Figure 1.
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.
Figure 2.
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.
Figure 3.
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).
Figure 4.
SPR analysis of the in vitro binding of chimeric HAdV5F35 vector to (A) surface-immobilized FX, or (B, C) immobilized HS with or without FX or FXGL.
(A), Representative sensorgrams for HAdV5wt vector (discontinuous lines) injected at 2×109 and 4×109 vp/ml, or for HAdV5F35 injected at the same doses (solid lines). (B), Comparison of binding to HS of HAdV5wt and HAdV5F35 vector particles (2×109 vp/ml) in the presence of FX or FXGL at 720 copies per vector particle. Controls shown are FX and FXGL alone. For better clarity, the sensorgrams for virions alone, which superimposed those of FX and FXGL, are not shown (refer to Fig. 1C). (C), Dose-response effect of FX on HAdV5F35 binding to immobilized HS. Note that a detectable signal was observed for 120 copies of FX per virion, and reached the maximal value for 480 copies/vp.
Figure 5.
Comparison of transduction efficiency of (a, b) CHO-K1 cells or (c) CHO-CD46 by (a) HAdV5wt, and (b, c) chimeric HAdV5F35 vectors at different MOI (200, 1,000 or 5,000 vp/cell) in the absence (w/o) or presence of (with) FX (8 µg/ml).
Results were expressed as (A) the percentage of GFP-positive cells, or (B) relative transduction efficiency (RTE; refer to the legend to Fig. 2). In B, the number on top of each bar corresponded to the fold increase in RTE, with the 1-value attributed to the TE of CHO-K1 or CHO-CD46 cells transduced by HAdV5F35 at MOI 200. Note that RTE of CHO-K1 cells with HAdV5F35 was lower in the presence of FX than in the absence of FX, at all MOI tested.
Figure 6.
Cellular uptake and extracellular release of HAdV5wt and HAdV5F35 vectors by CHO-K1 cells.
(A), Cell attachment of vector (MOI 5,000) was performed at 4°C for 1 h, and cellular internalization at 37°C for 1 h, respectively, with or without FX (8 µg/ml), as indicated on the x-axis. The number of viral genome copies was determined by qPCR of the fiber gene, normalized to the ß-actin gene. (B), Extracellular vectors associated with microvesicles (MVs) or exosomes (EXOs) recovered from the extracellular medium at 72 h post transduction, were determined as above.
Figure 7.
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).
Figure 8.
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.
Figure 9.
Electron microscopy of CHO-K1 cells (a–d) incubated with HAdV5F35 at 10,000 vp/cell in the presence of FX (8 µg/ml), and harvested after 2 h at 37°C.
(a), Representative CHO-K1 cell section showing a cytoplasmic vesicle containing abundant electron dense material. (b–d), Cell surface-bound HAdV5F35 particles. (e), CHO-CD46 cells incubated with HAdV5F35 in the absence of FX (w/o FX). Note the difference in size and sharpness of the viral contour between HAdV5F35 particles seen in (e) and in (b–d).
Figure 10.
Confocal microscopy of live cells (CHO-K1) incubated with (A) Alexa-555-labeled FX alone (8 µg/ml), or (B, C) in complex with Alexa-488-labeled HAdV5wt, or (D) in complex with Alexa-488-labeled HAdV5F35, both vectors used at 10,000 vp/cell.
Images were taken at 10-min intervals, until 3 h post incubation (pi). (i), phase contrast image; (ii) orange channel; (iii) green channel.
Figure 11.
SPR analysis of the pH-dependence of FX∶hexon protein interaction.
FX was immobilized on the sensorchip, and a hexon protein solution in 150 mM NaCl, 0.05 M sodium phosphate buffer of various pH values was injected into the flowcell.