A phylogenetic study of HAdV fiber shaft has revealed the absence of the putative heparan sulfate (HS)-binding site (KKTK motif) in all HAdVs other than species C [69]. The serotype 35 fibers carried by the chimeric HAdV5F35 vector lacked the KKTK motif, and species B HAdV35 has been shown to interact with cellular HSPG via capsid protein domains other than the fiber knob [42]. We therefore wanted to determine the capacity of our chimeric HAdV5F35 vector to bind to HS with or without a FX bridge. Firstly, we analyzed the binding of HAdV5F35 to immobilized FX in SPR assays, and found that HAdV5F35 bound to FX with a higher response compared to HAdV5wt used at the same particle input (Fig. 4 A). We also observed a higher FX-mediated binding of HAdV5F35 to surface-immobilized HS, compared to HAdV5wt (Fig. 4 B). No effect was observed with FXGL (Fig. 4 B). In dose- dependent binding assays, maximum binding was observed with 480 copies of FX per HAdV5F35 virion, i.e. a stoichiometric ratio of 2 FX molecules per hexon capsomere, with no further significant increase obtained using 3 copies of FX per hexon (720 FX copies per virion; Fig. 4 C). This differed from the sensorgrams of FX-mediated binding of HAdV5wt to immobi- lized HS, which was not maximal with 2 copies of FX per hexon, and still increased in the range 2 to 10 copies of FX per hexon (refer to Fig. 1 C). This difference could be due to the relatively higher accessibility of hexon capsomeres to FX in short fiber- carrying HAdV5F35 vector, compared to the long fiber-carrying vector HAdV5Fwt. The influence of FX-mediated bridging of HAdV5F35 to cellular HSPG on the transduction of CHO-K1 cell was then tested with increasing MOI, from 200 to 5,000 vp/cell. HAdV5wt was used at the same MOI for comparison and as control. As expected, the transduction efficiency with HAdV5wt vector was significantly higher when FX was added to the virus inoculum, from 14-fold to 30-fold higher at MOI 200 and 5,000 vp/cell, respectively (Fig. 5, A and B, panels a). With HAdV5F35 however, no enhancing effect on transduction of CHO-K1 cells was observed in the presence of FX, compared to the vector alone, but instead a 3- to 4-fold decreasing effect at high MOI (Fig. 5, A and B, panels b). Likewise, in control experiments using HAdV5F35 and permissive CHO-CD46 cells, no increasing effect of FX on cell transduction was observed, but a decreasing effect at high vector doses (Fig. 5, A and B, panels c). A similar phenomenon has previously been reported, and attributed to a blockage in postinternalization step(s) of chimeric vectors carrying serotype 35 fibers [37]. The fact that FX-HAdV5F35 complexes were less efficient than FX-HAdV5wt complexes in cell transduc- tion was paradoxical, considering (i) that both HAdV5wt and HAdV5F35 carried the same hexon capsomeres of serotype 5, (ii) which had the same ligand, FX, and (iii) that the FX-HAdV5F35 complexes were capable of binding to surface-immobilized HS and to cellular HSPG with an apparent higher affinity than that of the FX-HAdV5wt complexes. Cell attachment and internalization of FX-HAdV5F35 complex in HSPG-expressing CHO cells The following experiments were designed to explain the 2-log difference between FX-HAdV5wt and FX-HAdV5F35 complex- es in the transduction of CHO-K1 cells. We investigated the (i) cell attachment, (ii) cellular uptake (endocytosis and internaliza- tion), and (iii) intracellular trafficking of FX-HAdV5F35, in comparison with FX-HAdV5wt complexes, to determine which step(s) was blocked or altered in the infection pathway. The rationale for using CHO-K1 cells as target cells, was that they express neither CAR nor CD46, and thus made possible the Cellular Trafficking of FX-Ad5fiber35 Complex analysis of the cell entry pathway of the HAdV5wt and HAdV5F35 vectors mediated by their FX-bridging to cellular HSPG, while avoiding any bias due to adenovirus serotype- specific receptors. (i) Cell attachment. Samples of HAdV5wt and HAdV5F35 suspension were mixed with FX at a final concentration of 8 mg/ ml, and the mixture added to CHO-K1 cell monolayers at a MOI of 5,000 vp/cell. Incubation was carried out for 1 h at 4uC, which allowed for virus-cell attachment but not virus entry or endocytosis [70]. After extensive rinsing to remove unattached virus, cells were harvested and cell-bound virus determined by quantitative PCR analysis (qPCR) of viral genomes after DNA extraction, based on the fiber gene copy number normalized to the ß-actin gene. FX significantly increased the amounts of cell-bound virions, by a factor of 5 to 6 for HAdV5wt, and by a factor of 10 for HAdV5F35 (Fig. 6 A). This confirmed the apparent higher affinity of FX for HAdV5F35 as observed in vitro, compared to HAdV5wt (refer to Fig. 4). However, this was in apparent contradiction with the lower transduction efficiency of the FX- HAdV5F35 complex, compared to FX-HAdV5wt (refer to Fig. 5), and excluded the cell attachment as the limiting step for FX- HAdV5F35-mediated transduction. (ii) Cellular uptake. The subsequent step of endocytosis was then investigated. After an incubation period of 1 h at 4uC with FX-vector complexes at MOI 5,000, as above, samples of CHO- K1 cells were transferred to 37uC and harvested after 1 h. After a brief trypsin treatment, to remove vector particles remaining bound to the plasma membrane [71], cell samples were subjected to DNA extraction and viral genomes determined by qPCR, as above. In the absence of FX, the intracellular content was not significantly different between HAdV5wt and HAdV5F35. In the presence of FX, there was a 2-fold increase in HAdV5wt uptake, and a 4-fold increase for HAdV5F35 (Fig. 6 A). This suggested that the mechanism responsible for the lower cell transduction by the FX-HAdV5F35 complex did not involve the endocytic step of the vector. (iii) Extracellular release. The previous results incited us to explore the exocytic pathway of cell-internalized vector particles, i.e. the possibility that significant amounts of HAdV5F35 particles might be released in the extracellular medium, either by shedding of vector-containing microvesicles (MVs) budding from the plasma membrane, or by exocytosis, i.e. the release of exosomes (EXOs) segregated within the lumen of multivesicular bodies (MVBs) (reviewed in [72]). CHO-K1 cell culture supernatants were harvested at 2 h and 24 h post incubation with of HAdV5wt or HAdV5F35 at MOI 5,000, with or without FX. The extracellular MVs and EXOs from the culture supernatants were separated by sequential and differential ultracentrifugation. After DNA extraction, the amount of virus particles in each microparticle population was determined by qPCR quantification of the viral genomes. In the absence of FX, HAdV5wt genomes were recovered in significant amounts in MVs and EXOs, and these amounts decreased in both types of microparticles when transduction was performed in the presence of FX (Fig. 6 B). Interestingly, the profile was different for HAdV5F35, with and without FX: (i) HAdV5F35 genomes were undetectable in either microparticle population in the absence of FX, but became detectable in the presence of FX; (ii) HAdV5F35 was recovered in higher amounts in EXOs, compared to MVs (2-fold; Fig. 6 B). This pattern suggested that the intracellular trafficking was likely responsible for the low efficiency of FX-HSPG-mediated transduction of CHO-K1 cells by HAdV5F35, and the next experiments were designed to determine which subcellular compartment(s) were possibly implicated. PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e18205