Limits...
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.

Show MeSH

Related in: MedlinePlus

Cell transduction of CAR- and CD46-negative CHO cells by HAdV5wt in                            the absence of presence of FX.(A), Dose-response effect of FX on cell transduction. CHO-K1                            cells were transduced by GFP-expressing HAdV5wt vector in the presence                            of increasing concentration of FX. Both FXmax and                                FXGLmax corresponded to 8 mg/ml. Results were expressed                            as relative transduction efficiency (RTE). Transduction efficiency, in                            arbitrary units (AU), was given using the                            formula∶TE = (percentage of GFP-positive                            cells)×(MFI). The RTE was calculated using the                            formula∶RTE = (TE with FX)∶(TE without                            FX), with the 1-value attributed to TE in the absence of FX.                                (B), CHO-K1 (double CAR- and CD46-negative cells) and                            CHO-2241 (triple CAR- , CD46-, and HSPG-negative cells) were transduced                            by HAdV5wt vector at MOI 2,500 (left half of the bar graph) or MOI 5,000                            (right half of the bar graph) in the absence or presence of FX (8                            µg/ml). Results were expressed as RTE, with the 1-value attributed                            to the TE of CHO-K1 in the absence of FX.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102659&req=5

pone-0018205-g002: Cell transduction of CAR- and CD46-negative CHO cells by HAdV5wt in the absence of presence of FX.(A), Dose-response effect of FX on cell transduction. CHO-K1 cells were transduced by GFP-expressing HAdV5wt vector in the presence of increasing concentration of FX. Both FXmax and FXGLmax corresponded to 8 mg/ml. Results were expressed as relative transduction efficiency (RTE). Transduction efficiency, in arbitrary units (AU), was given using the formula∶TE = (percentage of GFP-positive cells)×(MFI). The RTE was calculated using the formula∶RTE = (TE with FX)∶(TE without FX), with the 1-value attributed to TE in the absence of FX. (B), CHO-K1 (double CAR- and CD46-negative cells) and CHO-2241 (triple CAR- , CD46-, and HSPG-negative cells) were transduced by HAdV5wt vector at MOI 2,500 (left half of the bar graph) or MOI 5,000 (right half of the bar graph) in the absence or presence of FX (8 µg/ml). Results were expressed as RTE, with the 1-value attributed to the TE of CHO-K1 in the absence of FX.

Mentions: We next assayed the transduction efficiency of HAdV5wt vector in the presence or absence of FX or FXGL in a CAR-negative cellular model, using CHO cells, which express HSPG at their surface (control CHO-K1), or CHO-2241, which are deficient in HSPG expression. The FX concentrations in the viral inoculum ranged from 0 to 8 µg/ml (FXmax). In HSPG-positive CHO-K1 cells, FX, but not the Gla domainless FXGL, enhanced the transduction efficiency (TE) of HAdV5wt in a dose-dependent manner (Fig. 2 A). Of note, the maximum enhancement of transduction was not reached with a concentration of FX in the medium corresponding to a ratio of 720 copies of FX per virion (i.e. with 3 FX per trimeric hexon capsomere), but with the highest FX concentration (FXmax; Fig. 2 A), a result consistent with sensorgrams shown in Fig. 1B. At FXmax, the increase of TE was 18-fold at 2,500 vp/cell, and 26-fold at 5,000 vp/cell (Fig. 2 B). No detectable effect of FX on TE levels was observed in HSPG-negative CHO-2241 cells (Fig. 2 B). This demonstrated that the surface expression of HSPG molecules was indispensable for the FX-mediated enhancing effect on the HAdV5wt cell binding and transduction, confirming previous studies [24], [37].


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)

Cell transduction of CAR- and CD46-negative CHO cells by HAdV5wt in                            the absence of presence of FX.(A), Dose-response effect of FX on cell transduction. CHO-K1                            cells were transduced by GFP-expressing HAdV5wt vector in the presence                            of increasing concentration of FX. Both FXmax and                                FXGLmax corresponded to 8 mg/ml. Results were expressed                            as relative transduction efficiency (RTE). Transduction efficiency, in                            arbitrary units (AU), was given using the                            formula∶TE = (percentage of GFP-positive                            cells)×(MFI). The RTE was calculated using the                            formula∶RTE = (TE with FX)∶(TE without                            FX), with the 1-value attributed to TE in the absence of FX.                                (B), CHO-K1 (double CAR- and CD46-negative cells) and                            CHO-2241 (triple CAR- , CD46-, and HSPG-negative cells) were transduced                            by HAdV5wt vector at MOI 2,500 (left half of the bar graph) or MOI 5,000                            (right half of the bar graph) in the absence or presence of FX (8                            µg/ml). Results were expressed as RTE, with the 1-value attributed                            to the TE of CHO-K1 in the absence of FX.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102659&req=5

pone-0018205-g002: Cell transduction of CAR- and CD46-negative CHO cells by HAdV5wt in the absence of presence of FX.(A), Dose-response effect of FX on cell transduction. CHO-K1 cells were transduced by GFP-expressing HAdV5wt vector in the presence of increasing concentration of FX. Both FXmax and FXGLmax corresponded to 8 mg/ml. Results were expressed as relative transduction efficiency (RTE). Transduction efficiency, in arbitrary units (AU), was given using the formula∶TE = (percentage of GFP-positive cells)×(MFI). The RTE was calculated using the formula∶RTE = (TE with FX)∶(TE without FX), with the 1-value attributed to TE in the absence of FX. (B), CHO-K1 (double CAR- and CD46-negative cells) and CHO-2241 (triple CAR- , CD46-, and HSPG-negative cells) were transduced by HAdV5wt vector at MOI 2,500 (left half of the bar graph) or MOI 5,000 (right half of the bar graph) in the absence or presence of FX (8 µg/ml). Results were expressed as RTE, with the 1-value attributed to the TE of CHO-K1 in the absence of FX.
Mentions: We next assayed the transduction efficiency of HAdV5wt vector in the presence or absence of FX or FXGL in a CAR-negative cellular model, using CHO cells, which express HSPG at their surface (control CHO-K1), or CHO-2241, which are deficient in HSPG expression. The FX concentrations in the viral inoculum ranged from 0 to 8 µg/ml (FXmax). In HSPG-positive CHO-K1 cells, FX, but not the Gla domainless FXGL, enhanced the transduction efficiency (TE) of HAdV5wt in a dose-dependent manner (Fig. 2 A). Of note, the maximum enhancement of transduction was not reached with a concentration of FX in the medium corresponding to a ratio of 720 copies of FX per virion (i.e. with 3 FX per trimeric hexon capsomere), but with the highest FX concentration (FXmax; Fig. 2 A), a result consistent with sensorgrams shown in Fig. 1B. At FXmax, the increase of TE was 18-fold at 2,500 vp/cell, and 26-fold at 5,000 vp/cell (Fig. 2 B). No detectable effect of FX on TE levels was observed in HSPG-negative CHO-2241 cells (Fig. 2 B). This demonstrated that the surface expression of HSPG molecules was indispensable for the FX-mediated enhancing effect on the HAdV5wt cell binding and transduction, confirming previous studies [24], [37].

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