Contrasting with the vast scientific information available on HAdV5 and its multiple interactions with host cell components, the clinical application of HAdV5, the most widely utilised serotype as gene transfer vector, has suffered from several drawbacks. The prevalence of anti-HAdV antibodies in the human population results in the rapid neutralisation of HAdV5 vector after in vivo administration. Secondly, intravenous delivery of HAdV5 vector results in the liver uptake of the vast majority of the virus particles, and therefore do not reach their target cells or tissues. Numerous strategies have been employed to overcome these hurdles, notably by engineering mutant or chimeric vectors to evade the neutralising antibodies and ablate the hepatotropism of the vector, but the results have been somewhat disappointing (reviewed in [24]). Recent breakthrough in the HAdV-host interactions showed that the vector particle accumulation in the liver is the result of HAdV5 binding to human blood coagulation factor X (FX) via the hexon capsomeres, followed by the interaction of the HAdV5-FX complexes to heparan sulfate proteoglycan (HSPG) molecules which are present in high concentration at the surface of Kupffer cells [25–34]. Further dissection of the molecular mechanism of the HPSG-mediated cellular uptake of HAdV5-FX complex revealed the importance of O- and N-sulfation of HPSG in this high affinity pathway, and the requirement of alphaV integrins as secondary receptors for an efficient internalization step [35]. In contrast to HAdV5, it has been observed in vivo that HAdV35 vectors have liver transduction efficiencies which are of four orders of magnitude lower than that of HAdV5 vectors [36]. Likewise, fiber-pseudotyped or chimeric fiber-carrying HAdV5 vectors showed less hepatotropism, compared to HAdV5. This was the case for HAdV5F35, which carried serotype 35 fibers [37], HAdV5/35 chimeric vector, which carried serotype 35 fiber knob domains [38], and HAdV5F2/BAdV4, carrying chimeric human- bovine fibers [39]. Intriguingly however, HAdV5, HAdV5F35 and HAdV5F2/BAdV4 vectors were all composed of the serotype 5 hexon capsomere, thus suggesting the contribution of factors other than hexon, FX and HSPG to the mechanism of liver uptake of FX-HAdV5 complex in vivo. Both HAdV35 and the HAdV5F35 chimera use CD46, one of the cell attachment receptors recognized by subspecies B HAdVs besides desmoglein-2 [40], and to a lesser degree HSPG molecules, to infect epithelial cells [41–44]. In the present study, we sought to determine the influence of capsid proteins other than the hexon, viz. penton base and/or fiber, on the interaction of FX with HAdV5-based vectors, and their FX- and HSPG-mediated cell entry pathway and gene transduction. To this aim, we analyzed the binding of wild type HAdV5 (HAdV5wt), penton base or fiber mutants of HAdV5, and fiber 35-pseudotyped HAdV5 vector (HAdV5F35) to heparan sulfate in the presence or absence of FX by surface plasmon resonance in vitro. We analyzed the effect of FX on the HAdV5wt- and HAdV5F35-mediated transduction of CHO cells expressing HSPG (CHO-K1), the alternative receptors for HAdV5 [45–47] and HAdV35 viruses [41–44], and HSPG-negative CHO cells (CHO-2241). Both cell lines lack the CD46 and CAR receptors for HAdV5F35 and HAdV5, respectively. We found that FX bound to HAdV5 hexon protein via its Gla- domain, and enhanced the binding of HAdV5wt and HAdV5F35 vector particles to surface-immobilized hypersulfated heparin (HS) in vitro, and to cellular HSPG in vivo. We also found that FX augmented the efficiency of cellular transduction by HAdV5, but had a negative effect on the transduction by HAdV5F35. Our experimental data showed that this negative effect did not involve the cell attachment or the endocytic step of the FX-HAdV5F35 Cellular Trafficking of FX-Ad5fiber35 Complex complex, but the intracellular trafficking. In the presence of FX, the HAdV5F35 particles accumulated in the late endosomal compartment, resulting in a delay in their vesicular release and nuclear import. Furthermore, HAdV5F35 were released in significant amounts in the extracellular medium via exocytosis, resulting in lower numbers of particles reaching the nucleus. Our results suggested that the serotype 35 fiber determined the cell internalization and trafficking pathway of the HAdV5F35 vector, despite the absence of known fiber receptor at the plasma membrane, and acted dominantly despite the interaction between hexon and cell surface HSPG mediated by FX. This observation has significant implications for the future design of target tissue- redirected adenoviral vectors. Results Gla domain-dependence of FX-mediated binding of serotype 5 hexon protein and adenovirions to heparan sulfate in vitro The interaction between soluble HAdV5wt hexon protein and heparan sulfate in vitro, directly or indirectly via FX, was investigated using surface plasmon resonance (SPR) analysis and a hypersulfated form of heparin (HS), recognized as the best structural model to mimic the heparan sulfate chains contained in HSPG [48,49]. HS was covalently immobilized onto the biosensor chip, and the binding of hexon to HS was assessed using FX in a stoichiometric ratio of 1:1 with hexon protein. A truncated version of FX devoid of its gamma-carboxylic acid (Gla) domain, FXGL, was assayed in parallel experiments. As expected from previous studies (reviewed in [24]), we found that hexon binding to HS was enhanced in the presence of FX, but not with FXGL (Fig. 1 A), and this enhancing effect occurred in a FX dose-dependent manner (not shown). The binding of HAdV5wt virions to immobilized HS with and without FX was also assessed by SPR, using various stoichiometric ratios of FX per hexon trimeric capsomere, as determined from the number of virus particles present in the samples. FX enhanced the binding of HAdV5wt virions to HS in a dose-dependent manner (Fig. 1 B). As for isolated hexon protein, the Gla domainless FXGL showed no significant enhancement of the binding of HAdV5wt virion to HS (not shown). Of note, a weak signal of binding was observed with control samples of FX alone (Fig. 1 A), used in amounts equivalent to its average physiological concentration in human adult serum (8 mg/ml), referred to as the maximum FX concentration (FXmax). This excluded the possibility that the signal of binding to HS observed with FX:hexon or FX:vector complexes were due to the binding of free FX. Two mutants of HAdV5 were then analyzed, HAdV5FTTAT, mutated in the KKTK motif of the fiber shaft, and HAdV5PbEGD, mutated in the RGD motif of the penton base. The KKTK tetrapeptide had been identified as a putative HSPG-binding motif [45,46,50], a function which is debatable [51]. FX used at 8 mg/ ml (FXmax) was found to enhance the binding of the two mutants to HS to equivalent levels (Fig. 1 C). As for HAdV5wt, no binding enhancement was observed with the Gla domainless version FXGL, indicating that the FX bridge between HAdV5wt hexon and HS required the integrity of its Gla domain. This confirmed previous reports which showed that the Gla domain of FX interacts with the hexon capsomere [26,33], whilst basic residues Arg240, Lys236, Lys169, Arg165, Lys96, Arg93, and Arg125 in the exosite of FX interact with HSPG [52]. Our results also indicated that the mutations in HAdV5FTTAT and HAdV5PbEGD had no effect on the FX-mediated binding function of HAdV5wt hexon to HS. PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e18205