Viruses and cell lines.
The Beaudette C strain of NDV was used throughout this study. In addition, the following strains were used for growth and replication studies: Australian-Victoria, California, Kansas, La Sota, and 73-T. All of the viral strains were propagated in DF1 chicken fibroblast cells (ATCC, Manassas, VA) with Dulbecco's modified Eagle medium (DMEM; BioWhittaker, Inc., Walkersville, MD) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) or 5% chicken allantoic fluid (for NDV strain La Sota).
The following cell lines and corresponding culture media were used: normal human skin fibroblast cells (CCD-1122Sk; ATCC, Manassas, VA), Iscove's modified Dulbecco's medium with 10% FBS; normal human lung fibroblast cells (MRC5; BioWhittaker), Eagle's minimal essential medium with 10% FBS; normal human prostate epithelial cells (PrEC; Clonetics, San Diego, CA), the manufacturer's instructions were followed; normal human skeletal muscle cells (SKMC; Clonetics), the manufacturer's instructions were followed; normal human mammary epithelial cells (HMEC; Clonetics), the manufacturer's instructions were followed; human prostate epithelial cells transfected with a plasmid carrying one copy of human papillomavirus 18 and transformed with Ki-Ras by using the Kirsten murine sarcoma virus (RWPE-2; ATCC), ATCC complete growth medium; human fibrosarcoma (HT-1080; ATCC), Eagle's minimal essential medium with 10% FBS; human cervical carcinoma (C-33A; ATCC), MEM with 10% FBS; human colorectal adenocarcinoma (SW-620; ATCC), Leibovitz's L15 medium with 10% FBS; human lung adenosquamous carcinoma (NCI-H596; ATCC), RPMI with 10% FBS.
Virus growth and production in vitro.
NDV was propagated and titrated in DF1 cells. For growth studies, the cells were grown to 80% to 90% confluence in a six-well plate and infected with NDV in DMEM with 0.15% bovine serum albumin at a multiplicity of infection (MOI) of 0.001. At 1 h postinfection, the inoculum was removed and the cells were washed twice with phosphate-buffered saline (PBS) and then covered with 2 ml of the recommended medium. Cells were then incubated in a 37°C incubator. For nonvirulent NDV strain La Sota, the medium was supplemented with 5% chicken allantoic fluid as a source of proteases that cleave the F protein. An aliquot of the culture supernatant was collected every 24 h for 4 days for estimation of the virus titer. The procedure was repeated three times, and the standard deviation was calculated.
NDV assay of culture supernatants.
Plaque assays were performed to determine the level of NDV infection in the culture supernatant. DF1 cells were adsorbed with different dilutions of the culture supernatants. At 1 h postinfection, the inoculum was removed and the cells were overlaid with Opti-MEM medium supplemented with 2% FBS and 0.9% (wt/vol) methylcellulose (Sigma) in the presence of 2% FBS. For NDV strain La Sota, the above-mentioned overlay included 5% chicken allantoic fluid. The cells were incubated at 37°C for 4 days or until plaques appeared. The cells were then stained with neutral red, and the plaques was counted.
Immunoflourescence of infected cells.
Cells were grown in glass chamber slides (Nalge Nunc International, Rochester, NY). The cells were infected with NDV at different MOIs. At 12 or 24 h postinfection, the medium was aspirated and the cells were rinsed in PBS and fixed with 3.7% formaldehyde. The slides were transferred to a coplin jar and treated with cold acetone for 7 min. The slides were then rinsed three times with PBS for 2 min per rinse. The cells were incubated with a monoclonal antibody against NDV hemagglutinin-neuraminidase (HN) for 45 min at 37°C. After the slides were rinsed in PBS thrice, the cells were incubated with fluorescein isothiocyanate-coupled secondary anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:3,000. The cells were then rinsed in PBS thrice and mounted with p-phenyldiamine (Sigma) mount. The cells were then examined under a confocal microscope.
Assessment of the anti-NDV factor(s) in supernatant from infected cells.
The supernatants from infected cells in culture were collected at 72 h postinfection. NDV was removed from the supernatants via ultrafiltration with Centricon YM-100 filters (Millipore, Billerica, MA) according to the manufacturer's protocol. Fresh cells were treated with the filtered supernatant for 15 h. After removal of the supernatant, the cells were infected with NDV at an MOI of 0.001. The culture supernatants were assayed for NDV at 24, 48, 72, and 96 h postinfection.
To identify the anti-NDV factor(s) in the supernatant, we performed an experiment similar to the one described above, except that the supernatant was incubated with a combination of antibodies that neutralize IFN-α and IFN-β (Βiosource, Camarillo, CA) for 1 h at room temperature before it was overlaid on the cells. Two concentrations, 300 U and 1,000 U, of each antibody were examined.
IFN assay of the supernatant.
NDV infection of different cell lines was performed as described above. The supernatant from the infected cells in culture was collected at different times postinfection, and an IFN-β enzyme-linked immunosorbent assay (ELISA) kit (Biosource) was used to detect IFN-β in the media according to the manufacturer's protocol.
Immunoblot analysis of NDV proteins.
The cells were grown to 80% to 90% confluence in a six-well plate and either pretreated with 500 IU of IFN-β (Βiosource) for 15 h before infection or infected directly with NDV at an MOI of 1. The infection medium used was DMEM with 0.15% bovine serum albumin. At 1 h postinfection, the inoculum was removed, the cells were washed twice with PBS, covered with 2 ml of the recommended medium, and then incubated at 37°C. The cells were lysed 48 h later with RIPA buffer (50 mM Tris-HCl [pH 7.4], 10 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% deoxycholate, 1% Triton X-100), and 5 μg of total protein was loaded onto a 10% SDS-polyacrylamide gel (precast gel from Invitrogen, Carlsbad, CA). The resolved proteins were transferred onto a polyvinylidene difluoride membrane and probed with polyclonal chicken antibodies specific for NDV and a horseradish peroxidase-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Laboratories). The membrane was exposed to a chemiluminescent substrate and detected by autoradiography.
Immunoblot analysis of IFN signaling intermediates.
The cells were treated with 500 IU/ml IFN-β for 15, 30, 60, 120, 240, or 360 min. The cells were harvested and lysed in RIPA buffer, and the protein concentration was determined by the Micro BCA method (Pierce, Rockford, IL). For analysis of STAT1, STAT2, and IRF9, we loaded equal amounts of protein onto a 10% SDS-polyacrylamide gel, transferred them onto a polyvinylidene difluoride membrane, and detected the protein with antibodies specific for STAT1 and STAT2 (rabbit polyclonal antibodies from Santa Cruz Biotechnologies, Santa Cruz, CA), phosphorylated forms of STAT1 (mouse monoclonal antibody to Tyr 701; Cell Signaling Technology, Danvers, MA), phosphorylated forms of STAT2 (mouse monoclonal antibody to Tyr 690; Cell Signaling Technology), and IRF9 (Santa Cruz Biotechnologies). For analysis of JAK1 and TYK2, the proteins were immunoprecipitated by antibodies specific to JAK1 kinase and TYK 2 kinase (mouse monoclonal antibody from BD Transduction Labs, Franklin Lakes, NJ). After resolution on the gel, the proteins were immunoblotted and probed with rabbit monoclonal antibodies against the JAK1 and TYK2 kinases or those against phosphorylated forms of the JAK1 kinase (Tyr 1022/1023) or the TYK2 kinase (Tyr 1054/1055) (Cell Signaling Technology). The blots were probed with the specific horseradish peroxidase-conjugated secondary antibody to determine the relative amounts of proteins.
Electrophoretic mobility shift assay (EMSA).
The biotinylated probe was prepared by end labeling the ISRE oligonucleotide sequences (AGGAAATAGAAACTG)2 with biotin via the Biotin 3′ End DNA Labeling Kit (Pierce). Cells were treated with IFN-β for 30 min, and then nuclear extracts were prepared with the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce). The LightShift Chemiluminescent EMSA Kit (Pierce) was used to perform the binding reaction between the extract and the labeled ISRE. The complexes were resolved on a precast native Tris-borate-EDTA polyacrylamide gel (Invitrogen) and transferred to a nylon membrane before cross-linking the DNA to the membrane. Biotinylated DNA was detected with streptavidin-horseradish peroxidase conjugate and chemiluminescent substrate. The membrane was visualized by autoradiography.
RNA isolation and preparation of cDNA for microarray analysis.
HT-1080 and CCD-1122Sk cells were grown in 10-cm dishes until 80% confluent and then treated with IFN-β (500 IU/ml) for 15 h. Control cells were left untreated. Total RNA was isolated by TRIzol reagent (Invitrogen) according to the manufacturer's protocol. To assess the integrity of the total RNA, we tested a small amount of the sample on a 2100 Bioanalyzer Lab-on-a-Chip system (Agilent Technologies, Palo Alto, CA). The RNA sample was confirmed to have two strong peaks representing the 18S and 28S rRNA fragments. The 28S/18S ratio was approximately 2, and there was very little low-molecular-weight degradation product. The RNA was reverse transcribed with dT primer, followed by synthesis of second-strand DNA. Double-stranded DNA was transcribed in vitro incorporating biotinylated ribonucleotides. The cRNA was fragmented to minimize steric hindrance during the hybridization step.
The labeled target was hybridized to the human gene chip UG133A (Affymetrix, Santa Clara, CA), which represents 22,283 human genes, according to the manufacturer's protocol. In brief, the hybridization was performed for 18 h at 45°C. The arrays were washed and stained with streptavidin conjugated to phycoerythrin in the Affymetrix automated fluidics station. The arrays were then scanned, and an image file was produced. The image file was converted with Affymetrix Microarray Suite software (version 5.0). Expression signals were scaled to a target intensity of 500 and log transformed. Arrays were omitted if the scaling factor exceeded 3 standard deviations of the mean or if the ratio of 3′ to 5′ mRNA for β-actin or glyceraldehyde-3-phosphate dehydrogenase was greater than 3. The data was analyzed with Spotfire DecisionSite software (Spotfire, Somerville, MA). The cDNA microarrays were analyzed by a statistical algorithm that calculated a signal value and a detection P value for each gene. A P value of <0.4 was considered as present call for a gene, 0.4 to 0.6 as marginal, and >0.6 as absent.