Cellular Trafficking of FX-Ad5fiber35 Complex Figure 11. SPR analysis of the pH-dependence of FX:hexon protein interaction. FX was immobilized on the sensorchip, and a hexon protein solution in 150 mM NaCl, 0.05 M sodium phosphate buffer of various pH values was injected into the flowcell. doi:10.1371/journal.pone.0018205.g011 intracellular mechanisms, as previously hypothesized [37]. Ac- cording to our data, (i) the primum movens was the fiber 35-mediated addressing of HAdV5F35 particles (bound or unbound to FX) to the late endosomal/lysosomal compartment; (ii) FX-bound HAdV5F35 particles remained sequestered in this compartment, due to the higher stability of the FX-HAdV5F35 complexes in an acidic environment, which in turn (iii) delayed the trafficking of HAdV5F35 particles to the nucleus. (iv) An additional mechanism involved the exocytic pathway: following the cellular uptake of FX-HAdV5F35 complexes, significant amounts of exosome- associated HAdV5F35 were released in the extracellular milieu, a phenomenon which was not observed in the absence of FX. This was not observed with HAdV5wt either, with or without FX. Our results therefore addressed the puzzling question why, in contrast to HAdV5 vectors, fiber-chimeric vectors such as HAdV5F35 do not efficiently transduce liver cells after intravenous injection, although they contain the same HAdV5 hexons, which mediate binding to heparan sulfate on liver cells via blood FX. Our data suggested that the retention of HAdV5F35 particles in late endosomal compartments, triggered by HAdV35 fibers, was further enhanced by the hexon:FX:heparan sulfate interaction. This mechanism led to an abortion of HAdV5F35 transduction in the presence of FX, and most likely explained why HAdV5F35 vectors do not transduce liver cells after intravenous injection in mice. The intracellular trafficking pathway of subspecies B HAdVs has been well studied [41,58]. It has been shown that, despite high levels of binding to cells and similar internalization kinetics, fiber- pseudotyped or full serotype species B HAdVs remain in late endosomes or lysosomes for relatively long periods of time after infection, and take significantly longer than species C HAdV to reach the nucleus [41,58]. This supposedly favored the recycling of the vectors to the cell surface and reduce their transduction efficiency, in contrast to HAdV5. Our experimental data on exosomal release of serotype 35 fiber-pseudotyped vector HAdV5F35 infection in the presence of FX supported the latter hypothesis. In conclusion, FX would be benefical to the infection of CAR-lacking cells by species C HAdVs, e.g. HAdV5, which transit via the early endosomal pathway, but detrimental to species B HAdVs, e.g. HAdV35, which follow the late endosomal pathway. In the light of the acid stability of the FX-HAdV5F35 complex that we observed, it remained to explain the relative efficient transduction of CHO-CD46 cells by HAdV5F35 in the presence of FX (refer to Fig. 5). We assume that in the absence of CD46, such as in CHO-K1 cells, only the alternative receptors HSPG via the intermediate ligand FX would mediate the cell binding and entry of HAdV5F35, and all or nearly all vector particles would be complexed with FX, and retarded or trapped within the late vesicular compartment. In the presence of CD46 receptors however, e.g. in CHO-CD46 cells, FX became dispensable, and a significant number of free, FX-unbound HAdV5F35 particles would be endocytosed via the CD46 pathway. These free particles would normally escape the late endosomal vesicles and reach the nucleus, unlike FX-complexed HAdV5F35 particles. Previous analyses of the intracellular fate of chimeric adenovi- ruses have shown that the fiber protein is the major determinant of the cell trafficking of incoming virions [57,58,81]. Thus, the targeting of HAdV5 pseudotyped with fibers of serotype 7 (a serotype belonging to species B HAdV) to the lysosomal pathway is under the control of the fiber serotype [57]. The present study provided another example of dominant effect of species B fiber, serotype 35 fiber, which occurred in the absence of CD46 and any other cognate fiber receptor at the plasma membrane. The endocytosis and retention of FX-HAdV5F35 complex in the late endosomal compartment, characteristic of species B HAdVs [41,58], implied that the nature of the adenoviral fiber was dominant over the high affinity serotype 5 hexon:FX interaction as a determinant of the choice of the endocytic compartment and intracellular trafficking pathway of HAdV particles. This con- firmed previous reports on the intracellular functions associated with the fibers carried by incoming adenovirions [57,58,70,81,82]. The molecular mechanism and factors involved in the cellular traffic determinism of adenoviral fiber remains an open question, PLoS ONE | www.plosone.org 14 May 2011 | Volume 6 | Issue 5 | e18205