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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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Transduction of CHO-K1 or CHO-2241 cells by GFP-expressing, fiber                            mutants of HAdV5-based vectors in the absence (w/o) or presence of                            (with) FX (8 µg/ml).(A), HAdV5wt, mutants HAdV5FTTAT and                                HAdV5PbEGD and serotype 35 fiber-pseudotyped HAdV5F35                            were all used at MOI 2,500, and transduction efficiency were expressed                            as arbitrary units (AU), as described in the legend to Fig. 2.                                (B), Dose-response effect of FX on cell transduction by                                HAdV5FTTAT mutant. CHO-K1 cells were transduced by                            GFP-expressing HAdV5FTTAT mutant vector in the presence of                            increasing concentration of FX                            (FXmax = 8 µg/ml). Results were                            expressed as relative transduction efficiency (RTE; refer to the legend                            to Fig. 2).
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pone-0018205-g003: Transduction of CHO-K1 or CHO-2241 cells by GFP-expressing, fiber mutants of HAdV5-based vectors in the absence (w/o) or presence of (with) FX (8 µg/ml).(A), HAdV5wt, mutants HAdV5FTTAT and HAdV5PbEGD and serotype 35 fiber-pseudotyped HAdV5F35 were all used at MOI 2,500, and transduction efficiency were expressed as arbitrary units (AU), as described in the legend to Fig. 2. (B), Dose-response effect of FX on cell transduction by HAdV5FTTAT mutant. CHO-K1 cells were transduced by GFP-expressing HAdV5FTTAT mutant vector in the presence of increasing concentration of FX (FXmax = 8 µg/ml). Results were expressed as relative transduction efficiency (RTE; refer to the legend to Fig. 2).

Mentions: It was shown that mutation of the putative HSPG binding site in the HAdV5wt shaft (91KKTK94) interfered negatively with the cellular trafficking of the virions to the nucleus [45], [51]. We therefore evaluated the influence of FX on the capacity of transducing CHO-K1 and CHO-2241 cells by the mutant vector HAdV5FTTAT. In the absence of FX, HAdV5FTTAT transduced CHO-K1 cells with a significantly lower TE, compared to HAdV5wt used at the same MOI (2,500 vp/cell ; Fig. 3 A). In the presence of increasing doses of FX, we observed a progressive augmentation of the TE, with a 35-fold enhancement for the FX∶virion ratio of 3∶1 for (viz. 720 copies of FX per 240 hexon capsomeres), and 65-fold for FXmax (Fig. 3 B). No enhancement, but instead a slight decreasing effect of FX on TE was observed in CHO-2241 (Fig. 3 A). This indicated that FX was able to rescue the loss of infectivity due to the KKTK-to-TTAT mutation in the fiber shaft, provided that HSPG molecules were present at the cell surface. This also indicated that the putative HSPG-binding motif KKTK was dispensable for the FX-mediated bridging of HAdV5wt virion to surface HSPG molecules, confirming the major role of hexon as the ligand of FX [24]. It could not be excluded however that other fiber interactions, besides the assumed interaction with HSPG, might be affected as a consequence of the TTAT mutation, although the TTAT mutant fibers folded as trimers and were incorporated at wild-type levels in the adenoviral capsid (data not shown).


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)

Transduction of CHO-K1 or CHO-2241 cells by GFP-expressing, fiber                            mutants of HAdV5-based vectors in the absence (w/o) or presence of                            (with) FX (8 µg/ml).(A), HAdV5wt, mutants HAdV5FTTAT and                                HAdV5PbEGD and serotype 35 fiber-pseudotyped HAdV5F35                            were all used at MOI 2,500, and transduction efficiency were expressed                            as arbitrary units (AU), as described in the legend to Fig. 2.                                (B), Dose-response effect of FX on cell transduction by                                HAdV5FTTAT mutant. CHO-K1 cells were transduced by                            GFP-expressing HAdV5FTTAT mutant vector in the presence of                            increasing concentration of FX                            (FXmax = 8 µg/ml). Results were                            expressed as relative transduction efficiency (RTE; refer to the legend                            to Fig. 2).
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pone-0018205-g003: Transduction of CHO-K1 or CHO-2241 cells by GFP-expressing, fiber mutants of HAdV5-based vectors in the absence (w/o) or presence of (with) FX (8 µg/ml).(A), HAdV5wt, mutants HAdV5FTTAT and HAdV5PbEGD and serotype 35 fiber-pseudotyped HAdV5F35 were all used at MOI 2,500, and transduction efficiency were expressed as arbitrary units (AU), as described in the legend to Fig. 2. (B), Dose-response effect of FX on cell transduction by HAdV5FTTAT mutant. CHO-K1 cells were transduced by GFP-expressing HAdV5FTTAT mutant vector in the presence of increasing concentration of FX (FXmax = 8 µg/ml). Results were expressed as relative transduction efficiency (RTE; refer to the legend to Fig. 2).
Mentions: It was shown that mutation of the putative HSPG binding site in the HAdV5wt shaft (91KKTK94) interfered negatively with the cellular trafficking of the virions to the nucleus [45], [51]. We therefore evaluated the influence of FX on the capacity of transducing CHO-K1 and CHO-2241 cells by the mutant vector HAdV5FTTAT. In the absence of FX, HAdV5FTTAT transduced CHO-K1 cells with a significantly lower TE, compared to HAdV5wt used at the same MOI (2,500 vp/cell ; Fig. 3 A). In the presence of increasing doses of FX, we observed a progressive augmentation of the TE, with a 35-fold enhancement for the FX∶virion ratio of 3∶1 for (viz. 720 copies of FX per 240 hexon capsomeres), and 65-fold for FXmax (Fig. 3 B). No enhancement, but instead a slight decreasing effect of FX on TE was observed in CHO-2241 (Fig. 3 A). This indicated that FX was able to rescue the loss of infectivity due to the KKTK-to-TTAT mutation in the fiber shaft, provided that HSPG molecules were present at the cell surface. This also indicated that the putative HSPG-binding motif KKTK was dispensable for the FX-mediated bridging of HAdV5wt virion to surface HSPG molecules, confirming the major role of hexon as the ligand of FX [24]. It could not be excluded however that other fiber interactions, besides the assumed interaction with HSPG, might be affected as a consequence of the TTAT mutation, although the TTAT mutant fibers folded as trimers and were incorporated at wild-type levels in the adenoviral capsid (data not shown).

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