Journal: Cellular microbiology

Article Title: Influenza A Virus-Induced Early Activation of ERK and PI3K Mediates V-ATPase-Dependent Intracellular pH Change Required for Fusion

doi: 10.1111/j.1462-5822.2010.01556.x

Figure Lengend Snippet: (A) Nuclear import of viral ribonucleoprotein (RNP) complexes (green) 2 h after infection with PR/8 (MOI = 200) in the presence of solvent, Baf-A1 (0.5 μM), U0126 (20 μM) or ZSTK474 (5.0 μM). RNPs were detected with anti-nucleoprotein (NP) antibody. Magnification 40x. (B) Quantification of NP-positive cells 60 min after virus addition (PR/8, MOI = 10) in the presence of solvent, Baf-A1 (0.5 μM), U0126 (20 μM) or ZSTK474 (5.0 μM). Stained cells were visualized and quantified by FACS. Results in solvent-control cells were assigned a value of 100%. Values are the mean (SE) of three individual experiments. *, P < 0.001, two-tailed unpaired t-test. (C) Effect of Baf A1 on replication and transcription of viral vRNA, cRNA, and mRNA. MDCK or A549 cells (upper panels) were infected with PR/8 (MOI = 2.5) and treated with Baf A1 at the indicated concentrations. Total RNA was isolated 6 h p.i. and analyzed by primer extension assay using PB1 gene-specific primers. 293T cells (lower panel) were treated with the indicated Baf A1 concentrations for 60 min before transfection with the pol I-CAT-RT plasmid encoding CAT in the negative sense, and plasmids expressing viral PB2, PB1, PA and NP. Total RNA was isolated 24 h post-transfection and analyzed by primer extension assay using CAT gene-specific primers. Expression of cellular 5s rRNA was used as a loading control. (D) MDCK cells were infected with PR/8 (MOI = 0.0025) and treated with 0.01 μM Baf A1 (i) 60 min before virus addition or (ii) 0 min, (iii) 15 min, (iv) 30 min, or (v) 60 min after virus addition. Supernatants were assayed 24 h p.i. ** P < 0.001, * P < 0.01, two-tailed unpaired t-test. (E) MDCK cells were simultaneously infected with PR/8 (MOI = 0.0025) and treated with the indicated concentrations of the H+/K+-ATPase inhibitor esomeprazole for 60 min. Virus was titrated in supernatants collected 24 h p.i. Values are the mean (SE) from three experiments. (F) Left panel: MDCK cells were transfected simultaneously with the indicated concentrations of 2 siRNAs specific for the E2 subunit isoform (ATP6V1E2) of the V-ATPase V1 domain for 24 h prior to virus infection (PR/8, MOI = 0.0025). Virus- containing supernatants were harvested 24 h p.i. Values are the mean (SE) of three independent experiments. * P < 0.05, 1-way ANOVA with Dunnet’s post-test. Right panel: the relative percentage of viable cells was measured by MTT cell proliferation assay to rule out cytotoxicity caused by the siRNA treatment. Each bar represents the means (SE) from three independent experiments.

Article Snippet: Fluorescence-activated cell sorting One hour after virus addition, cells were detached with trypsin, fixed and permeabilized with Cytofix/Cytoperm solution (BD), and incubated with FITC-conjugated mouse anti-NP monoclonal antibody (clone IA52, 1:500; Argene) in PBS/3% BSA for 15 min on ice.

Techniques: Infection, Solvent, Virus, Staining, Control, Two Tailed Test, Isolation, Primer Extension Assay, Transfection, Plasmid Preparation, Expressing, MTT Cell Proliferation

Journal: The Journal of General Virology

Article Title: The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways

doi: 10.1099/vir.0.032060-0

Figure Lengend Snippet: Tx/91 and Tx/91 NS1 : 1–126 infection of A549 and HTBE human lung cells. (a) NS1 transcript levels from A549 and HTBE cells infected with Tx/91 (empty bars) and Tx/91 NS1 : 1–126 (filled bars). A549 cells were infected at an m.o.i. of 2 and HTBE cells were infected at an m.o.i. of 20. Mean log10(ratio) values were calculated by using the equation 2-ΔΔCt. 18S rRNA was used as an endogenous control. (b) Viral NP and NS1 protein expression in A549 cells. Cells were infected at an m.o.i. of 2 and cell lysates were collected at 6 and 24 h p.i. (c) Immunofluorescence staining for viral NP protein in Tx/91- and Tx/91 NS1 : 1–126-infected A549 and HTBE cells. A549 cells were infected at an m.o.i. of 2 and HTBE cells were infected at an m.o.i. of 20. In infected A549 cells, FITC fluorescence corresponds to viral NP protein. Images shown for A549 infections are at 40× magnification. Detection of viral NP protein in HTBE cells is visualized with Texas red-conjugated secondary antibody; FITC fluorescence corresponds to ciliated cells. Images shown for HTBE infections are at 63× magnification.

Article Snippet: Following fixation, cells were permeabilized with 1× PBS containing 0.5 % NP-40 and 0.01 % NaN 3 , and then blocked in 1× PBS [0.5 % NP-40, 0.01 % NaN 3 containing 10 % FBS] for 30 min. FITC-conjugated mouse monoclonal anti-NP antibody (1 : 200) (Biodesign International) was incubated with cells overnight at 4 °C.

Techniques: Infection, Control, Expressing, Immunofluorescence, Staining, Fluorescence