- Open Access
Porcine adenovirus type 3 E1Blarge protein downregulates the induction of IL-8
© Zhou et al; licensee BioMed Central Ltd. 2007
- Received: 05 April 2007
- Accepted: 12 June 2007
- Published: 12 June 2007
Replication-defective (E1-E3 deleted) adenovirus vector based gene delivery results in the induction of cytokines including IL-8, which may contribute to the development of inflammatory immune responses. Like other adenoviruses, E1 + E3 deleted porcine adenovirus (PAdV) 3 induces the production of IL-8 in infected cells. In contrast, no IL-8 production could be detected in cells infected with wild-type or mutant PAdV-3s containing deletion in E1A + E3 (PAV211) or E1Bsmall + E3 (PAV212). Expression of PAdV-3 E1Blarge inhibited the NF-κB dependent transcription of luciferase from IL-8 promoter. Imunofluorescence and electrophoretic mobility shift assays suggested that constitutive expression of PAdV-3 E1Blarge inhibited the nuclear translocation of NF-κB and its subsequent binding to DNA. These results suggest that E1Blarge interacts with NF-κB to prevent transcription and down regulate proinflammatory cytokine IL-8 production.
- Electrophoretic Mobility Shift Assay
- Adenovirus Vector
- RNase Protection Assay
- African Swine Fever
- African Swine Fever Virus
Cytokines are important mediators of inflammation and regulators of the immune response. The inflammatory response including release of inflammatory cytokines is the first defense against viral infection. However, viruses have evolved a number of different strategies to avoid the host inflammatory responses. Large DNA viruses including poxviruses and herpes viruses [1–6] modulate cytokine action by encoding secreted forms of receptors for cytokines and chemokines. Adenoviruses modulate cytokine expression by encoding intracellular proteins, which counteract TNF-α [7, 8].
Although human adenovirus (HAdV) vectors have been utilized for gene transfer for functional studies in vivo [9, 10], their therapeutic use in delivering genes to the airways of humans is limited due to the transient gene expression . Earlier studies have shown that the airway administration of adenovirus vector results in the induction of non specific host responses consisting in part of neutrophil accumulation followed by mononuclear cell and macrophage accumulation. Adenovirus vector infection of airway epithelial A549 cells [12, 13] or airways of macaques  results in rapid induction of the inflammatory cytokine IL-8, which may contribute to the inflammatory host response . This induction of IL-8 production has been shown to be due to adenovirus induced activation of Raf/MAPK pathway . Thus, blocking these pathways may be required for developing an efficient adenovirus vector.
Porcine adenovirus (PAdV) 3, a non human adenovirus is being developed as a vector for gene delivery in animals and humans [16, 17]. Availability of the complete nucleotide sequence and transcription map of PAdV-3  genome has facilitated the construction of recombinant PAdV-3s [16, 17, 19, 20] and their use as vaccine delivery vehicles . Earlier, analysis of early region 1 (E1) of PAdV-3 suggested that while E1A  and E1Blarge  are essential for virus replication, E1Bsmall is not essential for virus replication . Here, we report that E1Blarge can impair the induction of inflammatory cytokine IL-8 by inhibiting the NF-κB dependent gene transcription.
RNase protection assay
Luciferase reporter assay
Since increased expression of proinflammatory chemokines including IL-8, in response to various stimuli including adenovirus vectors can be upregulated by NF-κB transcription factor , we employed luciferase reporter assay to examine the inhibition of transcriptional activation of IL-8 promoter (containing consensus sequence for NF-κB binding) by E1Blarge protein. As seen in Fig. 1B, reduced levels of the luciferase activity were obtained when phIL8-Luc DNA was cotransfected with pCDNA3.1-pE1BL DNA (expressing E1Blarge). In contrast, significant levels of luciferase activity were detected when phIL8-Luc DNA was cotransfected with pCDNA3.1 DNA showing that the competition for transcription factors binding to E1Blargeexpression vector did not nonspecifically reduce the activity of luciferase reporter gene. The results of the reporter gene expression indicated that E1Blarge reduced the NF-κB activated gene expression and was responsible for the observed inhibition of inflammatory cytokine IL-8 production.
E1Blarge inhibits the translocation of NF-κB to the nucleus
E1Blarge affects the NF-κB binding to oligonucleotides containing NF-κB consensus sequence
Viruses and cells
Recombinant PAdV-3 bearing deletions in the E1 region were generated as described previously . PAV211 contains deletions in the E1A + E3 regions, PAV212 contains deletions in the E1Bsmall + E3 regions, PAV227 contains deletions in E1 + E3 regions and PAV300 contains deletion in E3 region. Viruses were propagated and titrated as described [19, 20, 24]. HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).
RNase protection assays. Monolayers of HeLa cells (1X105/well) in 12 well plate were infected with wild-type or mutant PAdV-3s at a MOI of 100. At 6 h post infection, HeLa cells were harvested and processed for total RNA using TRIZOL (Invitrogen) as per manufacturer's protocol. RNase protection assay was performed with the RiboQuant Multi-Probe template (BD Biosciences) set hCK-5 as per manufacturer's protocol. Autoradiographs were analyzed by phosphoimaging with a Personal FX phosphoimager and Quantity One software (Bio-Rad).
The 181-bp human IL-8 promoter sequence (-135 to +46) was PCR amplified from the genomic DNA [26, 27] derived from HeLa cells using the primers: hIL8 (-135) Fw: 5'-CAATGCTAGC G AAGTGTGATGACTCAGG TT-3', which contains a NheI restriction enzyme site (bold letters), and hIL8 (+46) Bw: 5'-CGTTCTCGAG A AGCTTGTGTGCTCTGCTGT-3' containing a XhoI restriction enzyme site (bold letters). The PCR product was digested with NheI-XhoI and ligated to NheI-XhoI digested plasmid pGL3-Basic (Promega) creating plasmid phIL8-Luc. The plasmid phIL8-Luc contains luciferase gene under the control of IL-8 promoter. Similarly, the coding region of E1Blarge gene was PCR amplified using the primers: [PE1BL (NheI) Fw: 5'-CAGTGCTAG CATGTTCCCTGC TGGAGGCGC-3', which contains a NheI restriction enzyme site (bold letters), and PE1BL (XhoI) Bw: 5'-GTCA CTCGAG TC AGTCATC G TCATCGCTGAA-3' containing a XhoI restriction enzyme site (bold letters)] and PAdV-3 genomic DNA as a template. The PCR product was digested with NheI-XhoI and ligated to NheI-XhoI digested plasmid pCDNA3.1(-) (Invitrogen) creating plasmid pCDNA3.1-pE1BL. The plasmid pCDNA3.1-pE1BL contains E1Blarge gene under the control of human cytomegalovirus immediate early (HCMV IE) promoter.
HeLa cells (1x105 cells/well) were plated in 12-well plate and incubated overnight. Tansfections were carried out using 0.5 μg of each plasmid [(phIL8-Luc, pCDNA3.1-pE1BL] or [phIL8-Luc, pCDNA3.1])/well (in triplicate) using 5 μl of lipofectin (Invitrogen), followed by incubation for 5 h in Opti-MEM (Invitrogen). After adding FCS to each well to give a final concentration of 1%, the cells were incubated for 18 h at 37°C. Finally, the cells were washed with PBS and lysed in 200 μl of 1x lysis buffer (Luciferase reporter assay kit, BD Bioscience). Luciferase activity was determined using 50 μl of cell extract and was read using a TD-20/20 luminomitor (Turner Designs).
VIDO R1  and VR1BL  cells plated on glass coverslips were untreated or treated with 10 ng/ml TNF-α (R&D System). At 15 min post treatment, the cells were washed with PBS, fixed and permeabilized by incubating with methanol/acetone (1:1) at -20°C for 15 min. The cells were rehydrated with PBS and incubated for 1 hour in a 1:200 dilution of monoclonal antibody specific for the p65 subunit of NF-κB factor (Santa Cruz). The cells were washed three times with PBS and incubated with 1: 800 diluted Cy3-labeled goat anti-mouse antibody (Jackson Laboratory) for 30 min at room temperature. Finally, the cells were washed three times in PBS before incubating with DAPI at concentration of 1μg/ml (Roche) for 5 min. Fluorescence was examined and photographed using a Carl Zeiss Axiovert 200 M inverted fluorescent microscope.
Electrophoretic mobility shift assays (EMSA)
HeLa nuclear lysates were prepared as described previously (. Briefly, the cells were washed two times with phosphate-buffered saline, resuspended in 4 pellet volumes of buffer A [(10 mM TRIS (pH 7.9), 10 mM NaCl, 1.5 mM MgCl2, 5 mM dithiothreitol (DTT), 0.5 mM phenyl-methyl sulfanyl fluoride (PMSF), and 5 μg of aprotinin, leupeptin, and pepstatin (ALP) per ml)] and incubated at 4°C for 1 h. The cells were lysed by three freeze/thaw cycles and centrifuged for 5 min at 2000 × g at 4°C. The nuclei were washed once with buffer A, resuspended in 3 pellet volumes of buffer B [(20 mM TRIS (pH 7.9), 20% glycerol, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, and 5 μg of ALP per ml)] and incubated at 4°C for 30 min. The nuclear lysates were collected after centrifugation for 30 min at 12,000 × g at 4°C and stored at -80°C. The oligonucleotides containing wild-type NF-κB (shown in boldface) motif (5'-CGTAGCCATCAGTTGCAAA TCGTGGAATTTCCT CT-3') or mutant NF-κB (mutated residues underlined) motif (5'CTAGGCCATCAGTTGCAAATCGT TT AATTT AA T CT)  were end-labeled with [α-32P] dCTP using the Klenow fragment of DNA Polymerase I.
Each binding reaction was assembled on ice containing 0.2 ng of double-stranded labeled probe, 10 μg of HeLa nuclear lysate from indicated samples, 0.5 μg poly(dI-dC), 10 mM Tris (pH 7.8), 50 mM NaCl, 1 mM EDTA and 3.3 mM sodium acetate. DNA-protein complexes were electrophoresed for 2 h at 150 V through 5% acrylamide gels. The gels were dried for 60 min at 80°C and exposed to Phosphor screens. Images were analyzed with a Molecular phosphoimager FX and the Quantity One software package (BIO – RAD).
The work was supported by a grant from Natural Sciences and Engineering Research Council (NSERC) of Canada to S.K.T. Published as VIDO journal article # 457.
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