Li et al., 2007; Liu et
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al., 2010). GAPDH gene was utilized as the reference gene. All quantitative real-time
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PCR experiments were performed in triplicate. The qRT-PCR was performed in a 20μl volume containing 1μl of cDNA, 10μl of 2×SYBR green Premix Ex Taq (TaKaRa),
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and a 0.4μM of each gene-specific primers. Thermal cycling parameters were as
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follows: 94°C for 2 min; 40 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s; and 1 cycle of 95°C for 30 s, 60°C for 30 s, and 95°C for 30 s. The final step was to obtain a melt curve for the PCR products to determine the specificity of the
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amplification.
2.10. Statistical analysis. The significance of the differences between chRPL18 RNAi cells and controls in gene expressions and viral growth, and between pCMV-Myc-chpkr-transfected cells and 10
controls in viral growth was determined by the Mann-Whitney test or analysis of variance (ANOVA) accordingly. 3. RESULTS 3.1. IBDV VP3 enhanced expressions of type I interferon, and inhibited IBDV
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replication.
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As a major structural protein, IBDV VP3 plays a key role in virus assembly and pathogenesis (Ye et al., 2014; Maraver et al., 2003). To examine whether VP3 affects
type I interferon expression, we transfected DF-1 cells with pRK5-FLAG-vp3 plasmid
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or control plasmid. We found that transient expression of VP3-FLAG significantly
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enhanced expression of type I interferon, chIRF3, and NF-κB (Fig1A-D). As type I
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interferon plays a critical role in the host response against IBDV infection (Ragland et al., 2002; Li et al., 2013; O'Neill et al., 2010), we infected pRK5-FLAG-vp3 transfected
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DF-1 cells with IBDV, and examined the viral growth in pRK5-FLAG-vp3 transfected
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DF-1 cells in 24h after IBDV infection. As shown in Fig.1E&F, expression of VP3 inhibited IBDV replication in cell cultures and the supernatants compared to that of
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controls. Taken together, these results suggest that IBDV VP3 enhanced expressions of type I interferon and inhibited IBDV replication.
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3.2. Screening and identification of IBDV VP3-interacting cellular proteins in DF1 cells. Pathogen-host interaction is the basis for the pathogenesis of IBDV infection (Li et al., 2012; Li et al., 2013; Qin and Zheng, 2017; Lin et al., 2015). To investigate the host cellular proteins that might be targeted by IBDV VP3, we transfected DF-1 cells with 11
pRK5-FLAG-vp3 and performed a pull-down assay using anti-FLAG McAb. As a result, nine extra protein bands were observed in the FLAG-VP3 lane on SDS-PAGE compared to that of FLAG controls (Fig.2A), indicating that IBDV VP3 interacted with some cellular proteins in DF-1 cells. To determine the amino acid sequences of VP3-
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interacting proteins, the arrow-pointed protein bands in Fig. 2A were cut-down and subjected to Mass Spectrometry analysis. As shown in Fig.2B, among nine proteins
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potentially interacting with VP3, chRPL18 might be relevant to IBDV replication in host cells because RPL18 was required during DENV replication cycle, facilitated
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translational transactivation of CaMV, and prevented PKR activation by dsRNA
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(Cervantes-Salazar et al., 2015; Leh et al., 2000; Kumar et al., 1999b). Thus, we
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selected chRPL18 for further investigation. The sequence of RPL18 protein identified
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in band F by Mass Spectrometry analysis is shown in red (Fig.2C).
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3.3. VP3 interacts with chRPL18.
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To verify the interaction of chRPL18 with VP3, we constructed a plasmid that allows the expression of Myc-chRPL18 for analyzing its interaction with VP3 in HEK293T
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cells by immunoprecipitation assays. Since both chRPL18 and VP3 possess RNAbinding domains (Casanas et al., 2008; Woestenenk et al., 2002), RNase A was added
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into cell lysates before immunoprecipitation assays. When the lysates of cells expressing both FLAG-VP3 and Myc-chRPL18 were immunoprecipitated with antiFLAG antibody, Myc-chRPL18 was detected in the precipitate, indicating that VP3 interacted with ectopically expressed chRPL18 in mammalian cells (Fig. 3A). Similar results were obtained in an experiment using the DF-1 cells (Fig. 3B), indicating that 12
the interaction observed between these two proteins is not cell type specific. To further confirm the interaction, we transfected DF-1 cells with pRK5-FLAG-vp3 plasmids and performed an immunoprecipitation assay with anti-FLAG McAb. The binding of FLAG-VP3 with endogenous chRPL18 was readily detectable in cells expressing the
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viral protein VP3 (Fig. 3C). In addition, the interaction of endogenous chRPL18 with viral VP3 was also confirmed by pull-down assay in IBDV-infected DF-1 cells (Fig.
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3D) .These results clearly demonstrate that VP3 interacts with chRPL18 in host cells.
3.4. A domain that spans residues 87 to 172 of VP3 is involved in interacting with
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chRPL18.
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It was reported that VP3 prevents VP2-induced protein synthesis arrest and PCD
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dependent upon the presence of function Patch1 dsRNA-bing domain, and the substitution of positively charged K and R resides for negatively charged D residues
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(K99D, R102D, K105D, and K106D) within the Patch1 region results in loss of ability
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to binding dsRNA (Busnadiego et al., 2012). To determine the region of VP3 responsible for interacting with RPL18 and examine whether the interaction of VP3
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with RPL18 was related to its ability of binding dsRNA, we constructed VP3 mutant with replacement of residues (K99D, R102D, K105D, and K106D) and truncated VP3
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fused with GFP (Fig. 4A). These VP3 derivatives were individually expressed in HEK293T cells, and their ability to interact with RPL18 was examined by immunoprecipitation (Fig. 4B). Our results indicated that RPL18 interacted with WT, △1, △2 and VP3 mutant, indicating that the interaction of VP3 with RPL18 is not dependent on the presence of dsRNA and that the portion of VP3 from amino acids 87 13
aa to 172 aa is responsible for binding to RPL18. 3.5. VP3 colocalized with chRPL18 in IBDV infected cells. To determine the subcelluar localization of IBDV VP3 and chRPL18, we performed a confocal microscopy assay, in which we infected DF-1 cells with IBDV and examined
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the interaction of VP3 with endogenous chRPL18 using mouse anti-IBDV VP3 and/or rabbit anti-RPL18 antibodies. Under this condition, both chRPL18 and VP3 were
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primarily found in the cytoplasm (Fig.5D&E). The results show that the IBDV VP3
colocalized with endogenous chRPL18 in the cytoplasm of IBDV-infected cells (Fig.
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5D-I), indicating the interaction of VP3 with chRPL18 in the cytoplasm of host cells.
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3.6. Knockdown of chRPL18 inhibits IBDV growth in DF-1 cells.
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To examine the role of chRPL18 in IBDV replication, we made three chRPL18 RNAi constructs and found that one could effectively lower the cellular level of chRPL18
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compared with that of siRNA negative controls (Fig. 6A&B). We then knocked down
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the expression of chRPL18 in DF-1 cells and examined the viral replication in these cells by measuring viral titers in the culture of IBDV-infected cells at different time
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points post infection. As a result, virus titres in cells with chRPL18 knockdown were one log less compared to control cells in both supernatant and cell cultures (Fig.
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6C&D). These results suggest that knockdown of chRPL18 inhibits viral propagation. 3.7. Knockdown of chRPL18 enhances type I interferon expression in IBDV infected cells As type I interferon plays a critical role in the host response against IBDV infection (Li et al., 2013; O'Neill et al., 2010; Ragland et al., 2002). We hypothesized that inhibition 14
of IBDV replication by knockdown of chRPL18 might be due to the promotion of type I interferon expression. To test this hypothesis, we examined the expressions of type I interferon, chIRF3 and NF-κB in these cells receiving chRPL18 siRNA or control siRNA after infection with IBDV. As a result, knockdown of chRPL18 by RNAi
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markedly enhanced type I interferon in IBDV infected DF-1 cells (Fig7A&B). The similar results were obtained from the examination of chIRF3 (Fig.7C), and NF- κB
3.8. VP3, chRPL18, and chPKR form a complex.
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expression in host cells in response to IBDV infection.
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(Fig.7D) in those cells. These data suggest that chRPL18 inhibit type I interferon
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The fact that RPL18 interacts with PKR in mammalian cells prompted us to investigate
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the possibility that chRPL18 might interact with chPKR in chicken cell lines (Kumar et al., 1999a). Thus, we constructed a plasmid that allows the expression of Myc-chPKR
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for the analysis of its interaction with chRPL18 in DF-1 cells. When lysates of cells
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expressing both EGFP-chRPL18 and Myc-chPKR were immunoprecipitated with Myc antibody, EGFP-chRPL18 was readily detectable in the precipitate, indicating that
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chRPL18 interacted with ectopically expressed chPKR in HEK293T cells (Fig. 8A). Since chRPL18 interact with VP3 and chPKR, we examined the possible interaction of
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VP3 with chPKR. Lysates of cells expressing Myc-chPKR and FLAG-VP3 were immunoprecipitated with FLAG antibody, and Myc-chPKR was detected in the precipitate, indicating that chPKR interacted with ectopically expressed VP3 in DF-1 cells (Fig. 8B). Since chRPL18 interacts with VP3 and chPKR, we expanded our investigation to determine whether chPKR, chRPL18, and VP3 could form a complex. 15
Lysates of HEK293T cells expressing Myc-chPKR, FLAG-VP3, and Myc-chRPL18 were immunoprecipitated with FLAG antibody, and both Myc-chPKR and MycchRPL18 were detected in the precipitate (Fig. 8C), indicating that ectopically expressed VP3, chPKR, and chRPL18 formed a complex.
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To further determine whether the IBDV VP3, chRPL18, and chPKR could form a
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complex under a physiological condition, we infected DF-1 cells with IBDV and performed a pull-down assay using anti-VP3 monoclonal antibody. As a result, both
chRPL18 and chPKR could be detected in the precipitate of the lysates from IBDV-
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infected cells but not from mock infected controls (Fig. 8D). These data clearly show
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that chRPL18, chPKR, and IBDV VP3 formed a complex in host cells.
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4. Discussion
IBDV infection causes severe damages in the bursa of Fabricius (Becht and Muller,
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1991)，which result in immunosuppression of IBDV-infected chickens (Zachar et al.,
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2016)，leading to susceptibility of the chickens to other diseases (Muller et al., 2003). Although IBDV VP3 plays a critical role in viral infection (Chevalier et al., 2004;
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Maraver et al., 2003; Ye et al., 2014), the exact role of VP3 in host cells is not very clear. In this study, we found that IBDV VP3-induced expressions of type I interferon. Furthermore, we found that VP3 interacted with chRPL18, and that knockdown of chRPL18 by RNAi suppressed IBDV replication and enhanced type I interferon
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expression, suggesting that the interaction of VP3 with chRPL18 induced the expressions of type I interferon and suppression of viral replication. The ribosomal proteins, acting as critical components of ribosome function, were mainly thought to participate in protein synthesis and translational regulation (Graifer
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et al., 2014; Jiang et al., 2015; Merrick, 1992). Mounting evidence suggest that
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ribosomal proteins are involved in interaction between host cells and viruses (Cheng et
al., 2011; Ganaie et al., 2014; Sharma et al., 2015). It was reported that efficient MMTV (mouse mammary tumor virus) particle assembly in the nucleoli of infected cells is
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dependent upon the interaction of Gag and ribosomal protein L9 (Beyer et al., 2013).
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In addition, ribosomal protein S6 was identified as an indispensable host factor for HCV
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propagation, and knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation (Huang et al., 2012). Besides, interaction of
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Hantavirus Nucleocapsid Protein with Ribosomal Protein S19 facilitates ribosome
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loading on capped mRNAs during N-mediated translation initiation (Haque and Mir, 2010). Our data show that IBDV VP3 specifically interacted with chRPL18 under all
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tested conditions, and knockdown of chRPL18 by RNAi significantly suppressed IBDV replication in host cells.
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RPL18 is a component of ribosomal 60S subunit, playing a remarkable role in ribosomal assembly (Stelter et al., 2015). Besides, RPL18 has functions outside of the ribosome (Cervantes-Salazar et al., 2015; Leh et al., 2000). It was reported that RPL18 participates in translational transactivation of cauliflower mosaic virus by interacting with P6 (Leh et al., 2000). A recent study shows that Dengue virus NS1 protein interacts 17
with the ribosomal protein RPL18 and this interaction is required during DENV replication cycle (Cervantes-Salazar et al., 2015). Our data show that knockdown of chRPL18 significantly inhibited IBDV growth, suggesting that chRPL18, by interacting with VP3, promoted IBDV replication Furthermore, knockdown of
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chRPL18 markedly enhanced IFN expression, which accounts for the inhibition of viral
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growth in chRPL18 knockdown cells.
PKR, also known as eukaryotic translation initiation factor 2-alpha kinase 2, is a 551 amino acid protein (Kuhen et al., 1996). It is established that PKR plays a major role in
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the antiviral immunity (Balachandran et al., 2000; Iordanov et al., 2001; Khabar et al.,
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2000). . Our data show that VP3, chPKR, and chRPL18 formed a complex. It was
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reported that ribosomes are inhibitory for PKR activity since they compete with dsRNA for binding to PKR, inhibit the activation of the protein kinase by dsRNA, and prevent
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the phosphorylation of the PKR substrate eIF2α (Raine et al., 1998). However, what
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prevents PKR activation while it bound to ribosomes remains elusive. It was found that RPL18 competed with dsRNA for binding to PKR and inhibited both PKR
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autophosphorylation and PKR-mediated phosphorylation of eIF-2a, and mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding
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and RPL18 interaction, suggesting the molecular mechanism that RPL18 inactivate PKR (Kumar et al., 1999b). It was reported that IBDV VP3 inhibited PKR phosphorylation and mediated programmed cell death (Busnadiego et al., 2012). Our data show that RPL18, VP3 and PKR formed complex and that knockdown of chRPL18 enhanced type I interferon expression and inhibited viral replication in cells. It was 18
possible that chRPL18 facilitated IBDV replication via antagonizing chPKR. More efforts will be required to elucidate the molecular mechanisms underlying the signal transduction initiated by VP3 via chRPL18, and chPKR. In summary, our results revealed that IBDV VP3 interacted with chRPL18 in host cells.
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Knockdown of chRPL18 significantly promoted Type I interferon expression and
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inhibited IBDV replication. Furthermore, our data show that chPKR interacted with
VP3 and chRPL18 respectively, suggesting that the formation of VP3, chRPL18 and chPKR complex affects viral replication and host response to IBDV infection. These
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underlying the pathogenesis of IBDV infection.
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findings have provided insights for further studies of the molecular mechanism
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Acknowledgements
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We thank Dr. Jue Liu for his kind assistance. This work was supported by grants from the National Natural Science Foundation of China (#31430085) and Earmarked Fund
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Figure Legends
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Fig.1. Overexpression of IBDV VP3 enhanced expressions of type I interferon, and
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inhibited IBDV replication. DF-1 cells were transfected with pRK5-FLAG-vp3 or control plasmids. Twenty-four hours after transfection, cells were harvested for
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quantifying the expressions of chIFN-α (A),chIFN-β (B), chIRF3 (C) , and NF-κB (D)
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by qRT-PCR analysis, or infected with IBDV at an MOI of 1. Twenty-four hours after infection, the viral titers in the cell cultures (E) or supernatants (F) were determined by
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TCID50 analysis using 96-well plates. (Data were normalized to normal cells). Results are representative of three independent experiments. Data were presented as mean±SD; ***, p<0.001; **, <0.01; and *, p<0.05.
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Fig.2. Screening and identification of IBDV VP3-interacting cellular proteins in DF-1 cell. (A) DF-1 cells transfected with pRK5-FLAG or pRK5-FLAG-vp3 were lysed and incubated with protein A/G-anti-FLAG McAb complex or protein A/G-antibody control complex for pull-down assays. The pull-down pellets were examined by 12%
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SDS-PAGE and subjected to coomassie blue R-250 staining. (B) Analysis of the arrowpointed protein bands in (A) by Mass Spectrometry. (C) Alignment of the chRPL18
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sequences (GenBank: AAS49582.1) with its matched peptides as shown in red
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identified by Mass Spectrometry analysis.
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Fig.3. Interaction of IBDV VP3 with chRPL18 in cells. (A&B) Interaction of VP3 with ectopically expressed RPL18. HEK293T cells (A) and DF-1 (B) were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated (IP) with anti-FLAG antibody and
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immunoblotted with anti-FLAG or anti-Myc antibodies. (C) Interaction of VP3 with endogenous chRPL18. DF-1 cells were transfected with pRK5-FLAG-vp3 or empty
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vector as controls. Twenty-four hours after transfection, cell lysates were prepared and
immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG or
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anti-chRPL18 antibodies. (D) The interaction of IBDV VP3 with endogenous chRPL18
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in IBDV-infection cells. DF-1 cells were infected with IBDV at an MOI of 1. Twenty-
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four hours after infection, cell lysates were prepared and immunoprecipiated with anti-
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VP3 antibody and immunoblotted with anti-VP3 or anti-chRPL18 antibodies.
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FIG 4. The portion of VP3 from amino acids 87 aa to 172 aa is responsible for binding to RPL18. (A) Schematics represent the genes encoding the full-length VP3, VP3 mutant ( △3 with K99D, R102D, K105D and K106D), and truncated VP3 molecules ( △1 and △2). The numbers indicate the amino acid positions in the molecule. (B)
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RPL18 interacted with different truncated VP3 proteins. HEK293T cells were transfected with Myc-RPL18 and different truncated GFP-VP3 molecules (△1 and △
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2) or empty vectors or VP3 mutant. Thirty-six hours after transfection, cell lysates were prepared and immunoprecipitated with anti-Myc monoclonal antibody. The pellets
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were examined by Western Blot using anti-GFP monoclonal antibody.
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Fig.5. Colocalization of VP3 with chRPL18 in DF-1 cells. DF-1 cells were mock infected or infected with IBDV at an MOI of 1. Twenty-four hours after infection, cells were fixed and probed with mouse anti-VP3 and rabbit anti-RPL18 antibodies, followed by the TRITC-conjugated goat anti-mouse antibody (red) and FITC-
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conjugated goat anti-rabbit antibody (green). Nuclei were counterstained with DAPI
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(blue). The cell samples were observed with a laser confocal scanning microscope.
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Fig.6. Knockdown of chRPL18 suppressed the replication of IBDV. (A and B) Effects of chRPL18 RNAi on the expression of endogenous chRPL18. (A) DF-1 cells were transfected with siRNA (RNAi#1 to RNA i#3) or controls as described in Materials and Methods. Twenty-four hours after the second transfection, cell lysates were prepared
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and examined by Western Blot with anti-RPL18 antibody (A). Endogenous β-actin expression was used as an internal control. The band density of chRPL18 in (A) was
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quantitated by densitometry, and the relative levels of chRPL18 were calculated as
follows: band density of chRPL18 / band density of β-actin (B). (C&D) DF-1 cells
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receiving chRPL18 RNAi, control RNAi or Mock transfection were infected with
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IBDV at an MOI of 1. At different time points (12, 24, 48 and 72h) after IBDV
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infection, the viral titers in the cell cultures (C) or supernatants (D) were determined by
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TCID50 analysis using 96-well plates. The significance of the difference between
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chRPL18 RNAi and control RNAi treatment was performed by ANOVA. The graph
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shows the average of viral titers in DF-1 cells from three individual experiments.
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Fig.7. Knockdown of chRPL18 enhanced expressions of chIFN-α, chIFN-β, chIRF3, and NF-κB in IBDV infected DF-1 cells. DF-1 cells were transfected with siRNA against chRPL18 or control siRNA. Twenty-four hours after the second siRNA transfection, cells were infected with IBDV at an MOI of 1. Twenty-four hours after
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IBDV infection, cells were harvested for quantifying the expressions of chIFN-α (A),chIFN-β (B), chIRF3 (C) , and NF-κB (D) by qRT-PCR analysis (Data were
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normalized to control RNAi). Results are representative of three independent
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experiments. Data were presented as mean±SD; ***, p<0.001; **, <0.01; and *, p<0.05.
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Fig.8. VP3, chRPL18, and chPKR formed a complex. (A and B) Interaction of MycchPKR with exogenous chRPL18 (A) or VP3 (B). (A) HEK293T cells (1×106) were transfected with 4μg of pCMV-Myc-chpkr and/or pEGFP-chrpl18 or empty vector plasmids. Twenty-four hours after transfection, cell lysates were prepared,
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immunoprecipitated (IP) with anti-Myc antibody and immunoblotted with anti-Myc or anti-GFP antibodies. (B) DF-1 cells (1×106) were transfected with 4μg of pCMV-Myc-
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chpkr and/or pRK5-FLAG-vp3 or empty vector plasmids. Twenty-four hours after
transfection, cell lysates were prepared, immunoprecipitated (IP) with anti-FLAG
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antibody, and immunoblotted with anti-Myc or anti-FLAG antibodies. (C) VP3,
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exogenous chRPL18 and chPKR form a complex. HEK293T cells (1×107) were
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transfected with 4μg of pRK5-FLAG-vp3, and/or pCMV-Myc-chpkr and pEGFP-
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chrpl18 or empty vector plasmids.. Twenty-four hours after transfection, cell lysates
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were prepared, immunoprecipiated with anti-FLAG antibody and immunoblotted with anti-Myc or anti-FLAG antibodies. (D) VP3, chRPL18, and chPKR form a complex in
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IBDV infected cells. DF-1 cells were infected with IBDV at an MOI of 1. Twenty-four
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hours post infection, cell lysates were prepared, immunoprecipiated with anti-VP3 antibody and immunoblotted with anti-VP3, anti-chRPL18, or anti-chPKR antibodies.
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(E) The illustration of complex formed by IBDV VP3, chRPL18 and chPKR.
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