The HERO review: harnessing efficiencies, rethinking outcomes: the future of the defence estate

The HERO review: harnessing efficiencies, rethinking outcomes: the future of the defence estat

p53 expression and activity were not inhibited by 2′AP treatment of infected cells.

<p>(A) HepG2 and Hep3B cells were infected with Ad-dl309, Ad-dl1520 or Ad<b>Δ</b>E1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP for 2 days. p53 and E1a levels were detected by western blot analysis. (B) HepG2 and Hep3B cells were transfected with a p53-responsive firefly luciferase expression vector. The next day, transfected cells were mock infected or infected with Ad-dl309, Ad-dl1520 or Ad<b>Δ</b>E1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP. Luciferase expression was measured 2 days post-infection. Both HepG2 and Hep3B have high transfection efficiencies. Error bars correspond to +/−SD of triplicates.</p

2′AP increased virus-mediated death of HepG2 and Hep3B HCC cells but not normal fibroblasts.

<p>HepG2 and Hep3B HCC cells as well as MRC5 and WI-38 normal cells were infected at the indicated MOIs and then incubated with or without 2.5 mM 2′AP. Six days post-infection, cell survival was measured by Alamar Blue fluorescence measurements and normalized to uninfected controls. Error bars correspond to +/−SD of quadruplicates.</p

Schematic representation of the adenoviral genomes used in this study.

<p>Ad-dl309 encodes both E1a and E1b genes including their respective Ad promoters (pE1a and pE1b, respectively). The virus has a deletion in the E3 region spanning 30005–30750 bp <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065222#pone.0065222-Bett1" target="_blank">[81]</a>. Ad-dl309<b>Δ</b>VA and Ad-dl1520 have additional deletions in the VA-RNA genes (10667–10702 bp as well as 10929–10943 bp) or in the E1b-55K gene, respectively. Ad<b>Δ</b>E1b, Ad<b>Δ</b>E1b<b>Δ</b>VA and AdControl (<b>Δ</b>E1a<b>Δ</b>E1b) were constructed using the AdEasy-1 system and the pAdTrack shuttle plasmid. The pAd-Easy-1 plasmid has a larger deletion in the E3 region spanning 28130–30820 bp. The pAd-Track plasmid has E1 sequences spanning 480–3533 bp replaced with the EGFP gene under the control of the human cytomegalovirus immediate early (hCMV) promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065222#pone.0065222-He1" target="_blank">[53]</a>. The E1a gene under the control of the murine cytomegalovirus immediate early (mCMV) promoter was introduced back into the genomes of Ad<b>Δ</b>E1b and Ad<b>Δ</b>E1b<b>Δ</b>VA. Additionally, Ad<b>Δ</b>E1b<b>Δ</b>VA has the same VA-RNA deletions as Ad-dl309<b>Δ</b>VA.</p

2′AP increases the replication of adenoviruses with an E1b deletion or with both E1b and VA-RNA deletions.

<p>(A) Cells were infected in duplicate with Ad-dl309 and Ad<b>Δ</b>E1b at an MOI of 1 PFU/cell 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Cells and media were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined using plaques assays on HEK293 cells. (B) Cells were infected with Ad<b>Δ</b>E1b and Ad<b>Δ</b>E1b<b>Δ</b>VA at an MOI of 1 GFU/cell 1 hour prior to addition of medium containing no drug or 2.5 mM 2′AP. Cells and media were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined by titration in Hep3B cells. Error bars correspond to +/−SD.</p

Treatment of HepG2 cells with 2′AP increased expression of virally encoded E1a, fiber and GFP.

<p>(A) HepG2 or (B) MRC5 cells were mock infected or infected with the indicated viruses at MOI of 100 VP/cell and then incubated for 2 days or 4 days, with or without 2.5 mM 2′AP treatment. Cells were washed and lysed and western blot analysis was performed on 10 µg protein per lane using antibodies against E1a, fiber and β-actin. (C) HepG2 and MRC5 cells were infected with Ad<b>Δ</b>E1b, Ad<b>Δ</b>E1b<b>Δ</b>VA or AdControl at MOI of 100 VP/cell and then incubated in the absence or presence of 2.5 mM 2′AP. Green fluorescence intensity was measured 2 and 4 days post-infection and normalized to uninfected controls. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, one-way ANOVA).</p

2′AP increases the replication of an adenovirus with an E1b-55K deletion but not with a VA-RNA deletion.

<p>Cells were infected in duplicate with (A) Ad-dl309<b>Δ</b>VA, (B) Ad-dl1520, or (A and B) Ad-dl309 at a multiplicity of infection (MOI) of 1 plaque forming unit per cell (PFU/cell) 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Cells and media were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined using plaques assays on HEK293 cells. Error bars correspond to +/−SD.</p

Treatment of HepG2 and MRC5 cells with 2.5 mM 2′AP inhibited AdΔE1b and AdΔE1bΔVA release.

<p>Cells were infected with either Ad<b>Δ</b>E1b or Ad<b>Δ</b>E1b<b>Δ</b>VA at MOI of 1 GFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Four days following infection, infected cells combined with the media (total virus) or the media alone (released virus) were harvested. Virus yields were determined by titration in Hep3B cells. Error bars correspond to +/−SD of quadruplicates (*p<0.05, ***p<0.001, one-way ANOVA).</p

2′AP treatment increased both Ad-dl309 and AdΔE1b mediated HepG2 cell death.

<p>(A) parental HepG2, (B) HepG2-E1b-Mut and (C, D) HepG2-E1b-WT cells were infected with indicated viruses at MOI of 10 (D) or 100 (A, B, C) VP/cell and cultured in medium with or without 2.5 mM 2′AP. Survival was determined using an Alamar Blue assay 6 days post-infection. Fluorescence measurements were normalized to AdControl infected wells. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA).</p

2′AP rescued the C454S/C456S E1b-55K mutation in HepG2 cells.

<p>(A) Parental HepG2, HepG2-E1b-WT and HepG2-E1b-Mut were infected with Ad-dl309, Ad-dl1520 or Ad<b>Δ</b>E1b at MOI of 1 PFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Infected cells and media were harvested 4 days post-infection and virus yields were determine by plaque assays on HEK293 cells. (B) Cells were infected with Ad<b>Δ</b>E1b at MOI of 1 GFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Lysates from infected cells were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined by titration in Hep3B cells as described in the materials and methods. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ****p<0.0001, **p<0.01, one-way ANOVA).</p