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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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Confocal microscopy of live cells transduced by adenoviral vector                                particles or capsid components (penton dodecamers).(A–C), Confocal microscopy of live cells (CHO-K1)                                transduced by Alexa-488-labeled adenoviral vectors, used at 10,000                                vp/cell and complexed with FX (8 µg/ml). (A)                                HAdV5wt, 30 min pi; (B, C) HAdV5F35, 3 h pi. (i), Green                                channel image; (ii), phase contrast; (iii), merge of (i) and (ii).                                In (C), CHO-K1 cells were transduced by recombinant                                baculoviral vector expressing RFP-tagged, late endosome marker Lamp1                                protein, 24 h before incubation with HAdV5F35 vector. (i), Green                                channel image; (ii), phase contrast; (iii) orange channel; (iv),                                merge of (i) and (iii). (D) Live HeLa cells transduced                                by recombinant baculovirus expressing RFP-tagged, early endosome                                marker Rab5A protein, were incubated 24 h later with                                Alexa-488-labeled HAdV5wt particles without FX, at 10,000 vp/cell                                and 37°C. Picture shown was taken at 20 min after incubation                                with HAdV5wt. Note that most of the virus signal is weak and                                diffuse, but some green fluorescent dots are visible within the                                cytoplasm (white arrows). (E), Live CHO-CD46 cells                                transduced by recombinant baculovirus expressing RFP-tagged, late                                endosome marker Lamp1 protein, were incubated 24 h later with                                Cy5-labeled HAdV3 penton dodecahedrons (Pt-Dd) at 37°C. Picture                                shown was taken at 60 min after incubation with Pt-Dd. (i), Cy5                                channel; (ii), phase contrast image; (iii) RFP channel; (iv), merge                                of (i) and (iii).
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pone-0018205-g007: Confocal microscopy of live cells transduced by adenoviral vector particles or capsid components (penton dodecamers).(A–C), Confocal microscopy of live cells (CHO-K1) transduced by Alexa-488-labeled adenoviral vectors, used at 10,000 vp/cell and complexed with FX (8 µg/ml). (A) HAdV5wt, 30 min pi; (B, C) HAdV5F35, 3 h pi. (i), Green channel image; (ii), phase contrast; (iii), merge of (i) and (ii). In (C), CHO-K1 cells were transduced by recombinant baculoviral vector expressing RFP-tagged, late endosome marker Lamp1 protein, 24 h before incubation with HAdV5F35 vector. (i), Green channel image; (ii), phase contrast; (iii) orange channel; (iv), merge of (i) and (iii). (D) Live HeLa cells transduced by recombinant baculovirus expressing RFP-tagged, early endosome marker Rab5A protein, were incubated 24 h later with Alexa-488-labeled HAdV5wt particles without FX, at 10,000 vp/cell and 37°C. Picture shown was taken at 20 min after incubation with HAdV5wt. Note that most of the virus signal is weak and diffuse, but some green fluorescent dots are visible within the cytoplasm (white arrows). (E), Live CHO-CD46 cells transduced by recombinant baculovirus expressing RFP-tagged, late endosome marker Lamp1 protein, were incubated 24 h later with Cy5-labeled HAdV3 penton dodecahedrons (Pt-Dd) at 37°C. Picture shown was taken at 60 min after incubation with Pt-Dd. (i), Cy5 channel; (ii), phase contrast image; (iii) RFP channel; (iv), merge of (i) and (iii).

Mentions: The capsids of HAdV5wt and HAdV5F35 vectors were chemically labeled with Alexa-488, and mixed with FX, before incubation with CHO-K1 cells at high vector multiplicity (10,000 vp/cell). Intracellular virions were tracked in situ in live cells by time-lapse microscopy at early times of infection (0 to 4 h pi). As early as 20–30 min pi, most of the HAdV5wt signal was found in the vicinity of or within the nucleus (Fig. 7 A). This observation was consistent with the well-described rapid process of endocytosis, endosomal escape and intracellular trafficking of HAdV5wt virions [22], [73], and suggested that FX had no significant effect on the internalized HAdV5wt particles and the kinetics of their intracellular transit. This implied that FX acted at earlier steps, an hypothesis consistent with the role of molecular bridge between viral hexon capsomeres and cell surface HSPG played by FX. The fluorescence pattern of HAdV5F35 vector was however significantly different. At 30 min pi, no fluorescent signal was observed in the nucleus, instead multiple fluorescent dots were visible in the cytoplasm, and most of the fluorescence remained cytoplasmic until 3 h pi (Fig. 7 B). This suggested that HAdV5F35 particles were delayed in terms of intracellular trafficking, compared to HAdV5.


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)

Confocal microscopy of live cells transduced by adenoviral vector                                particles or capsid components (penton dodecamers).(A–C), Confocal microscopy of live cells (CHO-K1)                                transduced by Alexa-488-labeled adenoviral vectors, used at 10,000                                vp/cell and complexed with FX (8 µg/ml). (A)                                HAdV5wt, 30 min pi; (B, C) HAdV5F35, 3 h pi. (i), Green                                channel image; (ii), phase contrast; (iii), merge of (i) and (ii).                                In (C), CHO-K1 cells were transduced by recombinant                                baculoviral vector expressing RFP-tagged, late endosome marker Lamp1                                protein, 24 h before incubation with HAdV5F35 vector. (i), Green                                channel image; (ii), phase contrast; (iii) orange channel; (iv),                                merge of (i) and (iii). (D) Live HeLa cells transduced                                by recombinant baculovirus expressing RFP-tagged, early endosome                                marker Rab5A protein, were incubated 24 h later with                                Alexa-488-labeled HAdV5wt particles without FX, at 10,000 vp/cell                                and 37°C. Picture shown was taken at 20 min after incubation                                with HAdV5wt. Note that most of the virus signal is weak and                                diffuse, but some green fluorescent dots are visible within the                                cytoplasm (white arrows). (E), Live CHO-CD46 cells                                transduced by recombinant baculovirus expressing RFP-tagged, late                                endosome marker Lamp1 protein, were incubated 24 h later with                                Cy5-labeled HAdV3 penton dodecahedrons (Pt-Dd) at 37°C. Picture                                shown was taken at 60 min after incubation with Pt-Dd. (i), Cy5                                channel; (ii), phase contrast image; (iii) RFP channel; (iv), merge                                of (i) and (iii).
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pone-0018205-g007: Confocal microscopy of live cells transduced by adenoviral vector particles or capsid components (penton dodecamers).(A–C), Confocal microscopy of live cells (CHO-K1) transduced by Alexa-488-labeled adenoviral vectors, used at 10,000 vp/cell and complexed with FX (8 µg/ml). (A) HAdV5wt, 30 min pi; (B, C) HAdV5F35, 3 h pi. (i), Green channel image; (ii), phase contrast; (iii), merge of (i) and (ii). In (C), CHO-K1 cells were transduced by recombinant baculoviral vector expressing RFP-tagged, late endosome marker Lamp1 protein, 24 h before incubation with HAdV5F35 vector. (i), Green channel image; (ii), phase contrast; (iii) orange channel; (iv), merge of (i) and (iii). (D) Live HeLa cells transduced by recombinant baculovirus expressing RFP-tagged, early endosome marker Rab5A protein, were incubated 24 h later with Alexa-488-labeled HAdV5wt particles without FX, at 10,000 vp/cell and 37°C. Picture shown was taken at 20 min after incubation with HAdV5wt. Note that most of the virus signal is weak and diffuse, but some green fluorescent dots are visible within the cytoplasm (white arrows). (E), Live CHO-CD46 cells transduced by recombinant baculovirus expressing RFP-tagged, late endosome marker Lamp1 protein, were incubated 24 h later with Cy5-labeled HAdV3 penton dodecahedrons (Pt-Dd) at 37°C. Picture shown was taken at 60 min after incubation with Pt-Dd. (i), Cy5 channel; (ii), phase contrast image; (iii) RFP channel; (iv), merge of (i) and (iii).
Mentions: The capsids of HAdV5wt and HAdV5F35 vectors were chemically labeled with Alexa-488, and mixed with FX, before incubation with CHO-K1 cells at high vector multiplicity (10,000 vp/cell). Intracellular virions were tracked in situ in live cells by time-lapse microscopy at early times of infection (0 to 4 h pi). As early as 20–30 min pi, most of the HAdV5wt signal was found in the vicinity of or within the nucleus (Fig. 7 A). This observation was consistent with the well-described rapid process of endocytosis, endosomal escape and intracellular trafficking of HAdV5wt virions [22], [73], and suggested that FX had no significant effect on the internalized HAdV5wt particles and the kinetics of their intracellular transit. This implied that FX acted at earlier steps, an hypothesis consistent with the role of molecular bridge between viral hexon capsomeres and cell surface HSPG played by FX. The fluorescence pattern of HAdV5F35 vector was however significantly different. At 30 min pi, no fluorescent signal was observed in the nucleus, instead multiple fluorescent dots were visible in the cytoplasm, and most of the fluorescence remained cytoplasmic until 3 h pi (Fig. 7 B). This suggested that HAdV5F35 particles were delayed in terms of intracellular trafficking, compared to HAdV5.

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