Cellular Trafficking of FX-Ad5fiber35 Complex Figure 10. Confocal microscopy of live cells (CHO-K1) incubated with (A) Alexa-555-labeled FX alone (8 mg/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. doi:10.1371/journal.pone.0018205.g010 would overcome the serotype 5 hexon:FX interaction, resulting in reduced liver transduction by the chimeric vector HAdV5F35 in vivo, compared to HAdV5 [37]. However, this did not explain the limitation by FX of the transduction of CHO-CD46 cells in vitro by chimeric vectors carrying serotype 35 fibers, as previously reported [37], and observed in the present study (refer to Fig. 5), or the lower transduction of CD46 transgenic hepatocytes by a chimeric HAdV5/35 vector in vivo [38]. In the latter study, CD46- transgenic mice injected with a HAdV5/35-based chimeric vector carrying serotype 35 fiber knobs showed a two orders of magnitude lower liver transduction and 20-fold lower adenoviral genome content, compared to HAdV5-based vector [38]. Our analysis of the cell entry and trafficking pathway of HAdV5F35 vector in complex with FX provided some clues to reconcile these apparent contradictions, and to understand the possible mechanisms underlying the FX effect. We showed that FX augmented by 10-fold the attachment and cellular uptake of HAdV5F35, and confirmed that the negative effect of FX on HAdV5F35-mediated cell transduction did not result from a binding defect at the cell attachment step, but was due to PLoS ONE | www.plosone.org 13 May 2011 | Volume 6 | Issue 5 | e18205