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Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction.

Corjon S, Gonzalez G, Henning P, Grichine A, Lindholm L, Boulanger P, Fender P, Hong SS - PLoS ONE (2011)

Bottom Line: Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs.We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes.Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

View Article: PubMed Central - PubMed

Affiliation: University Lyon 1, INRA UMR 754, Retrovirus and Comparative Pathology, Lyon, France.

ABSTRACT
Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

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SPR analysis of the in vitro binding of HAdV5 hexon                            capsomeres and HAdV5-based vectors to immobilized HS with or without                            factor X (FX) bridging.Representative sensorgrams for (A) HAdV5 hexon capsomeres                            alone, or with FX or Gla domainless FXGL, (B) HAdV5wt                            virions alone or with FX, and (C) HAdV5 virion mutants                                HAdV5FTTAT and HAdV5PbEGD alone or with FX. In                            (A) and (B), the molecular ratio of FX to hexon protein (isolated                            capsomeres as in (A), or virion-encapsidated hexons, as in (B)) is                            indicated in parenthesis. The control sensorgrams with FX and FXGL alone                            were obtained at FX and FXGL concentrations of 8 µg/ml,                            corresponding to the concentration in human adult serum                                (FXmax). In (C), FX was also used at 8 mg/ml. Hexon                            capsomeres, HAdV5wt virions and HAdV5FTTAT and                                HAdV5PbEGD mutants bound to immobilized HS only in the                            presence of FX. RU, response units.
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pone-0018205-g001: SPR analysis of the in vitro binding of HAdV5 hexon capsomeres and HAdV5-based vectors to immobilized HS with or without factor X (FX) bridging.Representative sensorgrams for (A) HAdV5 hexon capsomeres alone, or with FX or Gla domainless FXGL, (B) HAdV5wt virions alone or with FX, and (C) HAdV5 virion mutants HAdV5FTTAT and HAdV5PbEGD alone or with FX. In (A) and (B), the molecular ratio of FX to hexon protein (isolated capsomeres as in (A), or virion-encapsidated hexons, as in (B)) is indicated in parenthesis. The control sensorgrams with FX and FXGL alone were obtained at FX and FXGL concentrations of 8 µg/ml, corresponding to the concentration in human adult serum (FXmax). In (C), FX was also used at 8 mg/ml. Hexon capsomeres, HAdV5wt virions and HAdV5FTTAT and HAdV5PbEGD mutants bound to immobilized HS only in the presence of FX. RU, response units.

Mentions: The interaction between soluble HAdV5wt hexon protein and heparan sulfate in vitro, directly or indirectly via FX, was investigated using surface plasmon resonance (SPR) analysis and a hypersulfated form of heparin (HS), recognized as the best structural model to mimic the heparan sulfate chains contained in HSPG [48], [49]. HS was covalently immobilized onto the biosensor chip, and the binding of hexon to HS was assessed using FX in a stoichiometric ratio of 1∶1 with hexon protein. A truncated version of FX devoid of its gamma-carboxylic acid (Gla) domain, FXGL, was assayed in parallel experiments. As expected from previous studies (reviewed in [24]), we found that hexon binding to HS was enhanced in the presence of FX, but not with FXGL (Fig. 1 A), and this enhancing effect occurred in a FX dose-dependent manner (not shown). The binding of HAdV5wt virions to immobilized HS with and without FX was also assessed by SPR, using various stoichiometric ratios of FX per hexon trimeric capsomere, as determined from the number of virus particles present in the samples. FX enhanced the binding of HAdV5wt virions to HS in a dose-dependent manner (Fig. 1 B). As for isolated hexon protein, the Gla domainless FXGL showed no significant enhancement of the binding of HAdV5wt virion to HS (not shown). Of note, a weak signal of binding was observed with control samples of FX alone (Fig. 1 A), used in amounts equivalent to its average physiological concentration in human adult serum (8 µg/ml), referred to as the maximum FX concentration (FXmax). This excluded the possibility that the signal of binding to HS observed with FX∶hexon or FX∶vector complexes were due to the binding of free FX.


Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction.

Corjon S, Gonzalez G, Henning P, Grichine A, Lindholm L, Boulanger P, Fender P, Hong SS - PLoS ONE (2011)

SPR analysis of the in vitro binding of HAdV5 hexon                            capsomeres and HAdV5-based vectors to immobilized HS with or without                            factor X (FX) bridging.Representative sensorgrams for (A) HAdV5 hexon capsomeres                            alone, or with FX or Gla domainless FXGL, (B) HAdV5wt                            virions alone or with FX, and (C) HAdV5 virion mutants                                HAdV5FTTAT and HAdV5PbEGD alone or with FX. In                            (A) and (B), the molecular ratio of FX to hexon protein (isolated                            capsomeres as in (A), or virion-encapsidated hexons, as in (B)) is                            indicated in parenthesis. The control sensorgrams with FX and FXGL alone                            were obtained at FX and FXGL concentrations of 8 µg/ml,                            corresponding to the concentration in human adult serum                                (FXmax). In (C), FX was also used at 8 mg/ml. Hexon                            capsomeres, HAdV5wt virions and HAdV5FTTAT and                                HAdV5PbEGD mutants bound to immobilized HS only in the                            presence of FX. RU, response units.
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Related In: Results  -  Collection

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pone-0018205-g001: SPR analysis of the in vitro binding of HAdV5 hexon capsomeres and HAdV5-based vectors to immobilized HS with or without factor X (FX) bridging.Representative sensorgrams for (A) HAdV5 hexon capsomeres alone, or with FX or Gla domainless FXGL, (B) HAdV5wt virions alone or with FX, and (C) HAdV5 virion mutants HAdV5FTTAT and HAdV5PbEGD alone or with FX. In (A) and (B), the molecular ratio of FX to hexon protein (isolated capsomeres as in (A), or virion-encapsidated hexons, as in (B)) is indicated in parenthesis. The control sensorgrams with FX and FXGL alone were obtained at FX and FXGL concentrations of 8 µg/ml, corresponding to the concentration in human adult serum (FXmax). In (C), FX was also used at 8 mg/ml. Hexon capsomeres, HAdV5wt virions and HAdV5FTTAT and HAdV5PbEGD mutants bound to immobilized HS only in the presence of FX. RU, response units.
Mentions: The interaction between soluble HAdV5wt hexon protein and heparan sulfate in vitro, directly or indirectly via FX, was investigated using surface plasmon resonance (SPR) analysis and a hypersulfated form of heparin (HS), recognized as the best structural model to mimic the heparan sulfate chains contained in HSPG [48], [49]. HS was covalently immobilized onto the biosensor chip, and the binding of hexon to HS was assessed using FX in a stoichiometric ratio of 1∶1 with hexon protein. A truncated version of FX devoid of its gamma-carboxylic acid (Gla) domain, FXGL, was assayed in parallel experiments. As expected from previous studies (reviewed in [24]), we found that hexon binding to HS was enhanced in the presence of FX, but not with FXGL (Fig. 1 A), and this enhancing effect occurred in a FX dose-dependent manner (not shown). The binding of HAdV5wt virions to immobilized HS with and without FX was also assessed by SPR, using various stoichiometric ratios of FX per hexon trimeric capsomere, as determined from the number of virus particles present in the samples. FX enhanced the binding of HAdV5wt virions to HS in a dose-dependent manner (Fig. 1 B). As for isolated hexon protein, the Gla domainless FXGL showed no significant enhancement of the binding of HAdV5wt virion to HS (not shown). Of note, a weak signal of binding was observed with control samples of FX alone (Fig. 1 A), used in amounts equivalent to its average physiological concentration in human adult serum (8 µg/ml), referred to as the maximum FX concentration (FXmax). This excluded the possibility that the signal of binding to HS observed with FX∶hexon or FX∶vector complexes were due to the binding of free FX.

Bottom Line: Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs.We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes.Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

View Article: PubMed Central - PubMed

Affiliation: University Lyon 1, INRA UMR 754, Retrovirus and Comparative Pathology, Lyon, France.

ABSTRACT
Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.

Show MeSH
Related in: MedlinePlus