FAST protein expression in A549 cells decreases cellular metabolic activity, promotes apoptosis and concomitant loss of membrane integrity.

<p><b>A)</b> A549 cells were infected at an MOI of 10 with AdEmpty or AdFAST. Cellular metabolic activity was assessed every 24 h until 96 hpi using an MTS assay. The average of three independent experiments (n = 3) done in triplicate is plotted with the standard error of the mean. Values were normalized to mock infected cells at 24 hpi. *p<0.05 compared to mock infected cells at their corresponding timepoints. **p<0.05 comparing AdFAST to AdEmpty infected cells. <b>B)</b> A549 cells were infected with AdEmpty or AdFAST at an MOI of 1, 10, 50 or 100. An MTS assay was conducted 72 hpi. The average of three independent experiments (n = 3) done in triplicate ± the standard error of the mean is shown with normalization to mock infected cells. *p<0.05 compared to mock infected cells. **p<0.05 comparing AdFAST to AdEmpty treated cells. <b>C)</b> A549 cells were treated with AdEmpty or AdFAST at an MOI of 10, or left uninfected, and crude protein extracts prepared 24 hr later and examined for total or cleaved caspase 3 by immunoblot. As positive controls, A549 cells were treated with 100 μM etoposide for 24 h, or 1 μM staurosporine for 6 h. Alpha-tubulin served as a loading control. <b>D)</b> A549 cells were infected with AdEmpty, AdFAST or VSVΔ51 using a range of MOI. Relative cell membrane integrity was measured based on the lactate dehydrogenase levels in the supernatant 72 hpi. The average of two independent experiments (n = 2) is shown with each experiment done in triplicate. Error bars denote the standard error of the mean. *p<0.05 compared to cells only. **p<0.05 when comparing AdFAST to AdEmpty infected cells.</p

FAST protein expression induces extensive cell fusion in 293 cells.

<p><b>A)</b> 293 cells were infected with AdEmpty or AdFAST at an MOI of 1 and stained with Giemsa stain 18 h later. Images were captured using bright field microscopy (20x objective). A region of fused cells for the AdFAST-treated cells is outlined with a dotted line, and several nuclei within the syncytium are indicated with asterisks (*) <b>B)</b> Fusion index of 293 cells infected with AdEmpty or AdFAST. The fusion index for two fields of view were determined, and the average with the standard deviation is depicted in the graph. <b>C)</b> 293 cells were infected with AdRFP or AdFAST/RFP at MOI 10 and observed using fluorescence microscopy at 48 h post infection. All images were taken using a 20x objective. <b>D)</b> 293 cells were infected at a MOI of 10 with AdEmpty or AdFAST and relative metabolic activity was determined using an MTS assay over a 96 h interval every 24 h. Experiments were completed in triplicate and the average of three independent experiments is shown (n = 3). Values were normalized to mock infected cells at 24 hpi. Error bars denote the standard error of the mean. *p<0.05 compared to mock infected cells. **p<0.05 when comparing AdFAST to AdEmpty infected cells.</p

Ad-mediated FAST protein expression from an E1-deleted vector does not affect virus growth or yield.

<p><b>A)</b> All viruses are early region 1 (E1) and early region 3 (E3) deleted. AdFAST-HA has a single HA tag linked to the C terminus through a glycine linker. ITR denotes inverted terminal repeats and Ψ is the packaging signal. RFP is the red fluorescent protein and CMV represents the cytomegalovirus enhancer/promoter. <b>B)</b> 293 and A549 cells were infected with the control virus AdEmpty or AdFAST-HA at an MOI of 10. Whole cell lysates were collected 48 hpi. Samples were probed for the HA tag, Ad5 fibre and alpha-tubulin for a loading control. The top band denoted by * in the A549 samples is non-specific. <b>C)</b> 293 cells were infected with AdEmpty or AdFAST at an MOI of 1 and whole cell lysates were collected at 6, 18, 24 and 30 hpi. Samples were probed for Ad5 fibre and alpha-tubulin was used as a loading control. <b>D)</b> Supernatants collected from 293 cells infected with AdEmpty or AdFAST at an MOI of 1 were used to conduct plaque forming assays to determine the number of viral progeny at various times post-infection.</p

AdFAST does not induce fusion or promote survival in immunodeficient CD-1 mice with subcutaneous A549 tumors.

<p><b>A)</b> CD-1 nude mice harbouring subcutaneous A549 tumors were intratumorally injected with PBS, 5x10⁸ pfu AdEmpty or AdFAST. Five days post injection, tumors were excised, fixed, sectioned and subjected to hematoxylin and eosin staining. Results are representative of 2–3 mice (10x objective). <b>B)</b> CD-1 mice with subcutaneous A549 tumors intratumorally injected with PBS, 5x10⁸ pfu AdEmpty or AdFAST were measured at day 20 post injection. The line in each column represents the average of the associated treatment group. <b>C)</b> A Kaplan-Meier survival curve shows the percentage survival of CD-1 mice with subcutaneous A549 tumors intratumorally injected with PBS, AdEmpty or AdFAST over time. Each treatment group consisted of 5 mice.</p

Analysis of MvaT_ctd-bound DNA conformation.

<p>(A) Selected helix parameters of the solution structures of MvaT_ctd-bound 3AT DNA (blue), a free DNA with ATATAT sequence (PDB 2LWG) (magenta) and an A-tract DNA with AAAAAA sequence (PDB 1FZX [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004967#ppat.1004967.ref052" target="_blank">52</a>]) (gold). Average minor groove width, roll and inclination angles including standard deviations are shown. Base steps of 3AT are indicated. (B) Mean structures of MvaT_ctd-bound 3AT (blue), 2LWG (magenta) and 1FZX (gold). Axes are shown by sticks.</p

Restraints and structural statistics for MvaT_ctd and its complex with DNA.

<p>*Pairwise RMSD was calculated among 20 refined structures over residues A79-G124 (MvaT_ctd) and/or C1-G24 (DNA).</p><p>Restraints and structural statistics for MvaT_ctd and its complex with DNA.</p

Sequence alignments and structural comparisons of MvaT_ctd with AT-hook-like motif containing DNA-binding domains.

<p>(A) Alignment of <i>Pseudomonas</i> MvaT with members of the H-NS family reveals regions of similarity outside of the canonical H-NS motif. Clustal Omega alignment of C-terminal domains of MvaT and MvaU from <i>P</i>. <i>aeruginosa</i> PAO1 with Bv3F from <i>Burkholderia vietnamiensis</i> G4 (Buv), H-NS from <i>S</i>. <i>typhimurium</i> (Sal), <i>Xanthomonas albilineans</i> (Xan), and <i>X</i>. <i>fastidiosa</i> (Xyl), and Lsr2 from <i>M</i>. <i>tuberculosis</i>. The canonical H-NS motif is boxed. In bold are the residues corresponding to the AT-hook-like motif in Lsr2 and H-NS. (B) Superimposing structures of MvaT_ctd (cyan) and <i>S</i>. <i>typhimurium</i> H-NS_ctd (magenta). Side chain conformations of K97 and N100 of MvaT_ctd are nearly identical to those of T110 and R114 of H-NS_ctd, respectively. MvaT_ctd lacks the corresponding residue of Q112, functioning as the first critical residue of the “Q/RGR” AT-hook-like structure in H-NS_ctd. When MvaT_ctd is complexed with DNA, side chain of R80 occupies similar position as the side chain of Q112 from H-NS_ctd. A blue circle is drawn to indicate the gap between the R80 side chain and the backbone of loop2. (C) MvaT_ctd/DNA complex structure viewed from one end of the double helix. The cavity above A6-T19 base pair is shown by red spheres.</p

Structure of MvaT_ctd and AT-rich DNA complex.

<p>(A) Superimposition for the ensemble of 20 structures of MvaT_ctd/DNA complex. Protein backbone is represented by ribbon and the target DNA is shown as lines. (B) Electrostatic potential of MvaT_ctd, calculated with APBS in the absence of DNA. (C) Close view of the binding interface. Residues of MvaT_ctd involved in DNA recognition are shown as sticks with one-letter amino acid code and residue number labeled. (D) Schematic representation of the intermolecular contacts. The DNA is drawn as a cylindrical projection viewed from the minor groove side. Hydrogen bonds between MvaT_ctd and DNA bases are indicated by red arrows. Green lines show the hydrophobic contacts. The interactions between positively charged lysine residues and DNA backbone phosphate groups are indicated by blue arrows.</p

DNA competition binding assay for MvaT binding to <i>cupA1</i> promoter DNA and <i>PA3900</i> fragment.

<p>Complexes of MvaT (300 nM) with 1 nM radiolabeled AT-rich <i>cupA1</i> promoter DNA fragment (upper panel) or a radiolabeled GC-rich <i>PA3900</i> DNA fragment (lower panel), were pre-formed prior to the addition of excess unlabeled competitor DNA as indicated.</p