Cellular Trafficking of FX-Ad5fiber35 Complex Figure 9. Electron microscopy of CHO-K1 cells (a–d) incubated with HAdV5F35 at 10,000 vp/cell in the presence of FX (8 mg/ml), and harvested after 2 h at 376C. (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). doi:10.1371/journal.pone.0018205.g009 [36], the serotype 35 fiber-pseudotyped vector HAdV5F35 [37], and the HAdV5/35 chimeric vector carrying serotype 35 fiber knobs [38] all showed a decreased hepatotropism, compared to HAdV5. Likewise HAdV5F2/BAdV4, another fiber-pseudotyped vector which carried human-bovine chimeric fibers, was able to bind to FX in vitro, but ineffective to promote liver transduction in vivo [39]. The fact that both HAdV5F35 and HAdV5F2/BAdV4 vectors carried the same hexon capsomere serotype and varied only in their fiber subtype incited us to explore the role of the fiber in the cell entry pathway and transduction mediated by HAdV5- based vectors in the presence of FX. Our results confirmed that FX bound to HAdV5 hexon protein via its Gla-domain and enhanced the binding of serotype 5 hexon protein and HAdV5wt virus particles to surface-immobilized HS. We also found that FX promoted the interaction of HS with HAdV5FTTAT and HAdV5PbEGD mutants in vitro, significantly enhanced the cell transduction by HAdV5wt, and was able to rescue the lower infectivity of HAdV5FTTAT and HAdV5PbEGD mutants. Since FX contains a RGD tripeptide motif at position 227–229, it was conceivable that in the case of HAdV5PbEGD mutant, FX might compensate for the integrin binding defect of penton base EGD mutant. However, this hypothesis could be excluded for two reasons: (i) activated FX (FXa) which had lost the activation peptide (amino acid sequence 183–234) including the RGD motif, showed the same effect on HAdV5PbEGD infectivity as RGD-containing FX (data not shown); (ii) the activity of FX required the presence of HSPG at the cell surface, whereas RGD- dependent integrins seemed to be dispensable. Intriguingly, however, we found that, although FX promoted the binding of fiber-pseudotyped vector HAdV5F35 to HS in vitro and to cellular HSPG in vivo, it failed to enhance, but instead decreased, the HAdV5F35-mediated transduction of CAR- and CD46-negative, but HSPG-positive CHO cells. It was recently hypothesized that the absolute levels of CAR, CD46, and HSPG on the surface of target cells would define the importance of FX in modulating cell binding and transduction mediated by HAdV5, HAdV35, and serotype 5/35 chimeric viruses: thus, in the presence of FX, the high affinity of serotype 35 fiber for CD46 PLoS ONE | www.plosone.org 12 May 2011 | Volume 6 | Issue 5 | e18205