Cellular Trafficking of FX-Ad5fiber35 Complex Figure 1. SPR analysis of the in vitro binding of HAdV5 hexon capsomeres and HAdV5-based vectors to immobilized HS with or without factor X (FX) bridging. Representative sensorgrams for (A) HAdV5 hexon capsomeres alone, or with FX or Gla domainless FXGL, (B) HAdV5wt virions alone or with FX, and (C) HAdV5 virion mutants HAdV5FTTAT and HAdV5PbEGD alone or with FX. In (A) and (B), the molecular ratio of FX to hexon protein (isolated capsomeres as in (A), or virion-encapsidated hexons, as in (B)) is indicated in parenthesis. The control sensorgrams with FX and FXGL alone were obtained at FX and FXGL concentrations of 8 mg/ml, corresponding to the concentration in human adult serum (FXmax). In (C), FX was also used at 8 mg/ml. Hexon capsomeres, HAdV5wt virions and HAdV5FTTAT and HAdV5PbEGD mutants bound to immobilized HS only in the presence of FX. RU, response units. doi:10.1371/journal.pone.0018205.g001 Requirement of HSPG expression at the cell surface for FX-mediated enhancement of cell transduction by HAdV5wt vector We next assayed the transduction efficiency of HAdV5wt vector in the presence or absence of FX or FXGL in a CAR-negative cellular model, using CHO cells, which express HSPG at their surface (control CHO-K1), or CHO-2241, which are deficient in HSPG expression. The FX concentrations in the viral inoculum ranged from 0 to 8 mg/ml (FXmax). In HSPG-positive CHO-K1 cells, FX, but not the Gla domainless FXGL, enhanced the transduction efficiency (TE) of HAdV5wt in a dose-dependent manner (Fig. 2 A). Of note, the maximum enhancement of transduction was not reached with a concentration of FX in the medium corresponding to a ratio of 720 copies of FX per virion (i.e. with 3 FX per trimeric hexon capsomere), but with the highest FX concentration (FXmax; Fig. 2 A), a result consistent with sensorgrams shown in Fig. 1B. At FXmax, the increase of TE was 18-fold at 2,500 vp/cell, and 26-fold at 5,000 vp/cell (Fig. 2 B). No detectable effect of FX on TE levels was observed in HSPG- negative CHO-2241 cells (Fig. 2 B). This demonstrated that the surface expression of HSPG molecules was indispensable for the FX-mediated enhancing effect on the HAdV5wt cell binding and transduction, confirming previous studies [24,37]. Absence of requirement of fiber KKTK motif and penton base RGD motif for FX- and HSPG-mediated enhancement of cell transduction by HAdV5 vectors It was shown that mutation of the putative HSPG binding site in the HAdV5wt shaft (91KKTK94) interfered negatively with the cellular trafficking of the virions to the nucleus [45,51]. We therefore evaluated the influence of FX on the capacity of transducing CHO-K1 and CHO-2241 cells by the mutant vector HAdV5FTTAT. In the absence of FX, HAdV5FTTAT transduced CHO-K1 cells with a significantly lower TE, compared to HAdV5wt used at the same MOI (2,500 vp/cell ; Fig. 3 A). In the presence of increasing doses of FX, we observed a progressive augmentation of the TE, with a 35-fold enhancement for the FX:virion ratio of 3:1 for (viz. 720 copies of FX per 240 hexon Figure 2. Cell transduction of CAR- and CD46-negative CHO cells by HAdV5wt in the absence of presence of FX. (A), Dose-response effect of FX on cell transduction. CHO-K1 cells were transduced by GFP-expressing HAdV5wt vector in the presence of increasing concentration of FX. Both FXmax and FXGLmax corresponded to 8 mg/ml. Results were expressed as relative transduction efficiency (RTE). Transduction efficiency, in arbitrary units (AU), was given using the formula:TE = (percentage of GFP-positive cells)6(MFI). The RTE was calculated using the formula:RTE = (TE with FX):(TE without FX), with the 1-value attributed to TE in the absence of FX. (B), CHO-K1 (double CAR- and CD46-negative cells) and CHO-2241 (triple CAR- , CD46-, and HSPG-negative cells) were transduced by HAdV5wt vector at MOI 2,500 (left half of the bar graph) or MOI 5,000 (right half of the bar graph) in the absence or presence of FX (8 mg/ml). Results were expressed as RTE, with the 1-value attributed to the TE of CHO-K1 in the absence of FX. doi:10.1371/journal.pone.0018205.g002 PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e18205