Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction Ste´ phanie Corjon1., Gae¨ lle Gonzalez1., Petra Henning 2 , Alexei Grichine3, Leif Lindholm4, Pierre Boulanger1, Pascal Fender5*, Saw-See Hong 1 * 1 University Lyon 1, INRA UMR 754, Retrovirus and Comparative Pathology, Lyon, France, 2 Department of Microbiology and Immunology, University of Go¨ teborg, Institute for Biomedicine, Go¨ teborg, Sweden, 3 Institut Albert Bonniot, CRI INSERM-UJF U-823, La Tronche, France, 4 Got-a-Gene AB, Kullavik, Sweden, 5 Unit for Virus-Host Interaction, UMI-3265, CNRS-EMBL-UJF, Grenoble, France Abstract Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG- mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35- mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors. Citation: Corjon S, Gonzalez G, Henning P, Grichine A, Lindholm L, et al. (2011) Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction. PLoS ONE 6(5): e18205. doi:10.1371/journal.pone.0018205 Editor: Robert J. Geraghty, University of Minnesota, United States of America Received November 17, 2010; Accepted February 28, 2011; Published May 26, 2011 Copyright: 2011 Corjon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work in France (Lyon and Grenoble) was supported by the French Cystic Fibrosis Foundation (Vaincre la Mucoviscidose, VLM contracts TG0801 and TG0901), the French Agency for AIDS Research (ANRS, DendrAde contract 2007–2008), the University of Lyon-1 and the Centre National de la Recherche Scientifique (FRE-3011), and the Re´gion Rhoˆ ne-Alpes (Cluster 10-Infectiology). SC was the recipient of an ANRS fellowship, and GG of a VLM fellowship. SSH is the recipient of a Contrat d’Interface HCL-INSERM (2008–2012). The funders in France had no role in our study design, data collection and analysis, decision to publish, or preparation of the manuscript. The work in Sweden for the construction of the chimeric adenoviral vector was supported by Got-a-Gene AB, and potential financial competing interests exist. Competing Interests: LL is affiliated with Got-a-Gene AB, but this affiliation does not alter the authors’ adherence to all the PLoS ONE policies on sharing data. * E-mail: saw-see.hong@univ-lyon1.fr (S-SH); pfender@embl.fr (PF) . These authors contributed equally to this work. Introduction The human adenovirus (HAdV) capsid is composed of eleven well identified structural proteins, of which the hexon is the major component with 240 copies forming the 20 facets and 30 edges of the icosahedral capsid. The penton is the second most represented capsid protein, with 12 copies of penton located at each apex. Each penton capsomere is made up of a fiber, a triple beta- stranded fibrous protein [1], anchored to a pentameric protein, the penton base, closing up the vertices of the icosahedron (reviewed in [2,3]). HAdVs are divided into subgroups or species A to F, covering 51 different serotypes. The members of species C (HAdV2, HAdV5) and species B (HAdV3, HAdV35) are the most studied and characterized in terms of capsid structure, cell entry mechanisms, cellular response and gene transfer (reviewed in [2,3]). The classical cell entry and trafficking pathway of HAdV5, as demonstrated by epithelial cell models of adenoviral infection in vitro, consists of (i) the fiber binding to CAR, the Coxsackie B and Adenovirus Receptor [4–11], followed by (ii) the interaction of the penton base RGD motifs with the cellular integrins alphaVbeta3 and alphaVbeta5, [12–15], which promotes virus endocytosis into clathrin-coated vesicles [16,17]. In step (iii), partially uncoated HAdV5 particles are released from the early endosomal compartment into the cytosol, a process involving the capsid protein VI [18,19]. (iv) Dynein and microtubules mediate the cytoplasmic transit of the residual HAdV5 capsid, which docks at the nuclear pore complex [20,21], before (v) the nuclear import of the viral nucleoprotein core [22,23]. PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e18205