Cells and viruses
We used Madin–Darby canine kidney (MDCK) cells for virus propagation and 50% tissue culture-infective dose (TCID50) titration assays. MDCK cells were in minimal essential medium (MEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C/5% CO2. The A549 human lung carcinoma, human bronchial epithelium (HBE), and human laryngeal epidermoid carcinome (HEp-2) cells were used for transfection experiments. These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C 5% CO2. We used a 2009 pandemic influenza A (H1N1) virus (pdmH1N1), strain A/Guangzhou/GIRD07/2009 (GenBank Accession No. HM_014326-HM_014333). A seasonal H3N2 influenza A virus (A/Guangdong/520/2009) was isolated from a patient with influenza-like symptoms. An influenza A H9N2 isolate (A/Chicken/Guangdong/SS/94) was kindly provided by South China Agricultural University. The pdmH1N1 and H3N2 viruses were grown in MDCK cells at 35°C, while the H9N2 virus was propagated in allantoic cavities of 10-day-old embryonated hens’ eggs at 37°C. All experiments with pdmH1N1 and H9N2 viruses were conducted under biosecurity level three conditions, and higher.
Preparation and transfection of siRNAs
The siRNAs against ST6GAL1 were designed using BLOCK-iT™ RNAi Designer  and synthesized by Invitrogen ( Invitrogen, Carlsbad, CA, USA). Sequences are available in Additional file 1: Table S1. As a negative control, we used non-targeting Allstars® siRNAs (Qiagen, Valencia, CA, USA). All siRNA duplexes were double-stranded RNA molecules comprising 21 nt with a dTdT overhang at the 3’ ends . Target sequences were subjected to a Basic Local Alignment Search Tool (BLAST) search against GenBank to ensure they were unique to ST6GAL1. Airway epithelial cell lines (A549, HBE, and HEp-2) were transfected with either ST6GAL1 or non-targeting Allstars® siRNAs using Lipofectamine® RNAiMax (Invitrogen) according to the manufacturer’s instructions. At different time points post-transfection, cells were either infected with influenza virus or harvested for downstream experiments.
Detection of ST6GAL1 and ST6Gal I
Expression of ST6GAL1 was detected using real-time reverse transcription polymerase chain reaction (qPCR) assays. Expression of the ST6Gal I protein was determined by western blotting. Respiratory epithelial cells (A549, HBE, and HEp-2) were transfected with control or ST6GAL1 siRNAs (2.5–50 nmol). At 48 h post-transfection we used an RNeasy Mini kit (Qiagen) for RNA extraction according to the manufacturer’s instructions. The extracted total RNA (500 ng/sample) was then used for cDNA synthesis. The resulting cDNA was amplified in a 20-μL reaction containing ST6GAL1-specific forward (0.25 μmol) and reverse (0.25 μmol) primers (Additional file 1: Table S2), and 1× Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Reactions were subjected to thermal cycling with an IQ5 System (Bio-Rad, Hercules, CA, USA) involving an initial 10-min denaturation step at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Fluorescence signals from these reactions were captured at the end of the 60°C extension step for each cycle. To determine the specificity of the assay, amplicons were subject to melting curve analysis after the 40th cycle (65–95°C, 0.1°C/s). Our data were analyzed using the 2-ΔΔCT method, according to the manufacturer’s instructions, with ST6GAL1 expression levels normalized to β-actin mRNA levels.
After transfection for 48 h, A549 cells were lysed in 50 mM Tris–HCl buffer (pH 7.4) containing 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 20 μg/mL leupeptin, 4 mM sodium fluoride, and 200 μM sodium pervanadate. Protein concentrations in the lysates were determined with a BCA assay kit (Pierce, Rockford, IL, USA). Proteins in lysates were resolved under reducing conditions for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We probed polyvinylidene fluoride (PVDF) membranes with 5 μg/mL of a rabbit antihuman ST6Gal Ι polyclonal antibody (Abcam, Cambridge, MA, USA) followed by a horseradish peroxidase (HRP)conjugated antirabbit IgG secondary antibody(Abcam). Specific signals were visualized using an ECL kit (Pierce). Protein concentrations between wells were normalized using HRP-conjugated β-actin-specific monoclonal antibodies (Sigma-Aldrich, St. Louis, MO, USA).
Cultured cells in the logarithmic growth phase were trypsinized, seeded into 96-well plates, and transfected with ST6GAL1 (2.5–50 nmol) or control (10 nmol) siRNAs. At 24, 48, and 72 h post-transfection, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays (Sigma-Aldrich). The absorbance at 492 nm was measured in a spectrophotometer (Molecular Devices, Palo Alto, CA, USA). Background values were subtracted from the average absorbance value obtained for each siRNA treatment and then compared with the value obtained when siRNAs were absent (100% viability). Each assay was performed in duplicate in at least four wells.
Detection of α-2,6 linked SA on the cell surface
The A549, HBE, and HEp-2 cells were treated with ST6GAL1 or control siRNAs and cultured for 72 h. Cells were washed with phosphate-buffered saline (PBS) and fixed in 3.7% formaldehyde in PBS for 30 min. For detection of sialic acid residues on the surface of cells, apical monolayers were blocked with 3% bovine serum albumin (BSA; Merck, Darmstadt, Germany) in PBS for 30 min and then incubated with 5 μg/mL fluorescein isothiocyanate (FITC)-conjugated Sambucus nigra lectin (SNA; Vector Laboratories, Burlingame, CA, USA) for 1 h. To confirm the specificity of lectin binding, monolayers were treated with 50 mU Vibrio cholerae neuraminidase (VCNA; Roche, Almere, Netherlands) for 1 h prior to fixation and then examined with a rapid-scanning confocal laser microscope (Nikon Corp, Tokyo, Japan).
Approximately 106 cells transfected with control or ST6GAL1 siRNAs were scraped from the culture surface and washed twice with PBS containing 10 mM glycine, and then washed once with buffer 1 (50 mM Tris–HCl, 0.15 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, pH 7.5). Cells were blocked with 3% BSA-PBS for 1 h on ice and washed in the same manner as described above. After centrifugation, the cell pellet was incubated with FITC-conjugated SNA at room temperature for 30 min, then washed and fixed with 1% paraformaldehyde. After another three washes with PBS, mean fluorescence intensities were determined on a fluorescence-activated cell sorter (FACS) Calibur flow cytometer (BD, San Jose, CA, USA) by counting a minimum of 10,000 events.
Receptor specificity of virus strains
To study the receptor-binding properties of the virus strains used, we enzymatically modified chicken red blood cells (CRBCs) to express either sialic acid (SA)-α2,6-Galactose (Gal) or SAα2,3Gal as previously described [38, 39] with minor modifications. Briefly, SA was removed from 100 μL of 10% CRBCs using 50 mU VCNA at 37°C for 1 h. Subsequent resialylation was performed using 50 μL of 0.5 mU α2,3-(N)-sialyltransferase (Calbiochem, La Jolla, CA, USA) or 125 μL of 2 mU α2,6-( N)-sialyltransferase (Japan Tobacco, Shizuoka, Japan), and 1.5 mM cytidine monophospho-N-acetylneuraminic (CMP) sialic acid (Sigma-Aldrich) at 37°C for 30 or 60 min, respectively. Receptor specificity of the virus strains was then determined using standard hemagglutination assays with the modified CRBCs.
Influenza virus challenge of ST6GAL1-siRNA transduced epithelial cells
All challenge experiments were carried out at a multiplicity of infection (MOI) of 0.01 for 1 h in the presence of N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich). Viral supernatants were harvested at various time points post-infection for TCID50 assays. To obtain dose–response curves, a dilution series of siRNAs were added to cells in 96-well plates in triplicate. Cells were challenged and supernatants were examined as described above .
The titer of viruses in supernatants were determined using TCID50 assays as previously described . Briefly, MDCK cells were seeded onto flat-bottom 96-well plates (3 × 104 cells/well); 24 h later, serum-containing medium was removed and 25 μL of virus-containing supernatants (serially diluted ten-fold from 10° to10 −8) was added to wells in triplicate. After incubation for 1 h, 175 μL of infection medium containing TPCK-trypsin (1.25 μg/mL) was added to each well. After incubation for 48 h at 37°C, the presence or absence of virus in culture supernatants was determined by hemagglutination of CRBCs. Virus titers were determined by interpolation of the dilution endpoint that infected 50% of wells. Virus titers are presented as log10 TCID50.
Cells were transfected with control or ST6GAL1 siRNAs, then infected with virus at an MOI of 50, and chilled at 4°C for 90 min. Infected cells were harvested and washed three times with PBS, then fixed with 3% glutaraldehyde for 45 min at room temperature, and post-fixed with 1% osmium tetroxide. Fixed cells were dehydrated with increasing concentrations of acetone from 30% to 100% and embedded in an epoxy resin. Polymerization was conducted at 60°C for 48 h. Ultrathin sections were stained with uranyl acetate and lead citrate, and sections viewed and photographed with a Hitachi H-800 transmission electron microscope (Hitachi Co., Tokyo, Japan).
Quantitation of viral genome copies by qPCR
We extracted RNA 2 h after virus infection using a QIAamp RNA isolation kit (Qiagen). First-strand cDNA was synthesized using RNAse H+ reverse transcriptase (Invitrogen) and random primers. We then used 2 μL of cDNA for each qPCR assay, along with primers (Additional file 1: Table S2), fluorescent probe, and Master Mix (Applied Biosystems). Samples were subjected to thermal cycling on an IQ5 System (Bio-Rad, Hercules, CA, USA): 42°C for 5 min; 95°C for 10 s; and 40 cycles of 95°C for 5 s and 60°C for 30 s. Expression levels of viral RNAs were normalized to the constitutive expression of ribonucleoprotein. All measurements were conducted three times for statistical analysis.
The A549, HBE, and HEp-2 cells were transfected with either control or ST6GAL1 siRNAs (10 nM). We measured the levels of IFN-β in culture supernatants 24 h later using an enzyme-linked immunosorbent assay (ELISA; PBL Biomedical Laboratories, Piscataway, NJ, USA). A long double-stranded RNA that induced the expression of IFN-β used as a positive control.
All statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The significance of variability among experimental groups was determined using one-way ANOVA, the paired t-test, or the Mann–Whitney U test. All differences were considered statistically significant if the P-value was less than 0.05.