Cell culture and virus infection
Primary human bronchial epithelial cell (PHBE) were grown in bronchial epithelial basal medium (BEBM) supplemented with growth factors (Lonza Walkersville, Walkersville, MD, USA) at 37°C in a 5% CO2 humidified chamber. BEAS-2B bronchial epithelial cell line was maintained in Dulbecco's Modification of Eagle's Medium/Ham's F-12 with 5% fetal calf serum. Cells, at 90% confluence, were treated with different RNA preparations and total cellular RNA or proteins were then extracted at indicated times.
Isolation of primary human alveolar macrophages was approved by a written consent by the University of North Carolina School of Medicine Committee on the protection of the rights of human subjects. Healthy, non-smoking male volunteers, 18 to 40 year of age, underwent fiberoptic bronchoscopy with lavage to procure human alveolar macrophages. Samples were put on ice immediately after aspiration and centrifuged at 300 x g for 10 min at 4°C. Cells were washed twice with RPMI-1640, and re-suspended in RPMI-1640 at 1×106 cells/ml.
For virus infection, BEAS-2B or PHBE cells were seeded and allowed to reach 70% confluency. Cells were then treated for 10% Ino-RNA, N-RNA, or IFN-α. After 24 hr, cells were infected with respiratory syncytial virus (RSV-A2) at multiplicity of infection (MOI) of 0.1 plaque forming unit (PFU)/cell. Following infection for 24 hr, cells were harvested for total RNA extraction.
Viral titers were measured by standard plaque assay using HEp-2 cells. Plaques were visualized by using goat anti-RSV antibody (Fitzgerald Industries International, Acton, MA, USA) and HRP-conjugated anti-goat secondary antibody (Sigma Chemicals, St. Louis, MO, USA).
In vitro RNA synthesis
pGEM-Luc (p-Luc) vector (Promega, Madison, WI, USA), was linearized with HindIII and purified by Wizard DNA Cleanup kit (Promega, Madison, WI, USA). Run-off transcription of normal p-Luc RNA (transcript a) was performed using the Megascript T7 kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol with nucleotide triphosphates at 7.5 mM each. p-Luc RNA containing 6% Inosine (transcript b) was transcribed by ATP, GTP, CTP and UTP concentrations to 5.6 mM each, and including 9.4 mM ITP in the reaction. RNA containing 10–11% Inosine (transcript c) was transcribed by adjusting ATP, GTP, CTP and UTP concentrations to 2.8 mM each, and including 9.4 mM ITP in the reaction. RNA containing 16% Inosine (transcript d) was transcribed by further reducing GTP concentration to 1.0 mM, where ATP, UTP, and CTP were kept at 2.8 mM each, ITP concentration was kept at 9.4 mM. All transcription reactions were performed at 37°C for 2 hr, followed by digestion of the template DNA and purification using Megascript purification kit according to manufacturer's protocol (Ambion, Austin, TX, USA). The transcripts were then quantitated by UV absorbance, and were analyzed by 1% agarose gel electrophoresis.
Inosine content analysis
Transcripts were characterized for inosine content by digestion of 3.5 mg of transcript with 2 units each of snake venom phosphodiesterase and calf Intestinal alkaline phosphatase in buffer containing 10 mM Tris pH 8.8, and 2 mM MgCl2 (40 ml total reaction volume) for 2 hr at 37°C. Digested products were analyzed by high performance liquid chromotography (HPLC) using C-18 reverse phase column. Relative nucleoside composition was determined by peak integration using Breeze software (Waters Corporation, Milford, MA, USA), and is represented as a ratio of area under each nucleoside peak.
RNA extraction and RT-PCR
TRIzol total RNA isolation reagent (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction. First strand cDNA was synthesized using superscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed using 0.5 µg total RNA in reverse transcription reaction. Following reverse transcription, 2 µl of cDNA was amplified by RT-PCR. Each experiment was performed in duplicate in 96 well plates using 1 X Sybr Green master mix (Bio-Rad, Hercules, CA, USA) in a final volume of 25 µl. The statistical analysis was performed using parametric two-tailed unpaired t-test with Welch's correction. The sequences of primers were as follows:
IFN-β forward, CAGCAGTTCCAGAAGGAGGA
IFN-β reverse, AGCCAGTGCTCGATGAATCT
TNF-α forward, GGAGAAGGGTGACCGACTCA
TNF-α reverse, TGCCCAGACTCGGCAAAG
IL-6 forward, ATT CTG CGC ACG TTT AAG GA
IL-6 reverse, ATC TGA GGT GCC CAT GCT AC
IL-8 forward, tctggcaaccctagtctgct
IL-8 reverse, gcttccacatgtcctcacaa
RANTES forward, TACCATGAAGGTCTCCGC
RANTES reverse, GACAAAGACGACTGCTGG
RSV-NS1 forward, CACAAACACAATGCCATTCA
RSV-NS1 reverse, AGAGATGGGCAGCAATTCAT
GAPDH forward, GGACCTGACCTGCCGTCTAG
GAPDH reverse, TAGCCCAGGATGCCCTTGAG
Cell extraction, protein kinase assay, protein purification and western blot analysis
For total cellular protein isolation, PHBE cells were washed 2X in phosphate-buffered saline (PBS) and equal numbers of cells were lysed using 1X SDS-sample buffer with 2.5% β-mercaptoethanol. Prior to electrophoresis, proteins were denatured by heating the samples at 95°C for 5 min. To shear the chromosomal DNA, samples were passed through a 26G needle several times. The proteins were then resolved on a 10% SDS-PAGE and were electrotransferred onto nitrocellulose membranes. The immunoblotted proteins were visualized using the enhanced chemiluminescence (ECL) western blot detection system (Amersham, Arlington Heights, IL, USA). For in vitro
kinase assays, cell extracts from BEAS-2B cells were prepared using a buffer containing nonidet-p40 detergent and were used for in vitro
PKR activation as previously described with minor modification 
. His-tagged human PKR, vaccinia virus dsRNA-binding protein E3L, reovirus σ3 protein and Galpha-i protein (as a negative control, a generous gift from Dr. Lutz Birnbaumer, NIEHS) were grown in E.Coli and purified by affinity chromatography.
Signaling antibodis were used according to the manufacturer's instructions. Rabbit antibodies were used to detect p38 MAPK, phospho-p38 MAPK, JNK, phospho-JNK, phospho-eIF-2α, MDA5 and RIG-I (Cell Signaling Technology, Beverly, MA, USA), TLR3 (Sigma Chemicals, St. Louis, MO, USA), PKR and phospho-PKR (Epitomics Inc., Burlingame, CA, USA).
The sequences targeted by chemically synthesized small interfering RNAs (Thermo Scientific, Asheville, NC, USA) in transient knockdown experiments were as follows:
TLR3 gene, GGTATAGCCAGCTAACTAG
PKR gene, GCAGGGAGTAGTACTTAAATA
RIG-I gene, GGAAGAGGTGCAGTATATT
MDA5 gene, GGTGAAGGAGCAGATTCAG
PHBE cells were transfected with TLR3-siRNA, RIG-I-siRNA, MDA5-siRNA, or control-siRNA (30 nM) using DharmaFECT transfection reagent (Thermo Scientific, Asheville, NC, USA) according to the manufacturer's instructions. 72 hr after the transfection, cells were stimulated with N-RNA or 10% Ino-RNA. Then, cytokine production was investigated at mRNA level and the phosphorylation state of MAPKs was determined by western blot analysis after.
RNA structure analysis
Non-denaturing gel electrophoresis of RNA was performed using 0.6% agarose gel in the presence or absence of 120 mM KCl, 4 mM MgCl2. Loading buffer consisted of the running buffer with 40% sucrose. For Denaturing gel electrophoresis, we used 1% agarose gel with 2% formaldehyde and 10 mM MOPS. Prior to loading, samples were heated for 10 min at 65°C. For RNA structural analysis using fluorescence spectrum we used acridine orange RNA-binding dye. RNA at 5 µg/ml was dissolved in buffer containing 12 mM Hepes (PH 7.5), 120 mM KCl, 4 mM MgCl2. Spectral analysis was performed on a SpectraMax Gemini XS (Molecular Devices, Sunnyvale, CA, USA) with excitation at 460 nm and emission maximum at 530 nm.
Animal experiments and flow cytometry
All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review of the protocol (ASP# 05-53, LRB) by the NIEHS animal care and use committee. 6–10 week old male C57BL/6 mice (The Jackson Labs, Bar Harbor, ME, USA) were used in all experiments. Mice were housed and maintained in specific pathogen-free conditions. For treatments, anesthetized mice were made to aspirate intratracheally N-RNA, Ino-RNA at indicated concentration or vehicle control (PBS). After indicated times, bronchoalveolar lavage (BAL) was performed using phosphate buffered saline. ELISA was then used to measure the levels of cytokines in the BAL fluid. For PKR knockout experiments, 8–12 week old male mice genetically-deficient in PKR and wild-type control C57BL/6/SV129 were used. After 4 hr treatments, lung tissues were harvested for RT-PCR.
For flow cytometry, BAL cells were first treated with rat anti-mouse CD16/CD32 (BD Pharmingen, San Jose, CA, USA) and as immunoglobulin receptor blockers (Jackson ImmunoResearch, West Grove, PA, USA). Then, one microgram of each monoclonal antibody specific to CD11c (APC), Gr-1 (PE-Cy7) and major histocompatibility complex Class II (MHC II)- FITC (eBiosciences, San Diego, CA, USA), were added and incubated for 30 min on ice. Cell acquisition and analysis were performed on a BD LSR-II flow cytometer using FACSDiva software (version 4.1.2, Beckton Dickinson).
10% Ino-RNA was labeled with Cy3 using Mirus RNA labeling kit (Madison, WI, USA). Labeled RNA was then purified with RNAeasy mini kit (Qiagen, Valencia, CA, USA). Cells were grown in glass-bottom microwell dishes (MatTek Corp., Ashland, MA, USA) and were treated with 10 µg/ml of Cy3-labeled 10% Ino-RNA alone or were first treated with 10 µg/ml of dextran sulfate or feutin (Sigma Chemicals, St. Louis, MO, USA). After 5 min, cells were washed 3 X in PBS, and mounted with mounting medium (Vector Lab, Burlingame, CA, USA).
Gene array analysis
Gene expression analysis was conducted using Agilent Whole human Genome 4x44 multiplex format oligo arrays (Agilent Technologies, Santa Clara, CA, USA) following the Agilent 1-color microarray-based gene expression analysis protocol. Starting with 500 ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer's protocol. For each sample, 1.65 µg of Cy3 labeled cRNA was fragmented and hybridized for 17 hr in a rotating hybridization oven. Slides were washed and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v9.5), using the 1-color defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (version 7.2) (Rosetta Biosoftware, Kirkland, WA, USA).