Detection of Ad14 PtDd by Western blot analysis.

<p>HeLa cells were infected with wild-type Ad3, Ad14, and Ad5 at an MOI of 500 vp/cell for 7 hours. Thirty-six hours after infection, cell lysates were subjected to ultracentrifugation on a sucrose gradient. Fractions were analyzed by Western blot using a polycloncal antibody against Ad3 PtDd, which also cross-reacts with Ad5 and Ad14 penton base.</p

Detection of PtDd in infected cells.

<p>HeLa cells were infected with wt-Ad3GFP or mu-Ad3GFP at an MOI of 500 VP/cell. Thirty six hours later cells were collected and cell lysates subjected to ultracentrifugation in a 15–40% sucrose step gradient. Fractions with different densities were then analyzed by Western blot using polyclonal antibodies raised against purified PtDd. The antibody recognizes Ad3 penton base (61.8 kDa) and fiber (34.8 kDa). The band at ∼50 kDa is most likely a fiber derivative. Blots were also hybridized with anti-Ad antibodies that recognize Ad3 hexon (∼90 kDa) and with anti-actin antibodies (loading control).</p

Penton-Dodecahedral Particles Trigger Opening of Intercellular Junctions and Facilitate Viral Spread during Adenovirus Serotype 3 Infection of Epithelial Cells

<div><p>Human adenovirus serotypes Ad3, Ad7, Ad11, and Ad14 use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. During Ad infection, the fiber and penton base capsid proteins are produced in vast excess and form hetero-oligomers, called pentons. It has been shown for Ad3 that pentons self-assemble into penton-dodecahedra (PtDd). Our previous studies with recombinant purified Ad3 PtDd (produced in insect cells) showed that PtDd bind to DSG2 and trigger intracellular signaling resulting in the transient opening of junctions between epithelial cells. So far, a definitive proof for a function of Ad3 PtDd in the viral life cycle is elusive. Based on the recently published 3D structure of recombinant Ad3 PtDd, we generated a penton base mutant Ad3 vector (mu-Ad3GFP). mu-Ad3GFP is identical to its wild-type counterpart (wt-Ad3GFP) in the efficiency of progeny virus production; however, it is disabled in the production of PtDd. For infection studies we used polarized epithelial cancer cells or cell spheroids. We showed that in wt-Ad3GFP infected cultures, PtDd were released from cells before viral cytolysis and triggered the restructuring of epithelial junctions. This in turn facilitated lateral viral spread of <i>de novo</i> produced virions. These events were nearly absent in mu-Ad3GFP infected cultures. Our <i>in vitro</i> findings were consolidated in mice carrying xenograft tumors derived from human epithelial cancer cells. Furthermore, we provide first evidence that PtDd are also formed by another DSG2-interacting Ad serotype, the newly emerged, highly pathogenic Ad14 strain (Ad14p1). The central finding of this study is that a subgroup of Ads has evolved to generate PtDd as a strategy to achieve penetration into and dissemination in epithelial tissues. Our findings are relevant for basic and applied virology, specifically for cancer virotherapy.</p></div

PCR primers for mu-Ad3GFP plasmid construction.

<p>PCR primers for mu-Ad3GFP plasmid construction.</p

Viral spread in polarized T84 cells.

<p><b>A and B</b>) Viral spread in T84 cells cultured in transwell chambers. Cells were cultured as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003718#ppat-1003718-g004" target="_blank">Fig. 4</a>. Ad vectors were added to the inner chamber at an MOI of 10 vp/cell. <b>A</b>) Confocal microscopy images (XY planes) of the cell surface and of a layer that is 20 µm beneath the cell surface were taken 4 days after infection. Transduced cells express GFP (green). The scale bar of all images is 20 µm. <b>B</b>) Titration of progeny viruses in the inner chamber and in the outer chamber. T84 cells, which had been cultured in 0.4 µm transwell inserts for 16 days, were infected from the apical side with Ad3 vectors at the indicated MOIs for 2 hours. Supernatants were collected from the inner chambers at days 4, 7, 9 and 10 post-infection, and from the outer chambers at days 9 and 10, and titered for GFP-expressing units on 293 cells. The medium were changed at days 4, 7, 9 and 10 post-infection. Shown are data from samples collected at day 9 post-infection. N = 3. The differences between wt-Ad3GFP and mu-Ad3GFP titers in the inner chamber are not significant (n.s.). *P<0.01. Virus titers in inner chamber were about 5 higher on day 7 than on day 4. Viral titers in the inner chamber remained stable from day 7 on (data not shown). Note that, the filter pore size of the chambers is 400 nm and should allow for the passage of 100 nm Ad virions). <b>C</b>) Viral spread in the presence of recombinant PtDd added to the inner chamber. T84 cells were cultured as desrcibed in B) and infected (in triplicate) with mu-Ad3GFP and wt-Ad3GFP at an MOI of 1vp/cell. Recombinant PtDd (5 µg/ml) was added to to the inner chamber on days 3, 5, and 7 post-infection. Medium in both chambers was changed on days 4, 6, 8, and 10 p.i. Day 8 and 10 medium samples from the outer chamber were then titered on 293 cells for progeny virus that has passed through the layer of T84 cells based on GFP epressing/infectious units. N = 6, *- p<0.01, n.s.-not significant.</p

Ad3 penton base mutations that prevent the formation of PtDd.

<p><b>A</b>) Localization of D100 (in red) and R425 (in blue) in the Ad3 dodecahedron context (PDB accession 4AQQ). The panel shows a representative view of contacts taking place at the interface between adjacent pentamers in a slab view. <b>B</b>) Two out the five monomers constitutive of a penton base are coloured in blue and green with D100 and R425 highlighted. The salt bridge between these monomers is shown. <b>C</b>) Amino acids sequence of Ad3 penton base. Substitutions of 58-SELS-61→58-SDVA-61, 100-D→R and 425-R→E were introduced into wild-type Ad3 penton base. The mutated residues are shown in red letters. <b>D</b>) Ad3 vectors. Both vectors contain the same GFP expression cassette inserted into the E3 region of Ad3 in front of the fiber sequence. Both vectors contain the complete E1 regions and are therefore replication-competent. mu-Ad3GFP contains both 100-D→R and 425-R→E mutations in the penton base.</p

Production of wt-Ad3GFP and mu-Ad3GFP.

<p><b>A</b>) 293 cells were infected at indicated MOIs (vp/cell) for 2 hours. Medium samples were collected at indicated time points. Viruses were titrated on 293 cells based on GFP-expressing units (“Infectious Units”). N = 3. <b>B</b>) Subconfluent T84 cells were infected and progeny virus production was measured as described in A). <b>C</b>) Comparison of penton, fiber, and hexon expression. HeLa cells infected with wt-Ad3GFP or mu-Ad3GFP were collected at 36 hours post-infection and total cell lysates (without prior ultracentrifugation) were subjected to Western blot with anti-PtDd antibodies (recognize Ad3 penton base and fiber), anti-Ad antibodies (recognize Ad3 hexon, penton base, and fiber) and anti-actin (loading control) antibodies.</p

Viral spread in epithelial tumors in vivo.

<p><b>A</b>) Scheme of experiment. Immunodeficient CB17-SCID beige mice with pre-established subcutaneous T84 or A549 tumors (∼100 mm³ diameter) were intravenously injected with 2×10⁹ IU of wt-Ad3GFP or mu-Ad3GFP. Fourteen days later mice were sacrificed and tumors harvested. <b>B</b>) Tumor sections were analyzed for GFP and DSG2. Representative sections are shown. The scale bar of all images is 20 µm. <b>C</b>) Tumors were digested with collagenase and dispase, and single cell suspensions were then analyzed for GFP expression by flow cytometry. N = 5. Shown is the percentage of GFP expressing cells and the mean GFP fluorescence intensity. The differences between the two viruses were significant (p<0.01). <b>D</b>) Immunofluorescence analysis of A549 tumor sections. The first (left) panels show GFP expression (green) and staining for the junction marker E-cadherin (red). The second and third panels show E-cadherin and GFP staining individually. The 4th panel shows nuclei stained with DAPI. Representative sections are shown. The staining pattern suggests that junctions around wt-Ad3GFP infected cells are absent while they are visibly present in areas of mu-Ad3GFP transduced cells. For the immunofluorescence experiments, cellular nuclei were counterstained with DAPI (blue).</p

PtDd release from infected cells and effect on epithelial junctions.

<p>T84 cells were cultured in transwell chambers until the TEER was constant, i.e. epithelial junctions had formed. <b>A</b>) Confocal immunofluorescence microscopy of T84 cells. The left panel shows an XY image of the cell surface. The right panel shows stacked XZ images. Antibodies against E-cadherin (red) and DSG2 (green) visualize epithelial junction proteins. <b>B</b>) Release of PtDd from mu-Ad3GFP or wt-Ad3GFP-infected T84 cells. Polarized T84 cells were infected with mu-Ad3GFP or wt-Ad3GFP at an MOI of 10 vp/cell (added into the inner chamber) for 2 hours. 24 hours post infection, cells were fixed in 4% PFA for 15 minutes and then permeabilized with cold methanol for 10 minutes. Immunofluorescence staining was performed with anti-Ad3 PtDd polyclonal antibody (red). Nuclei were counterstained with DAPI (blue). <b>C</b>) Cells were infected as described for B). Cells were stained for the junction protein E-cadherin (red) 72 hours pos-tinfection. Virally infected cells express GFP (green). The scale bar of all images is 20 µm.</p

Spread of viruses in T84 cell spheroids.

<p>T84 spheroids were infected with mu-Ad3GFP or wt-Ad3GFP at an MOI of 100 vp/cell for 2 hours. Viruses were removed by washing. <b>A</b>) Spheroids were fixed with 4% PFA at day 4 post-infection. The periphery of spheroids was visualized with rhodamine-labeled Concanavalin A (Con A, red), and cellular nuclei were counterstained with DAPI (blue). Shown are XY and XZ sections. Transduced cells are GFP positive. The scale bars are 50 µm. <b>B</b>) Culture medium aliquots of the virus-infected T84 spheroids were collected at days 3 and 4 post-infection and titrated on 293 cells. N = 3 **p<0.01.</p