Interaction between NP of pandemic Influenza A/Malaysia/854/2009(H1N1) virus with human THO complex 4 (THOC4) or Aly/REF (Experiment III).

<p>Mammalian two hybrid assay was conducted in 24-well plate. HEK293T cells were transfected with respective plasmids (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-t002" target="_blank">Table 2</a>) in triplicates, incubated at 37°C, 5% CO₂, for 48 hours. Culture media were harvested and tested for SEAP activity using GreatEscAPe™ SEAP Chemiluminescence Detection Kit (Clontech). The values reported are averages from 3 independent transfections (±S.D.). SEAP activity directly reflects the interaction between pandemic H1N1 NP and human THO complex 4 (THOC4). Results show that NP- human Aly/REF interaction was also relatively strong and statistically comparable (p-value >0.05) to when compared to the positive control pM3-VP16 and other 2 experiments conducted with different subtypes of Influenza.</p

Set-up for mammalian-two hybrid assays.

<p>Set-up for mammalian-two hybrid assays.</p

High-quality double-stranded cDNA generated using SMART™ cDNA synthesis and library complexity check <i>via</i> colony PCR amplification on randomly picked yeast colonies.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g001" target="_blank">Figure 1A</a> illustrates the agarose gel of total cDNA generated from whole lung of 4 weeks old specific pathogen free (SPF) white leghorn chicken using the Make Your Own “Mate & Plate” Library System. Lane M: 1kb ladder molecular weight standard. Lane 1: Purified total chicken lung cDNA before size selection with CHROMA SPIN + TE-400 columns. Lane 2: Reduced abundance of purified cDNA below 400 bp compared to lane 1 after selection with CHROMA SPIN + TE-400 columns. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g001" target="_blank">Figure 1B</a> shows the agarose gel of yeast colony PCR screenings of 10 colonies revived from a 1-ml stock of cDNA library cultured on SD/-Leu plates. Inserts of different sizes indicate the complexity of the library created. Lane M: 1 kb ladder molecular weight standard. Lane 1: positive control using pGADT7 vector as template, Lane 2: negative control. Lane 3 to 12: 10 randomly picked yeast recombinant clones from the constructed chicken lung cDNA library.</p

Molecular docking using crystal structures of human UAP56 (PDB: 1XTI) and H5N1 nucleoprotein (PDB: 2Q06).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g008" target="_blank">Figure 8A</a> shows ribbon representation of docking between H5N1 NP and human UAP56. Both the molecules shows favorable and tight binding with binding energy of 35.42 kcal/mol, which interestingly seems stronger than the previous one (3ULH and 2Q06). There are several residues of 1XTI that interact with 2Q06; they are marked red: Arg123, Gly150, Gly151, Glu298, Glu366, Ser382, andAsp393. Residues of 2Q06 that interact with 1XTI are marked blue: Ile41, Thr45, Lys48, Ser50, Asp51, Lys87, Asp101, Leu108, Leu110, Tyr111, Ala286, Ser287, and Gly288. Magnified version of the interacting residues are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g008" target="_blank">Figure 8C</a>.</p

Interaction between NP of Influenza A/New Jersey/1976/H1N1 virus with human THO complex 4 (THOC4) or Aly/REF (Experiment II).

<p>Mammalian two hybrid assay was conducted in 24-well plate. HEK293T cells were transfected with respective plasmids (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-t002" target="_blank">Table 2</a>) in triplicates, incubated at 37°C, 5% CO₂, for 48 hours. Culture media were harvested and tested for SEAP activity using GreatEscAPe™ SEAP Chemiluminescence Detection Kit (Clontech). The values reported are averages from 3 independent transfections (±S.D.). SEAP activity directly reflects the interaction between H1N1 NP and human THO complex 4 (THOC4). Results show that NP-Aly/REF interaction was relatively strong and statistically comparable (p-value >0.05) to when compared to the positive control pM3-VP16.</p

Molecular docking of human THOC4 or Aly/REF (PDB: 3ULH) and H5N1 nucleoprotein (PDB: 2Q06).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g003" target="_blank">Figure 3A</a> shows ribbon representation of docking between H5N1 NP and human Aly/REF. Both the molecules shows favorable and tight binding with binding energy of −22.81 kcal/mol. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g003" target="_blank">Figure 3B</a> shows the surface representation of the both interacting molecules. Aly/REF (3ULH) residues that interact with H5N1 nucleoprotein (2Q06) are red, and H5N1 nucleoprotein (2Q06) residues that interact with Aly/REF (PDB: 3ULH) are blue. Amino acid residues of 2Q06 that directly interact with 3ULH are, Ser69, Asp72, Arg74, Tyr78, Lys87, Asp88, Pro173, Ala366, and Ser367. Meanwhile, 3ULH residues that interact with 2Q06 are Leu119, Glu124, Leu125, Glu128, Gly130, Thr131, Ala157, Lys161, Lys164, Gln165, Asn167, Gly168, Asp172, and Gly173. Magnified version of the interacting residues are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g003" target="_blank">Figure 3C</a>.</p

Insert Check primers (Clontech).

<p>Insert Check primers (Clontech).</p

Knockdown of UAP56 impairs A/chicken/Malaysia/5858/2004 H5N1 virus growth.

<p>A549 cells were transfected with siRNA targeting UAP56 or with a non-targeting siRNA control as indicated. Fourty-eight hours later, depleted cells were infected with A/chicken/Malaysia/5858/2004 H5N1 at MOI of 5. Cells were harvested 48 hours after infection and viral titers were determined by plaque assays on Vero cells. Overall, knockdown of UAP56 decreased viral replication by nearly 10 fold when compared to non-treated. The mean ± SD of three independent experiments is shown.</p

Co-localization of HPAI H5N1 NP from A/chicken/Malaysia/5858/2004 and Aly/REF in nucleus of mammalian cells.

<p><b><i>A and B</i></b> Vero cells were seeded onto cover slips in a 24 well plate at a density of 10⁴/well. The cells are then infected with influenza A/chicken/Malaysia/5858/2004 virus at a multiplicity of infection (MOI) of 5 (<b><i>For A only</i></b>). A low (4°C) temperature pre-incubation method was used to allow synchronized infection for 1 h in DMEM medium supplemented with 2% BSA (GIBCO). After 1 h incubation, the cells were washed with DMEM once and then grown with DMEM supplemented with 0.2% BSA and 1 µg/ml N-p-tosyl-1-phenyl alanine chloromethyl ketone (TPCK) (Sigma Aldrich). At 12 hour p.i., cells were fixed and processed for immunostaining. NP was stained using anti-NP monoclonal primary antibody and Alexa594 conjugated secondary antibody (Red). Aly/REF was stained using Aly/REF specific primary antibody and Alexa488 conjugated secondary antibody (Green). Nuclei were stained with DAPI (Blue). <b><i>A</i></b> shows H5N1 infected cells whereas <b><i>B</i></b> shows control mock infected cells. Panels are labeled for their respective staining. Lower right panel in <b><i>A</i></b> shows primarily nuclear co-localization of NP and Aly/REF.</p

Knockdown of Aly/REF do not impair A/chicken/Malaysia/5858/2004 H5N1 virus growth.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g009" target="_blank">Figure 9A</a> shows the knockdown efficiency, briefly, low-passage A549 cells were transfected with siRNA directed at Aly/REF at a concentration of 100 nM at varying time points; 24, 48 and 96 hour according to the manufacturer's instructions, and the knockdown efficiency was checked by Aly/REF specific antibody, beta actin was used as loading control. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072429#pone-0072429-g009" target="_blank">Figure 9B, A</a>549 cells were transfected with siRNA targeting Aly/REF or with a non-targeting siRNA control as indicated. Fourty-eight hours later, depleted cells were infected with A/chicken/Malaysia/5858/2004H5N1 at MOI of 5. Cells were harvested 48 hours after infection and viral titers were determined by plaque assays on Vero cells. Overall, no significant reduction in virus yields was found in siRNA treated compared to non-treated infected cells.</p