Cell line and viruses
Human embryonic kidney (293T) cells obtained from National Centre For Cell Science, Pune, India were grown in modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA,USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies),100 units/ml penicillin, 100 μg/ml streptomycin in tissue culture flasks (Corning, USA) at 37°C in a CO2 incubator. Influenza A/Chicken/India/WBNIV2653/2008(H5N1)  and A/Aquaticbird/India/NIV-17095/2007(H11N1)  viruses were grown and propagated in the allontoic cavities of 10-day old embryonated chicken eggs.
Cloning of NS1 genes
Total RNA was isolated from influenza A H5N1 and H11N1 strains using viral RNA isolation kit (Qiagen, Germany) following manufacturer’s instructions. The RNA was reverse transcribed and the NS1 genes were amplified using subtype specific primers. Amplicons cloned into pcDNA 3.0(-) expression vector (Invitrogen, Carlsbad, CA) were screened by sequencing. Competent E.Coli DH5α cells were transformed with the plasmids. The plasmids containing NS1 inserts were isolated and purified using plasmid midi kit (Qiagen, Germany).
Transient transfection and protein expression system
293T cells were transfected with pcDNA 3.0(-) control vector, pcDNA3.0-H5N1-NS1 and pcDNA3.0-H11N1-NS1 plasmid constructs using TransIT-LT1 (Mirus biosciences) transfection reagent according to the manufacturer’s protocol. Briefly, cells were cultured to monolayer in T-25 tissue culture flasks 24 hours before transfection. Plasmid DNA (6 μg/flask) and transfection reagent were mixed in serum-free medium and incubated for 30 minutes at room temperature following manufacturer’s protocol. Transfection complexes were then gently added into individual flasks. Cells were analyzed for gene and protein expression at 24 hours post transfection. Untransfected cells served as controls and cells transfected with the expression vector served as mock controls.
Western blot analysis
Total cellular protein from control and transfected cells was isolated using RIPA lysis buffer [10X-20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA,1 mM EGTA, 1% NP-40, Protease-Inhibitor cock-tail(Qualigens). Equal amount of proteins (10 μg) from cell extracts were separated by 12.5% SDS-polyacrylamide gel electrophoresis (12.5% SDS-PAGE) and transferred to Hybond-C (Amersham Biosciences) membrane with an electrotransfer apparatus (Cleaver Scientific Ltd) at 10 Volts (100 mA) for 1 h. The membrane was probed with specific monoclonal antibodies against Influenza A-NS1 protein (Santa Cruz Biotechnology, Inc. Santa Cruz, CA). Primary and secondary antibody interaction was performed in phosphate-buffered saline (pH 7.5). Proteins were visualized using the ECL detection system (Amersham Biosciences, USA).
Total RNA was extracted from the control, mock transfected and NS1- transfected cells using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and purified by the RNeasy kit (Qiagen, Germany) following standard methodology as described earlier [23, 24]. Amplification of RNA and indirect labeling of Cy-dye was done by Amino Allyl MessageAmp II aRNA amplification kit (Ambion, Austin, TX, USA) using manufacturer’s instruction. One microgram of total cellular RNA from control and transfected cells was used for the experiments. The RNA was reverse transcribed and amplified. The purified amino allyl aRNA was labeled with Cy3 and Cy5 (Amersham Biosciences, USA) for control and experimental samples respectively. Purified samples were lyophilized, resuspended in hybridization buffer (Pronto Universal Hybridization kit, Corning, USA) and hybridized on the Discover human chip (Arrayit Corporation, Sunnyvale, CA). Hybridization was carried out in a Hybstation (Genomic Solutions, Ann Arbor, MI) and the conditions used were 55°C for 6 h, 50°C for 6 h, and 42°C for 6 h. Scanning was performed at 5-mm resolutions with the Scan array express (PerkinElmer, Waltham, MI). Grid alignment was done using gene annotation files and raw data were extracted into MS EXCEL.
Data was analyzed using GENOWIZ Microarray and Pathway analysis tool (Ocimum Biosolutions, Hyderabad, India). Data analysis was performed as described earlier . In order to detect highly expressed genes, fold change analysis was done. Genes with 2 folds up/down-regulation were considered as differentially expressed at a p-value < 0.05, Student’s t-test. Functional classification of the genes was performed using gene ontology and pathway analysis. Microarray experiments were carried out in triplicates. The data is MIAME compliant and the raw data has been deposited in Gene Expression Omnibus (GEO) database No GSE39155.
Quantitative RT-PCR using SYBR green I
The mRNA levels for IFN-β, STAT1, CCL5, CXCL10, CASP8, BAK1, HSP90 and BCL2 genes in control and transfected cells were analyzed by real-time RT-PCR. Total RNA was prepared from the control and transfected cells using RNeasy kit (Qiagen). One hundred nanograms (100 ng) of total RNA was used for quantitative RT-PCR analysis. Reaction was performed using the QuantiTect SYBR Green RT-PCR kit (Qiagen, Germany) according to the manufacturer’s instructions. Reactions were carried out on an ABI 7300 realtime PCR system (Applied Biosystems, Foster City, CA, USA) and the thermal profile used was Stage 1: 50°C for 30 min; Stage 2: 95°C for 15 min; Stage 3: 94°C for 15 sec, 55°C for 30 sec; and 72°C for 30 sec, repeated for 30 cycles. Melting curve analysis was performed to verify product specificity. Reactions were performed in triplicates. All quantitations (threshold cycle [CT] values) were normalized to that of β-Actin to generate ΔCT, and the difference between the ΔCT value of the sample and that of the reference was calculated as ΔΔCT. The relative level of gene expression was expressed as 2-ΔΔCT. Primer sequences for the genes of interest have been described earlier [23, 24].
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL Assay)
The assay was carried out using TUNEL assay kit (Invitrogen) according to the instructions of the manufacturer. Briefly, Equal number of control, NS1-transfected and mock (vector) transfected cells(2 X 106) were fixed in 1%(w/v) paraformaldehyde in 1XPBS(Phosphate buffer saline) for 15 minutes, washed in PBS and resuspended in 0.5 ml of PBS added with 70% Ethanol. The cells were kept at -20°C for 1hour. Labeling reactions were performed with BrdUTP using TdT enzyme for 60 min at 37°C in a humidified chamber. The labeled DNA was detected using Alexa Fluor 488 dye–labeled anti-BrdU antibody. Apoptosis was evaluated microscopically as flurescent cells per field at high-power magnification.
Sequence analysis of NS1 proteins
H5N1 NS1 and H11N1 NS1 clones were directly used for cycle sequencing reactions. Sequencing was done on an automated Applied Biosystems’ 3130 XL system using cycle sequencing big dye terminator. The sequence of the NS1 genes cloned for the experiment are available in NCBI sequence database [Accession No CY055179 (H11N1 NS1) and CY046074 (H5N1 NS1)]. Alignment of H5N1 and H11N1 NS1 protein sequences was carried out using ClustalW program. The amino acid sequences were used to generate protein data bank (.pdb) files using SWISS-MODEL server (http://swissmodel.expasy.org/). The protein data bank (.pdb) files were used for visualization and generation of protein 3D structure using UCSF Chimera program (http://www.cgl.ucsf.edu/chimera).