What Happened? An Instructional Model for Promoting Student Research Into World War II

List of immune-related genes showing a significantly attenuated expression 3 and 6 dpi in p53KO mice as compared to p53WT mice. (XLS 104 kb

Additional file 4: of Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection

GO analysis for biological process. (PPT 403 kb

Nonstructural Protein 1 of Influenza A Virus Interacts with Human Guanylate-Binding Protein 1 to Antagonize Antiviral Activity

<div><p>Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner <em>in vitro</em>. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1.</p> </div

Overexpression of hGBP1 inhibited PR8 replication.

<p><b><i>A.</i></b> A549 cells were transfected with plasmid Myc-hGBP1-wt or empty vector (Vec) and infected with PR8 at MOI = 1 after 24 h. Viral titers in transfectants were measured by plaque assay at 24 hpi. <b><i>B</i></b> and <b><i>C.</i></b> A549 cells were transfected with increasing amounts of plasmid Myc-hGBP1-wt and infected with PR8 at MOI = 1 after 24 h. Empty vector (Vec, 3 µg) was transfected in parallel as a control. <b><i>B.</i></b> HA and NP mRNA in transfectants was analyzed at 24 hpi by qRT-PCR. <b><i>C.</i></b> Myc-hGBP1-wt and NP were detected at 24 hpi by western blot. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055920#s3" target="_blank">Results</a> are means with SD from three independent experiments. *, <i>p</i><0.05 compared with cells transfected with empty vector.</p

Mapping the hGBP1-binding region of NS1.

<p><b><i>A.</i></b> Schematic representation of Flag-tagged wild-type NS1 and truncated mutants. <b><i>B.</i></b> H1299 cells were transfected with the indicated plasmids in the presence of plasmid Myc-hGBP1-wt. Transfectants were collected after 36 h and immunoprecipitated with anti-Myc antibody. <b><i>C.</i></b> H1299 cells were transfected with plasmid Flag-NS1-wt or Flag-NS1-Δ123/144 in the presence of plasmid Myc-hGBP1-wt. Transfectants were collected after 36 h and immunoprecipitated with anti-Myc antibody. IP, immunoprecipitation. IB, western blot.</p

Additional file 3: Table S3. of Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection

List of genes expressed 3 and 6 dpi in p53KO mice with an expression significantly higher than in p53 WT mice. (XLS 324 kb

Detection of interaction between NS1 and hGBP1.

<p><b><i>A.</i></b> Immunoprecipitation assay for detecting interaction between NS1 and hGBP1. H1299 cells were transfected with the indicated plasmids. Transfectants were harvested after 36 h and subjected to immunoprecipitation and western blot with anti-Myc or anti-Flag. IP, immunoprecipitation. IB, western blot. <b><i>B.</i></b> BiFC assay for detecting interaction between NS1 and hGBP1. A549 cells were transiently transfected with indicated plasmids and incubated for 20 h. Fluorescence emission and brightfield were visualized. <b><i>C.</i></b> Indirect immunofluorescence assay for detecting colocalization of NS1 and hGBP1. A549 cells were transfected with plasmid Myc-hGBP1-wt or Myc-vector and incubated for 12 h before infection with PR8 at MOI = 1 or mock-infection and incubation for 24 h. Cells were double-immunostained for Myc-hGBP1 (red) and NS1 (green). Nuclei were counterstained with DAPI (blue). <b><i>D.</i></b> Percentage of cells with cytoplasmic localization of NS1. The number of cells with cytoplasmic localization of NS1 in ten randomly selected visual fields was counted. The percentage of cells with cytoplasmic localization of NS1 was calculated and plotted. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055920#s3" target="_blank">Results</a> are presented as the means ± S.E. from three independent experiments. **, <i>p</i><0.01 compared with cells transfected with PR8+Myc-hGBP1-wt.</p

Detection of interaction between hGBP1-K51A and NS1.

<p>H1299 cells were transfected with plasmid Myc-hGBP1-wt or Myc-hGBP1-K51A in the presence of plasmid Flag-NS1-wt. Transfectants were collected after 36 h and immunoprecipitated with anti-Myc and anti-Flag antibodies. IP, immunoprecipitation. IB, western blot.</p

Upregulated expression of hGBP1 in PR8-infected cells.

<p>A549 cells were infected with PR8 strain or mock-infected (Mock) and collected at 0, 6, 12, 24 and 48 h post-infection (hpi). <b><i>A.</i></b> The hGBP1 mRNA was detected by qRT-PCR. <b><i>B.</i></b> Viral HA and NP mRNA in PR8-infected A549 cells was detected by qRT-PCR. <b><i>C.</i></b> The hGBP1, viral NS1 and NP in PR8-infected A549 cells was detected by western blot. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055920#s3" target="_blank">Results</a> are means with SD from three independent experiments. *, <i>p</i><0.05 compared with sample collected at 0 hpi.</p

Overexpression of hGBP1-K51A did not inhibit IAV replication.

<p><b><i>A.</i></b> A549 cells were transfected with plasmid Myc-hGBP1-wt, Myc-hGBP1-K51A or Myc-vector and infected with PR8 at MOI = 1 after 24 h. Viral titers in transfectants were measured by plaque assay at 24 hpi. <b><i>B</i></b> and <b><i>C.</i></b> A549 cells were transfected with increasing amounts of plasmid Myc-hGBP1-K51A and infected with PR8 at MOI = 1 after 24 h. Empty vector (Vec, 3 µg) was transfected in parallel as a control. <b><i>B.</i></b> HA and NP mRNA in transfectants was analyzed at 24 hpi by qRT-PCR. <b><i>C.</i></b> Myc-hGBP1-K51A and NP were detected at 24 hpi by western blot. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055920#s3" target="_blank">Results</a> are means with SD from three independent experiments. *, <i>p</i><0.05 compared with cells transfected with Myc-hGBP1-wt.</p