Cellular Trafficking of FX-Ad5fiber35 Complex 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 mg/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. doi:10.1371/journal.pone.0018205.g005 Intracellular trafficking and compartmentalization of FX-HAdV5F35 complexes in HSPG-expressing CHO cells (i) Live cell imaging. 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. (ii) Retention of HAdV5F35 particles in the late endosomal compartment. To determine the nature of the subcellular compartment in which HAdV5F35 was retained, CHO-K1 cells were transduced by baculovirus vectors expressing fluorescent markers designed for live-imaging of different cellular organelles or compartments. We found that the green fluorescent signal of HAdV5F35 colocalized with red fluorescent Lamp1 protein [74,75], a marker of the lysosomal/late endosomal compartment (Fig. 7 C). This result suggested that the retarded trafficking to the nucleus of HAdV5F35 particles was due to their segregation into the late endosomal compartment. (iii) Evaluation of the baculovirus-mediated labeling of cellular organelles and compartments for the study of adenoviral vector pathway in living cells. To verify the validity of our observation of late endosomal compartment- alization of HAdV5F35 particles, HeLa, HEK-293, CHO-K1 or CHO-CD46 cells were transduced with recombinant baculo- viruses expressing Lamp1-RFP, as above, or Rab5A-RFP [76], a marker of the early endosomes, 24 h prior to incubation with Alexa-488-labeled HAdV5wt particles or Cy5-labeled HAdV3 penton dodecahedrons (Pt-Dd). No image of colocalisation of Alexa-488 labeled HAdV5wt with RFP-labeled early endosomes could be captured, even after short incubation period: as early as after 10–15 min incubation with living cells at 37uC, green PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e18205