A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

<div><p>Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes.</p></div

Western blotting using anti-protein VII antibodies.

<p>U2OS cells were either mock-infected (lanes 1, 3, 5, and 7) or infected with Ad5 (lanes 2, 4, 6, and 8) and collected at 24 hpi. Cell lysates were prepared and subjected to 12% SDS-PAGE. Proteins were transferred to membranes and subjected to western blot analyses using mouse (lanes 1 and 2), rabbit (lanes 3 and 4), or rat anti-protein VII antibodies (lanes 5 and 6) or Ponceau Red staining as loading control (lanes 7 and 8).</p

Live-cell imaging using EGFP-TAF-Iβ.

<p>(A) Live-cell imaging with EGFP-TAF-Iβ constructs. U2OS cells were transiently transfected with the expression vectors for either EGFP-TAF-IβWT (green, left) or PME mutant (green, right panels) and at 24 hpt were either mock-infected or infected with Alexa594-labeled Ad5-GFP (magenta). At 3 hpi, live-cell imaging was performed, frames were taken every 3 s for 3 min, and snapshots from the movies are shown. Full movies are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137102#pone.0137102.s004" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137102#pone.0137102.s005" target="_blank">S2</a> Movies for TAF-IβWT and TAF-IβPME, respectively. (B) Series of snapshots of EGFP-TAF-IβWT. Selected images from the time series are shown. An image of maximum intensity projection generated by superimposing the frames of the time series is shown below, showing limited movement of EGFP-TAF-IβWT dots.</p

IF analyses using anti-protein VII antibodies.

<p>(A) IF analyses with mouse and rat anti-protein VII antibodies. U2OS cells were either mock-infected or infected with Ad5 and at 3 hpi subjected to IF analyses with mouse (green) and rat anti-protein VII antibodies (red). Merged images with DAPI staining (gray) are also shown. (B) IF analyses with rabbit and rat anti-protein VII antibodies. IF analyses were carried out as described in (A) but using rabbit (cyan) and rat anti-protein VII antibodies (red). Arrows indicate cytoplasmic protein VII signals. (C) IF analyses with Alexa-labeled viruses. U2OS cells were first incubated with Alexa488-labeled Ad5-GFP (green) at 4°C for adsorption, and then transferred to 37°C. After 0, 20, and 60 min, cells were collected for IF analyses using rabbit (cyan) and rat anti-protein VII antibodies (red). Asterisks indicate protein VII signals at the nuclear rim. (D) IF analyses using cells at late phases of infection. U2OS cells were either mock-infected or infected with Ad5, and at 24 hpi subjected to IF analyses using either mouse and rat (green and red, left panels) or rabbit and rat anti-protein VII antibodies (cyan and red, right panels). DAPI staining (gray) and merged images were shown for mock and infected cells, respectively. For right panels, magnified images of the regions marked by squares are also shown.</p

IF analyses using EGFP-TAF-Iβ.

<p>(A) IF analyses with anti-TAF-Iβ antibody. U2OS cells were either mock-infected or infected with Ad5. At 3 hpi, cells were subjected to IF analyses using mouse anti-TAF-Iβ (green) and rat anti-protein VII antibodies (red). (B) IF analyses with EGFP-TAF-Iβ. U2OS cells were transiently transfected with the expression vector for EGFP-TAF-Iβ (green) and at 24 hpt (hours post transfection) were either mock-infected or infected with Ad5. At 3 hpi, cells were either immediately fixed (first and second rows) or pre-extracted with Triton X-100 and then fixed (third row, + Triton) and subjected to IF analyses using rat anti-protein VII antibody (red). Nuclear shapes are indicated by dashed lines. (C) IF analyses with TAF-IβPME mutant. U2OS cells were transiently transfected with the expression vectors for either EGFP-TAF-IβWT (left) or PME mutant (right panels), together with the one for histone H2B-tdiRFP (gray), and at 24 hpt IF analyses were carried out as described above.</p

<i>Tb</i>SAXO RNAi knockdowns exhibit impaired flagellar motility.

<p>Inducible RNAi<i>^TbSAXO</i> in PCF (<b>a, b, c, d</b>) and BSF (<b>e, f, g, h</b>) cells. Growth curves of PCF (<b>a</b>) and BSF (<b>e</b>) RNAi<i>^TbSAXO</i> cell lines. Corresponding WBs (PCF in <b>b</b>, BSF in <b>f</b>) of WT (parental), RNAi non-induced (-), and 24 h and 96 h induced cells probed with mAb25 and L8C4 (anti-PFR2). For PCF 5.10⁶ cells were used and 1.25×10⁵ cells for BSF. <b>c</b>. Sedimentation assay of PCF RNAi. WT (closed squares). RNAi non-induced (−TET) (closed triangles) and induced (+TET) (open circles). <b>d</b>. Mobility graph obtained from Movie S1. The positions of individual cells are plotted at 2.5 s intervals. Open circle: starting position of each cell. Arrowhead: ending position. Number in parentheses: time in seconds of a given cell was within the field of view. Bar, 10 µm. <b>g</b>. Graph of cell populations with orthodox and unorthodox kinetoplast number in BSF RNAi cultures (72 h of induction). K: kinetoplast. N: nucleus. Asterisks indicate statistical significance compared with the WT population, and −TET <i>versus</i> +TET condition (*<i>P<0.1</i>; ** <i>P<0.05</i>; ***<i>P<0.01</i>). <b>h</b> Electron-micrograph of a thin section of an aberrant BSF RNAi induced cell (72 h). (*) indicates a flagellum. Scale bar, 2 µm. Error bars in a, c, e, and g represent the standard error from 3 independent experiments.</p

<i>Tb</i>SAXO is an axoneme-associated protein in <i>T. brucei</i>.

<p><b>A</b>. Immuno-labeling and localization of <i>Tb</i>SAXO on PCF cytoskeletons. Left panel: <i>Tb</i>SAXO localization in the flagellum was identified by the mAb25 antibody (green). Labeling extends along the entire length of the flagellum from the flagellar transition zone (*, labeled with the FTZC antibody) to the distal tip. The PFR is labeled red and begins where the flagellum exits the cell (antibody L8C4). Right panel: a merge of IF and phase contrast. N: nucleus. K: kinetoplast. F: flagellum. Bar, 5 µm. <b>B</b>. Images of the proximal flagellar regions of PCF cytoskeletons from cells through mitosis and cytokinesis. In each row, the left panel shows the PFR and FTZC (*) (red), the center panel <i>Tb</i>SAXO (green), and the right panel shows merged images. The cell cycle stages are defined as 1K1N1F (1 Kinetoplast, 1 Nucleus, 1 Flagellum), 1K1N2F, 2K1N2F and 2K2N2F in rows a–d respectively. <i>Tb</i>SAXO labeling is present immediately distal to the FTZC and is clearly distinct from PFR staining. Bar, 1 µm. <b>C</b>. Immuno-gold electron microscopy reveals that <i>Tb</i>SAXO is localized in the axoneme. Mab25 immuno-gold particles can be seen mainly on the axoneme and not on the PFR of flagella of PCF WT cells. Bars, 100 nm.</p

Identification of SAXO proteins, a MAP6-related protein family.

<p><b>A</b>. Motif 1. The N-terminal domain and its cysteine consensus sequence. Left panel: alignment of the N-terminal sequences of the proteins used for the MEME analysis in C. The boxed sequences correspond to motif 1. Amino acids corresponding to the regular expression of motif 1 are shown in blue. Right panel: motif 1 is represented as a position-specific probability matrice derived from the MEME analysis in C. <b>B</b>. Motif 2. Mn domains in mouse Map6-1, Map6d1, and Mn-like domains and inter-repeats in mouse Saxo1, <i>Plasmodium</i> SAXO and <i>Trypanosoma</i> SAXO. Left panel: characters in blue correspond to the regular expression of the Mn and Mn-like domains identified by the MEME analysis in C; the Mn-like domains identified manually are in italics. The underlined sequences in Map6-1 and Map6d1 correspond to the experimentally identified Mn domains in mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031344#pone.0031344-GoryFaure1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031344#pone.0031344-Bosc2" target="_blank">[6]</a>. CP motifs are boxed. IR: inter-repeat regions. Right panel: the Mn-like regular expression is represented as position-specific probability matrix derived from MEME analysis in C. <b>C</b>. Identification of a family of proteins containing Mn-Like domains. MEME analysis using mouse Map6s, mouse Saxo1, and only protozoan SAXO sequences identified a characteristic N-terminal motif (motif 1, dark blue boxes) in SAXO proteins and Mn-like domains (motif 2, light blue boxes) common to the SAXO and MAP6 proteins. Manually identified supplementary motifs 2 are in grey boxes (Motif 2 manual). The asterisk indicates a conserved CP sequence in the last Mn-like domain.</p

<i>Tb</i>SAXO is a microtubule-associated protein and a microtubule-stabilizing protein.

<p>Mammalian cells (U-2 OS) expressing either MAP6-1-GFP (row a), <i>Tb</i>SAXO-Myc (row b), or various truncated versions of <i>Tb</i>SAXO-Myc (rows c–j) (constructs are represented on the schemes on the right panel). In each case, the transfected cells were incubated at 37°C or 4°C to test for MTs cold stability. Anti-tubulin (TAT1) and anti-Myc antibodies provided the images in left and centre columns respectively at each temperature. The right columns for each temperature set are merged images. The cells were subjected to short extraction before fixation and immuno-labeling. <i>Tb</i>SAXO MT stabilization is seen in images b, c, e, h and j. MT stabilization is also observed in the positive control MAP6-1-GFP expressing cells (a). Nuclei were labeled with DAPI. Bar, 20 µm.</p

Analysis of <i>T. b. gambiense</i> organs and central nervous system invasion in BALB/c-infected mice.

<p>A. Immunohistochemical detection of trypanosomes in the brains of mice (n = 2, only results from one mouse are shown) infected for 4 months with 10⁶ parasites of the <i>Tbg</i>945b isolate (1, 2, 3) and of paralyzed mice (n = 2) treated with cyclophosphamide before infection with either 5×10⁶ parasites of subchronic <i>Tbg</i>1122c or <i>Tbg</i>1166c isolates. Paralysis occurred 10 and 6 months PI with <i>Tbg</i>1122c and <i>Tbg</i>1166c isolates respectively. Only results from <i>Tbg</i>1122c are shown (4, 5, 6). A1 and A4 correspond to an olfactory bulb coronal section, A2 to a forebrain section, A3 and A5 to brain stem sections and A6 to a cerebellum section. Whole brain invasion was observed with the chronic isolate at an advanced stage of the disease (4 months PI, death within 6–8 months). Invasion was restricted to the olfactory bulb and the brain stem (including the cerebellum for <i>Tbg</i>1122c) in paralyzed mice infected with the sub-chronic isolates after treatment with cyclophosphamide. No invasion was observed in mice (n = 2) infected for 9 months with 5×10⁶ parasites of subchronic <i>Tbg</i>1166c isolate (data not shown). B. Spatial distribution of R-Luc activity in animals developing a sub-chronic or a silent infection and treated with or without cyclophosphamide (+/−cyclo). BALB/c mice were either directly infected with 10⁶ LucR-<i>Tbg</i>1135b (n = 6) or LucR-<i>Tbg</i>1135c (n = 2) or treated 24 h before infection with cyclophosphamide (n = 2). At different time PI, mice were anaesthetized and injected intravenously (i.v., retro-orbital) or intraperitoneally (i.p.) with coelenterazine and BLI signals were recorded in real time with a Biospace Imaging System. The panels show dorsal and ventral images of 2 representative mice infected for 8–11 weeks: LucR-<i>Tbg</i>1135b (11 weeks), LucR-<i>Tbg</i>1135c (10 weeks), LucR-<i>Tbg</i>1135b+cyclo (9 weeks), LucR-<i>Tbg</i>1135+cyclo (8 weeks). C. Spatial distribution of R-Luc activity in organs removed from LucR-Tbg1135 infected BALB/c mice. The different organs shown in this figure were isolated from mice: (1) non infected (control) (2) infected for 18 weeks with 10⁶ LucR-<i>Tbg</i>1135b, (3) infected for 18 weeks with 10⁶ LucR-<i>Tbg</i>1135c, (4) pre-treated with cyclophosphamide and infected for 16 weeks with 10⁶ LucR-<i>Tbg</i>1135c. Quantification data of light emission signals for ROI delimitating each organ are given in photons/second/cm²/steradian (p/sec/cm²/sr).</p