Cellular Trafficking of FX-Ad5fiber35 Complex 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 mg/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 37uC. 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 37uC. 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). doi:10.1371/journal.pone.0018205.g007 were found to localize at the nuclear periphery or inside the nucleus, and less colocalization with FX was visible (Fig. 10 C). In CHO-K1 cells incubated with double labeled FX-HAdV5F35 complex, FX and virions remained colocalized within the cytoplasm until late times pi (3 h pi; Fig. 10 D). This confirmed the rapid intracellular transit of HAdV5wt, in contrast to the slow trafficking and vesicular retention of HAdV5F35. We next investigated whether the vesicular retention or sequestration of HAdV5F35 could be explained by a higher stability of FX-HAdV5F35 complex in the acidic environment of the late endosomal compartment, compared to the higher pH of early endosomes, the endocytic compartment of HAdV5wt. This was determined indirectly, using SPR analysis of the apparent affinity between FX and serotype 5 hexon, the capsomeres which were in common between HAdV5F35 and HAdV5wt and the targets of FX. FX was covalently immobilized onto the biosensor chip, and hexon protein diluted in the same 0.05 M phosphate buffer but with the different Na2HPO4:NaH2PO4 ratios required to obtain the desired pH values. The sensorgrams obtained showed that the profile of binding of serotype 5 hexon to FX was significantly higher at pH 5.7, compared to neutral pH, with an intermediate interaction observed at pH 6.3 (Fig. 11). This suggested that the FX-HAdV5F35 complex dissociated at slower rate in the acidic environment of late endosomal vesicles, compared to that of FX-HAdV5 complex in early endosomes. This vesicular sequestration would account for the lower PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e18205