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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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Detection of Ad14 PtDd by Western blot analysis.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.
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ppat-1003718-g008: Detection of Ad14 PtDd by Western blot analysis.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.

Mentions: So far, we have worked with Ad3-based vectors. The subgroup of DSG2-interacting Ad serotypes comprises Ad3, Ad7, Ad14 and Ad11. We hypothesized that these serotypes have evolved a similar strategy for viral dissemination in epithelial cells that involve the formation of PtDd. To test this, we infected HeLa cells with wild-type Ad14 virus and analyzed PtDd production in comparison to wild-type Ad3 and Ad5 virus (Figure 8, Figure S12). In this study, we capitalized on the fact that the anti-serum raised against purified Ad3 PtDd crossreacted with penton base from other serotypes. As seen before with wt-Ad3GFP, wild-type Ad3 PtDd appeared in sucrose fractions 30–40%. The signals in fractions with lower sucrose concentrations originated from free capsid proteins and defective particles. Importantly, PtDd signals were detectable for Ad14 but not for Ad5. The latter is in agreement with earlier studies demonstrating that Ad2 and Ad5 are unable to form stable pentondodecahedra [9].


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)

Detection of Ad14 PtDd by Western blot analysis.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.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003718-g008: Detection of Ad14 PtDd by Western blot analysis.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.
Mentions: So far, we have worked with Ad3-based vectors. The subgroup of DSG2-interacting Ad serotypes comprises Ad3, Ad7, Ad14 and Ad11. We hypothesized that these serotypes have evolved a similar strategy for viral dissemination in epithelial cells that involve the formation of PtDd. To test this, we infected HeLa cells with wild-type Ad14 virus and analyzed PtDd production in comparison to wild-type Ad3 and Ad5 virus (Figure 8, Figure S12). In this study, we capitalized on the fact that the anti-serum raised against purified Ad3 PtDd crossreacted with penton base from other serotypes. As seen before with wt-Ad3GFP, wild-type Ad3 PtDd appeared in sucrose fractions 30–40%. The signals in fractions with lower sucrose concentrations originated from free capsid proteins and defective particles. Importantly, PtDd signals were detectable for Ad14 but not for Ad5. The latter is in agreement with earlier studies demonstrating that Ad2 and Ad5 are unable to form stable pentondodecahedra [9].

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