Reverse-phase ODS elution profiles of PA-glycans obtained from three different fractions separated by the DEAE column.

<p>The neutral, mono-, and di-sialyl fractions were individually applied onto the ODS column and gave elution profiles according to their hydrophobicity.</p

Structures and relative quantities of neutral, mono- and di-sialyl PA-oligosaccharides derived from the porcine upper trachea, lower trachea and lungs of a pig.

<p>a, mol % was calculated from the peak area in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016302#pone-0016302-g003" target="_blank">Figure 3</a> by comparison with total <i>N</i>-glycan content in each porcine tissue. b, Structures of PA-oligosaccharides are represented by symbols as follows: red diamond, NeuAcα2-6; purple diamond, NeuAcα2-3; green diamond, NeuGcα2-6; light blue diamond, NeuGcα2-3; yellow circle with α, α-galactose(Gal); yellow circle, galactose; blue square, N-acetylglucosamine (GlcNAc); green circle, mannose (Man), red triangle, fucose (Fuc). c, Units of glucose (GU) were calculated from the elution times of the peaks obtained from the ODS column in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016302#pone-0016302-g003" target="_blank">Figure 3</a>.</p

Anion exchange DEAE elution profiles of PA-glycans derived from the porcine trachea (upper and lower) and lungs.

<p>The PA-glycans were fractionated according to their sialic acid content as neutral, mono-, and di-sialyl oligosaccharide fractions as indicated.</p

Amounts of α2,3- and α2,6-sialyl receptors in comparison with those of the other outer residues present on <i>N</i>-glycans derived from the porcine trachea (upper and lower) and lungs of a pig.

<p>Amounts of α2,3- and α2,6-sialyl receptors in comparison with those of the other outer residues present on <i>N</i>-glycans derived from the porcine trachea (upper and lower) and lungs of a pig.</p

A predominant quantity of Siaα2-6Gal receptor on <i>N</i>-glycans in the trachea (upper and lower) and lungs of a pig.

<p>A predominant quantity of Siaα2-6Gal receptor on <i>N</i>-glycans in the trachea (upper and lower) and lungs of a pig.</p

Distribution of Siaα2-3Gal and Siaα2-6Gal receptors in the porcine respiratory tract detected by lectin staining.

<p>Sections containing Siaα2-3Gal and Siaα2-6Gal receptors were reacted with DIG-labeled MAA and biotinylated SNA, respectively. DIG-MAA- and biotin-SNA-exposed sections were then reacted with anti-DIG-rhodamine (red) and avidin-fluorescein (green), respectively. Sections from representative tissues are shown at original magnification of x400. E =  epithelium, CC  =  ciliated cells, GC  =  goblet cells, BM  =  basement membrane, LP  =  lamina propria, TBC  =  terminal bronchiole.</p

A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

<div><p>Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV <i>in vitro</i> and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus <i>in vivo</i>. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. <i>In silico</i> docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, <i>in vitro</i> nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.</p></div

Model for potential binding of RK424 to NP.

<p><i>In silico</i> docking analysis was used to predict potential binding sites for RK424 on NP. The configuration with the highest binding energy was visualized using PyMol. (A) Crystal structure of NP. The nuclear export signal (NES) (yellow: amino acid (aa) 256–266), RNA binding grove (orange: aa 1–180), and dimer interface (purple: aa 482–489) are shown on the surface representation. The small pocket is highlighted by red circles. (B) Close-up of the NP small pocket. An electrostatic surface representation of the potential binding site on NP. Amino acids are colored blue (R162), pink (S165), red (L264), and green (Y487). (C) Purified wild-type NP-Flag (WT) and purified mutant NP-Flag (Mut) proteins harboring alanine substitutions at all four potential binding sites (162 165, 264, and 487) were added to RK424 cross-linked affinity beads or uncross-linked beads. Isolated NP-Flag (WT) and mutant NP-Flag (Mut) proteins were run in 10% SDS-PAGE gels and purity was checked by Coomassie Brilliant Blue (CBB) staining (upper panel). The binding of NP to RK424 beads was detected by western blotting with an anti-Flag MAb (lower panel). The positions of the NP-Flag proteins are indicated. Three independent experiments were performed and one representative result is shown. (D) Structure-activity relationship (SAR) analysis of RK424. The <i>in vitro</i> antiviral activity (IC₅₀) and cell toxicity (CC₅₀) of four different structural compounds derived from RK424 were evaluated in a plaque assay and in a WST-1 assay based on MDCK cells. Data are expressed as the mean ± SD of three samples in each of three independent experiments.</p

Effect of RK424 on viral transcription, replication, and translation.

<p>(A) HEK293T cells were transfected with pCAGGS expression plasmids encoding PB2, PB1, PA, NP, and vNP-luc in the absence or presence of RK424 (0.2, 0.5, and 2 μM). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h post-transfection. Data are expressed as the mean ± SD of three samples in each of three independent experiments. *;p<0.005 and **;p<0.001. (B) MDCK cells were infected with A/WSN/1933 (H1N1) at an MOI of 5 in the absence or presence of RK424 (0.2 and 2 μM) and then fixed at 6 h post-infection. The cells were probed with Quasar 670-labeled probes against the PB2 segment (magenta) and nuclei were stained with Hoechst (blue). Two independent experiments were performed and one representative result is shown. (C) Virus-infected cells were treated with RK424 (0.2 μM and 2 μM) for 18 h and the cell lysates subjected to western blotting with anti-WSN virus serum and an anti-β-actin monoclonal antibody (MAb). Bands representing HA, NP, NA, M1, M2, and actin are indicated. Oseltamivir phosphate (Os) was used as the negative control. Two independent experiments were performed and one representative result is shown.</p

Effect of RK424 on nuclear export of NP and the binding of NP to CRM1.

<p>(A) <i>In vitro</i> nuclear export assay incorporating NP-mRFP-Flag. Cells expressing NP-mRFP-Flag were permeabilized with digitonin and incubated for 1 h without (Buffer) or with HeLa cell lysate (+Lys) in the presence of the RK424 at the indicated concentrations (upper panels). Leptomycin B (LMB) was used as a positive control (LMB inhibits nuclear export of NP). Nuclei were stained with Hoechst 33342 (lower panels). The export of NP-mRFP-Flag was monitored by measuring mRFP fluorescence in the nucleus. Three independent experiments were performed and one representative result is shown. (B) Quantification of mRFP fluorescence in the nucleus. The nuclear localization of NP-mRFP-Flag was determined by monitoring mRFP fluorescence in the nucleus (> 100 cells). Data are expressed as the mean ± SD of three independent experiments. *;p<0.05 and **;p<0.005. (C) RK424 inhibits the binding of NP-mRFP-Flag to CRM1. FLAG agarose beads coupled to NP-Flag (NP-flag beads) were subjected to SDS-PAGE and the purity of the NP-Flag protein was checked by Coomassie Brilliant Blue (CBB) staining (left panel). HeLa cell lysate was incubated with NP-Flag beads in the absence or presence of RK424 (0.5, 5, or 10 μM). LMB was used as a positive control. Binding of CRM1 to NP was detected by western blotting with an anti-CRM1 MAb (right panel). EtOH and DMSO were used as vehicle controls. The positions of NP-Flag and CRM1 are indicated. Two independent experiments were performed and one representative result is shown.</p