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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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Production of wt-Ad3GFP and mu-Ad3GFP.A) 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) Subconfluent T84 cells were infected and progeny virus production was measured as described in A). C) 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.
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ppat-1003718-g002: Production of wt-Ad3GFP and mu-Ad3GFP.A) 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) Subconfluent T84 cells were infected and progeny virus production was measured as described in A). C) 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.

Mentions: A previous study using recombinant penton base dodecahedra showed that the deletion of the first 60 amino acids or the substitution of amino acid residues 58-SELS-61 to 58-SDVA-61 prevented dodecamerization. Furthermore, penton base mutants 100-D→R or 425-R→E drastically reduced the yield and stability of penton base dodecamers [9]. We therefore attempted to generate Ad3 penton base mutants that contained the SELS→SDVA mutation, the 100-D→R mutation, and the 425-R→E mutation individually or in combination (Figures 1A–C). The mutations were introduced into a vectorized Ad3 genome, where the parental Ad3 vector (wt-Ad3GFP) contained a CMV promoter-driven GFP gene inserted into the E3 region [2]. Mutations were introduced into the penton base sequence of wt-Ad3GFP. The correctness of the penton base gene modifications was confirmed by PCR and DNA sequencing (Figure S2). The recombinant viral genomes were transfected into 293 cells for virus rescue. Single plaques were then amplified and viral genomes sequenced. We could only rescue viruses that contained the D100R and R425E mutations individually or in combination. In our further studies, we used the Ad3 mutant that contained both the D100R and R425E mutations (mu-Ad3GFP) (Figure 1D). Progeny virus yields during the virus amplification were comparable between wt-Ad3GFP and mu-Ad3GFP (Figure S3). To ensure that the introduced mutations did not affect viral entry, DNA replication, assembly, and release of progeny virus, we performed virus growth curve assays in 293 cells (Figure 2A) and T84 cells that were cultured at relative low cell density to avoid the formation of intercellular junctions (Figure 2B). These studies showed that the kinetics and yields of progeny virus production did not significantly differ for wt-Ad3GFP and mu-Ad3GFP. This is further supported by the analysis of protein levels of Ad3 penton base, fiber, and hexon in infected cells by Western blot, which did not show differences between wt-Ad3GFP and mu-Ad3GFP at 36 hours after infection (Figure 2C).


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)

Production of wt-Ad3GFP and mu-Ad3GFP.A) 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) Subconfluent T84 cells were infected and progeny virus production was measured as described in A). C) 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814681&req=5

ppat-1003718-g002: Production of wt-Ad3GFP and mu-Ad3GFP.A) 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) Subconfluent T84 cells were infected and progeny virus production was measured as described in A). C) 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.
Mentions: A previous study using recombinant penton base dodecahedra showed that the deletion of the first 60 amino acids or the substitution of amino acid residues 58-SELS-61 to 58-SDVA-61 prevented dodecamerization. Furthermore, penton base mutants 100-D→R or 425-R→E drastically reduced the yield and stability of penton base dodecamers [9]. We therefore attempted to generate Ad3 penton base mutants that contained the SELS→SDVA mutation, the 100-D→R mutation, and the 425-R→E mutation individually or in combination (Figures 1A–C). The mutations were introduced into a vectorized Ad3 genome, where the parental Ad3 vector (wt-Ad3GFP) contained a CMV promoter-driven GFP gene inserted into the E3 region [2]. Mutations were introduced into the penton base sequence of wt-Ad3GFP. The correctness of the penton base gene modifications was confirmed by PCR and DNA sequencing (Figure S2). The recombinant viral genomes were transfected into 293 cells for virus rescue. Single plaques were then amplified and viral genomes sequenced. We could only rescue viruses that contained the D100R and R425E mutations individually or in combination. In our further studies, we used the Ad3 mutant that contained both the D100R and R425E mutations (mu-Ad3GFP) (Figure 1D). Progeny virus yields during the virus amplification were comparable between wt-Ad3GFP and mu-Ad3GFP (Figure S3). To ensure that the introduced mutations did not affect viral entry, DNA replication, assembly, and release of progeny virus, we performed virus growth curve assays in 293 cells (Figure 2A) and T84 cells that were cultured at relative low cell density to avoid the formation of intercellular junctions (Figure 2B). These studies showed that the kinetics and yields of progeny virus production did not significantly differ for wt-Ad3GFP and mu-Ad3GFP. This is further supported by the analysis of protein levels of Ad3 penton base, fiber, and hexon in infected cells by Western blot, which did not show differences between wt-Ad3GFP and mu-Ad3GFP at 36 hours after infection (Figure 2C).

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