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 4uC for 1 h, and cellular internalization at 37uC for 1 h, respectively, with or without FX (8 mg/ 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. doi:10.1371/journal.pone.0018205.g006 fluorescent dots of HAdV5wt were already found within the cytoplasm, outside of the red fluorescent endosomal compartment (Fig. 7 D). This confirmed the scenario described by Greber et al., which showed that incoming HAdV5wt particles escaped very rapidly from early endosomes [73]. By contrast, Cy5-labeled Pt- Dd localized in the late endosomal compartment of HeLa or CHO cells after incubation at 37uC for 1 h (Fig. 7 E), as expected for the HAdV3 pathway [40,77–79]. The usefulness of baculovirus- mediated labeling of specific organelles for tracking adenovirus particles or adenoviral components within living cells was therefore validated using our in-house model of HAdV3 Pt-Dd. Electron microscopy (EM) of cell-internalized HAdV5wt and HAdV5F35 particles in the presence of FX 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 mg/ml). Cells were harvested at 2 h pi at 37uC, and Cellular Trafficking of FX-Ad5fiber35 Complex 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 2564 nm (m 6 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). The pattern was different for CHO-K1 cells incubated with FX- HAdV5F35 complex for 2 h at 37uC. No particle with the regular shape of adenovirions was observed in any of the cellular compartments, but each cell section showed large vesicles containing abundant, electron dense material (Fig. 9 a). This pattern was consistent with the results of confocal microscopy showing the accumulation of FX-HAdV5F35 complexes in lysosomes (refer to Fig. 7). Interestingly at the cell surface, HAdV5F35 particles were frequently seen connected to the plasma membrane via a bridge consisting of filamentous material (Fig. 9 b– d). The length of these bridges varied from 80 to 140 nm, with an average at 105 nm, a value compatible with the thickness of the fibrilllous glycocalyx coat (77 to 201 nm; [80]). By contrast, in control CHO-CD46 cells incubated with HAdV5F35 vector in the absence of FX, cell-bound particles were seen at a distance of 1362 nm from the plasma membrane outer leaflet (Fig. 9 e), a value compatible with the short-shafted serotype 35 fiber bound to its CD46 receptor. Enlargements of cell-bound HAdV5F35 particles showed fuzzy material decorating the viral capsid (Fig. 9 d), conferring the whole complex a diameter of 10766 nm, instead of 80–85 nm for free virions (Fig. 9: compare the sharp contour of the control HAdV5F35 virion in panel e to the blurred contour of FX-HAdV5F35 complex in panel d). We hypothesize that the filaments emanating from the cell which bridged the vector to the cell surface represented HSPG, components of the cell glycocalyx [80], whereas the fuzzy material which coated the capsid corresponded to FX molecules bound to hexon. Cellular uptake and retention of FX with or without adenoviral particles Our observation that FX enhanced the vector-cell binding for both HAdV5wt and HAdV5F35, but failed to augment the FX- HAdV5F35-mediated cell transduction, raised the question of the fate of FX after the cell attachment of the FX-vector complex: was FX coendocytosed with HAdV5wt or HAdV5F35, or did it remain outside of the cell? To address this issue, Alexa-555-labeled FX was preincubated with Alexa-488-labeled vector particles, and the complex added to CHO-K1 cells. As control, Alexa-555- labeled FX was added to CHO-K1 cells alone, without preincubation with the vector. Both types of samples were followed by live cell imaging. We found that FX alone could bind to CHO-K1 cells and was rapidly endocytosed: intracellular FX was detected as early as 15–20 min post incubation (Fig. 10 A). No detectable cellular uptake of FX was observed in HSPG- deficient CHO-2241 cells (not shown). In CHO-K1 cells incubated with the double labeled FX-HAdV5wt complex, both fluorescent signals were observed within the cell at 15–20 min pi, and most signals colocalized in cytoplasmic dots and patches (Fig. 10 B). At later times pi (45–60 min), HAdV5wt particles PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e18205