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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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Electron microscopy of CHO-K1 cells incubated with HAdV5wt at 10,000                            vp/cell, (A) in the absence (w/o), or (B) presence of FX (8 µg/ml)                            for 2 h at 37°C.(A), (i) and (ii): General views of cell sections showing                            (i) intravesicular and (ii) cytoplasmic vector particles. In (iii) and                            (iv), a vector particle (Vir) is seen within a clathrin-coated vesicle                            (CCV); (iv), enlargement of the CCV shown in (iii), with measurements of                            the space between the vector particle and the inner leaflet of the                            vesicular membrane. N, nucleus; NPC, nuclear pore complexes viewed in a                            tangential section. (B), (i): vector particle within an                            endocytic vesicle in the vicinity of a nuclear pore; (ii), viral core                            seen in the process of traverse of the nuclear pore.
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pone-0018205-g008: Electron microscopy of CHO-K1 cells incubated with HAdV5wt at 10,000 vp/cell, (A) in the absence (w/o), or (B) presence of FX (8 µg/ml) for 2 h at 37°C.(A), (i) and (ii): General views of cell sections showing (i) intravesicular and (ii) cytoplasmic vector particles. In (iii) and (iv), a vector particle (Vir) is seen within a clathrin-coated vesicle (CCV); (iv), enlargement of the CCV shown in (iii), with measurements of the space between the vector particle and the inner leaflet of the vesicular membrane. N, nucleus; NPC, nuclear pore complexes viewed in a tangential section. (B), (i): vector particle within an endocytic vesicle in the vicinity of a nuclear pore; (ii), viral core seen in the process of traverse of the nuclear pore.

Mentions: To further explore the cellular localization of internalized vector particles in the presence of FX, CHO-K1 cells were incubated with HAdV5wt or HAdV5F35 vector (MOI 10,000) alone or complexed with FX (8 µg/ml). Cells were harvested at 2 h pi at 37°C, and processed for EM. As expected for CAR-negative cells transduced in the absence of FX, rare HAdV5wt virions were observed within the cells, in vesicles (Fig. 8 A, i) or free in the cytoplasm (Fig. 8 A, ii). Occasionally, HAdV5wt virions were seen attached to the invaginated plasma membrane forming clathrin-coated vesicles: in such cases, the average distance of the capsid to the plasma membrane was found to be 25±4 nm (m ± SEM), a value consistent with the length of serotype 5 fiber (Fig. 8 A, iii and iv). This suggested that in the absence of CAR and FX, HAdV5wt bound directly to components of the CHO-K1 plasma membrane acting as alternative receptors. In the presence of FX however, HAdV5wt found in vesicles seemed to be associated with electron lucent material (Fig. 8 B, i). Very rare HAdV5wt particles were found within the cells at 2 h pi, likely due to their rapid transit to the nucleus and traverse of the nuclear pore (Fig. 8 B, ii).


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)

Electron microscopy of CHO-K1 cells incubated with HAdV5wt at 10,000                            vp/cell, (A) in the absence (w/o), or (B) presence of FX (8 µg/ml)                            for 2 h at 37°C.(A), (i) and (ii): General views of cell sections showing                            (i) intravesicular and (ii) cytoplasmic vector particles. In (iii) and                            (iv), a vector particle (Vir) is seen within a clathrin-coated vesicle                            (CCV); (iv), enlargement of the CCV shown in (iii), with measurements of                            the space between the vector particle and the inner leaflet of the                            vesicular membrane. N, nucleus; NPC, nuclear pore complexes viewed in a                            tangential section. (B), (i): vector particle within an                            endocytic vesicle in the vicinity of a nuclear pore; (ii), viral core                            seen in the process of traverse of the nuclear pore.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0018205-g008: Electron microscopy of CHO-K1 cells incubated with HAdV5wt at 10,000 vp/cell, (A) in the absence (w/o), or (B) presence of FX (8 µg/ml) for 2 h at 37°C.(A), (i) and (ii): General views of cell sections showing (i) intravesicular and (ii) cytoplasmic vector particles. In (iii) and (iv), a vector particle (Vir) is seen within a clathrin-coated vesicle (CCV); (iv), enlargement of the CCV shown in (iii), with measurements of the space between the vector particle and the inner leaflet of the vesicular membrane. N, nucleus; NPC, nuclear pore complexes viewed in a tangential section. (B), (i): vector particle within an endocytic vesicle in the vicinity of a nuclear pore; (ii), viral core seen in the process of traverse of the nuclear pore.
Mentions: To further explore the cellular localization of internalized vector particles in the presence of FX, CHO-K1 cells were incubated with HAdV5wt or HAdV5F35 vector (MOI 10,000) alone or complexed with FX (8 µg/ml). Cells were harvested at 2 h pi at 37°C, and processed for EM. As expected for CAR-negative cells transduced in the absence of FX, rare HAdV5wt virions were observed within the cells, in vesicles (Fig. 8 A, i) or free in the cytoplasm (Fig. 8 A, ii). Occasionally, HAdV5wt virions were seen attached to the invaginated plasma membrane forming clathrin-coated vesicles: in such cases, the average distance of the capsid to the plasma membrane was found to be 25±4 nm (m ± SEM), a value consistent with the length of serotype 5 fiber (Fig. 8 A, iii and iv). This suggested that in the absence of CAR and FX, HAdV5wt bound directly to components of the CHO-K1 plasma membrane acting as alternative receptors. In the presence of FX however, HAdV5wt found in vesicles seemed to be associated with electron lucent material (Fig. 8 B, i). Very rare HAdV5wt particles were found within the cells at 2 h pi, likely due to their rapid transit to the nucleus and traverse of the nuclear pore (Fig. 8 B, ii).

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