Cellular Trafficking of FX-Ad5fiber35 Complex Figure 3. Transduction of CHO-K1 or CHO-2241 cells by GFP-expressing, fiber mutants of HAdV5-based vectors in the absence (w/ o) or presence of (with) FX (8 mg/ml). (A), HAdV5wt, mutants HAdV5FTTAT and HAdV5PbEGD and serotype 35 fiber-pseudotyped HAdV5F35 were all used at MOI 2,500, and transduction efficiency were expressed as arbitrary units (AU), as described in the legend to Fig. 2. (B), Dose-response effect of FX on cell transduction by HAdV5FTTAT mutant. CHO-K1 cells were transduced by GFP-expressing HAdV5FTTAT mutant vector in the presence of increasing concentration of FX (FXmax = 8 mg/ml). Results were expressed as relative transduction efficiency (RTE; refer to the legend to Fig. 2). doi:10.1371/journal.pone.0018205.g003 capsomeres), and 65-fold for FXmax (Fig. 3 B). No enhancement, but instead a slight decreasing effect of FX on TE was observed in CHO-2241 (Fig. 3 A). This indicated that FX was able to rescue the loss of infectivity due to the KKTK-to-TTAT mutation in the fiber shaft, provided that HSPG molecules were present at the cell surface. This also indicated that the putative HSPG-binding motif KKTK was dispensable for the FX-mediated bridging of HAdV5wt virion to surface HSPG molecules, confirming the major role of hexon as the ligand of FX [24]. It could not be excluded however that other fiber interactions, besides the assumed interaction with HSPG, might be affected as a consequence of the TTAT mutation, although the TTAT mutant fibers folded as trimers and were incorporated at wild-type levels in the adenoviral capsid (data not shown). Human adenovirus serotype 35 (HAdV35) utilizes CD46 and av-integrins as primary and secondary receptors, respectively [41,53]. We next investigated the possible influence of RGD- dependent integrins on the FX+HSPG-mediated enhancing effect on transduction by HAdV5 vectors. To this aim, the HAdV5P- bEGD penton base mutant was used in transduction assays of CHO-K1 and CHO-2241 cells. In the absence of FX, HAdV5PbEGD showed a lower TE of both CHO-K1 and CHO-2241 cells, compared to HAdV5wt used at the same MOI (Fig. 3 A). In the presence of FXmax, we observed an enhancement of transduction of CHO-K1 cells, at levels similar to those of HAdV5wt or HAdV5FTTAT (50- to 100-fold), whereas no effect was detectable in CHO-2241 (Fig. 3 A). This showed that FX was able to rescue the negative effect of the penton base RGD-to-EGD mutation in HSPG-expressing CHO-KI cells, but not in HSPG-negative CHO cells. Similar to the HAdV5FTTAT fiber shaft mutant, this result implied that the penton base RGD motifs and the integrins played no significant role in the FX+HSPG-mediated enhancement of cell transduction using HAdV5 vectors. Interestingly, CHO-2241 seemed to be slightly more permissive to HAdV5wt compared to CHO-K1 infected at the same MOI in the absence of FX, which could suggest a higher accessiblity of alternative virus attachment receptor(s) other than HSPG for the primary binding of HAdV5wt to CHO-2241 cells, e.g. integrins [12,54,55]. The possibility of integrins acting as alternative attachment receptor of Ad5 to CHO cells in the absence of CAR and HSPG were envisaged, based on the results obtained with our RGD-mutant vector : HAdV5PbEGD transduced CHO- 2241 cells in the absence of FXmax with a 10-fold lower efficiency, compared to HAdV5wt (Fig. 3 A). However, since CHO cells lack ß-integrins and fail to express aVß3/5 integrin heterodimers at their surface, other types of RGD-interactors/ligands might be responsible for the low levels of HAdV5PbEGD-mediated trans- duction. Paradoxical behavior of serotype 35 fiber-pseudotyped HAdV5F35 vector in the context of FX and HSPG Fiber swapping between adenovirus serotypes has been widely used as a rational strategy to (i) explore the various functions associated with fibers in fundamental virology [56–58], (ii) to allow fiber-pseudotyped vectors to evade prexisting neutralizing anti- bodies [59,60], or (iii) to ablate the natural tropism of the virus and confer a novel transductional retargeting capacity to the pseudotyped vectors [24,27,34,37,61–67]. Novel and sometimes unexpected properties, e.g. nonnative entry pathway and/or aberrant cellular trafficking, have been observed with such chimeric adenoviruses. This was the case for the serotype 35 fiber-pseudotyped HAdV5F35 vector [68], or for HAdV5F2/ BAdV4, which carried bovine-human chimeric fibers [39]. PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e18205