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Penton-dodecahedral particles trigger opening of intercellular junctions and facilitate viral spread during adenovirus serotype 3 infection of epithelial cells.

Lu ZZ, Wang H, Zhang Y, Cao H, Li Z, Fender P, Lieber A - PLoS Pathog. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Washington, Division of Medical Genetics, Seattle, Washington, United States of America.

ABSTRACT
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 de novo produced virions. These events were nearly absent in mu-Ad3GFP infected cultures. Our in vitro 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.

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Viral spread in polarized T84 cells.A and B) Viral spread in T84 cells cultured in transwell chambers. Cells were cultured as described for Fig. 4. Ad vectors were added to the inner chamber at an MOI of 10 vp/cell. A) 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) 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). C) 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.
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ppat-1003718-g005: Viral spread in polarized T84 cells.A and B) Viral spread in T84 cells cultured in transwell chambers. Cells were cultured as described for Fig. 4. Ad vectors were added to the inner chamber at an MOI of 10 vp/cell. A) 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) 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). C) 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.

Mentions: Transwell cultures of polarized T84 cells form layers with an average thickness of five cells. Addition of wt-Ad3GFP and mu-Ad3GFP vectors resulted in the infection of the top cell layer with comparable efficiency. Confocal microscopy performed 5 days after infection revealed transduction of cells in deeper cell layers for wt-AdGFP but not for mu-Ad3GFP (Figure 5A). This suggests that wt-Ad3GFP produced de novo in primarily infected cells was able to penetrate deeper into the cell layer. Over time, progeny virus should completely spread through the multi-cell layer and be detectable at the basal side, i.e. the outer chamber of the transwell cultures. We therefore measured the titer of infectious units in the inner and outer chamber at different time points. Virus became detectable in the outer chamber only at day 9 after infection (Figure 5B). In the outer chamber, the titers of mu-Ad3GFP were significantly lower than those of wt-Ad3GFP. In contrast, the titers of both viruses in the inner chamber were comparable, indicating that release of de novo produced virus from initially infected cells in the top cell layer is not impaired for mu-Ad3GFP. The process of transepithelial spread was similar at MOI 2 and MOI 50, indicating that the cells with accessible DSG2 on the apical side were already saturated with virus at MOI 2. To further prove a role of PtDd in viral spread we added recombinant PtDd to the inner chamber of T84 cells at days 3, 5, and 7 after infection (Figure 5C). As in the previous experiment, less mu-Ad3GFP progeny virus was found in the outer chamber at day 8 and 10 after infection indicating that mu-Ad3GFP is impaired in viral spread. Importantly, while “exogenous” recombinant PtDd had no significant effect on the spread of wt-Ad3GFP through the layer of T84 cells, spread of mu-Ad3GFP was significantly increased. This indicates that recombinant PtDd, to some degree, compensates for the reduced PtDd production from mu-Ad3GFP.


Penton-dodecahedral particles trigger opening of intercellular junctions and facilitate viral spread during adenovirus serotype 3 infection of epithelial cells.

Lu ZZ, Wang H, Zhang Y, Cao H, Li Z, Fender P, Lieber A - PLoS Pathog. (2013)

Viral spread in polarized T84 cells.A and B) Viral spread in T84 cells cultured in transwell chambers. Cells were cultured as described for Fig. 4. Ad vectors were added to the inner chamber at an MOI of 10 vp/cell. A) 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) 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). C) 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814681&req=5

ppat-1003718-g005: Viral spread in polarized T84 cells.A and B) Viral spread in T84 cells cultured in transwell chambers. Cells were cultured as described for Fig. 4. Ad vectors were added to the inner chamber at an MOI of 10 vp/cell. A) 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) 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). C) 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.
Mentions: Transwell cultures of polarized T84 cells form layers with an average thickness of five cells. Addition of wt-Ad3GFP and mu-Ad3GFP vectors resulted in the infection of the top cell layer with comparable efficiency. Confocal microscopy performed 5 days after infection revealed transduction of cells in deeper cell layers for wt-AdGFP but not for mu-Ad3GFP (Figure 5A). This suggests that wt-Ad3GFP produced de novo in primarily infected cells was able to penetrate deeper into the cell layer. Over time, progeny virus should completely spread through the multi-cell layer and be detectable at the basal side, i.e. the outer chamber of the transwell cultures. We therefore measured the titer of infectious units in the inner and outer chamber at different time points. Virus became detectable in the outer chamber only at day 9 after infection (Figure 5B). In the outer chamber, the titers of mu-Ad3GFP were significantly lower than those of wt-Ad3GFP. In contrast, the titers of both viruses in the inner chamber were comparable, indicating that release of de novo produced virus from initially infected cells in the top cell layer is not impaired for mu-Ad3GFP. The process of transepithelial spread was similar at MOI 2 and MOI 50, indicating that the cells with accessible DSG2 on the apical side were already saturated with virus at MOI 2. To further prove a role of PtDd in viral spread we added recombinant PtDd to the inner chamber of T84 cells at days 3, 5, and 7 after infection (Figure 5C). As in the previous experiment, less mu-Ad3GFP progeny virus was found in the outer chamber at day 8 and 10 after infection indicating that mu-Ad3GFP is impaired in viral spread. Importantly, while “exogenous” recombinant PtDd had no significant effect on the spread of wt-Ad3GFP through the layer of T84 cells, spread of mu-Ad3GFP was significantly increased. This indicates that recombinant PtDd, to some degree, compensates for the reduced PtDd production from mu-Ad3GFP.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Washington, Division of Medical Genetics, Seattle, Washington, United States of America.

ABSTRACT
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 de novo produced virions. These events were nearly absent in mu-Ad3GFP infected cultures. Our in vitro 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.

Show MeSH
Related in: MedlinePlus