squares containing 5 to 10 cell sections each were examined for counting virions in different cell compartments. Statistics Results were expressed as mean 6 SEM. of n observations. Sets of data were compared with an analysis of variance (ANOVA) or a Student’s t test. Differences were considered statistically significant when P,0.05. Symbols used in figures were (*) for P,0.05, (**) for P,0.01, (***) for P,0.001, and ns for no significant difference, respectively. All statistical tests were performed using GraphPad Prism version 4.0 for Windows (Graphpad Software). Acknowledgments We are grateful to Elisabeth Errazuriz-Cerda, Jonathan Girard, Chantal Thevenon and Christophe Vanbelle (Centre Commun d’Imagerie de Laennec, Faculty of Medicine, Lyon), for their valuable assistance in our References 1. Mitraki A, Papanikolopoulou K, Van Raaij MJ (2006) Natural triple beta- stranded fibrous folds. Adv Protein Chem 73: 97–124. 2. Russell WC (2000) Update on adenovirus and its vectors. J Gen Virol 81: 2573–2604. 3. Russell WC (2009) Adenoviruses: update on structure and function. J Gen Virol 90: 1–20. 4. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, et al. (1997) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275: 1320–1323. 5. Bergelson JM, Krithivas A, Celi L, Droguett G, Horwitz MS, et al. (1998) The murine CAR homolog is a receptor for Coxsackie B viruses and adenoviruses. J Virol 72: 415–419. 6. Kirby I, Davison E, Beavil AJ, Soh CP, Wickham TJ, et al. (2000) Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein. J Virol 74: 2804–2813. 7. Roelvink PW, Lizonova A, Lee JG, Li Y, Bergelson JM, et al. (1998) The coxsackievirus-adenovirus receptor protein can function as a cellular attach- ment protein for adenovirus serotypes from subgroups A, C, D, E, and F. J Virol 72: 7909–7915. 8. Roelvink PW, Mi Lee G, Einfeld DA, Kovesdi I, Wickham TJ (1999) Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science 286: 1568–1571. 9. Santis G, Legrand V, Hong SS, Davison E, Kirby I, et al. (1999) Molecular determinants of adenovirus serotype 5 fibre binding to its cellular receptor CAR. J Gen Virol 80: 1519–1527. 10. Tomko RP, Johansson CB, Totrov M, Abagyan R, Frisen J, et al. (2000) Expression of the adenovirus receptor and its interaction with the fiber knob. Exp Cell Res 255: 47–55. 11. Tomko RP, Xu R, Philipson L (1997) HCAR and mCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B Coxsackieviruses. Proc Natl Acad Sci USA 94: 3352–3356. 12. Nemerow GR (2000) Cell receptors involved in adenovirus entry. Virology 274: 1–4. 13. Wickham TJ, Carrion ME, Kovesdi I (1995) Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs. Gene Ther 2: 750–756. 14. Wickham TJ, Filardo EJ, Cheresh DA, Nemerow GR (1994) Integrin alphavbeta5 selectively promotes adenovirus mediated cell membrane permeabilization. J Cell Biol 127: 257–264. 15. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR (1993) Integrins alphavbeta3 and alphavbeta5 promote adenovirus internalization but not virus attachment. Cell 73: 309–319. 16. Meier O, Greber UF (2003) Adenovirus endocytosis. J Gene Med 5: 451–462. 17. Wang K, Huang S, Kapoor-Munshi A, Nemerow GR (1998) Adenovirus internalization and infection require dynamin. J Virol 72: 3455–3458. 18. Wiethoff CM, Wodrich H, Gerace L, Nemerow GR (2005) Adenovirus protein VI mediates membrane disruption following capsid disassembly. J Virol 79: 1992–2000. 19. Wodrich H, Henaff D, Jammart B, Segura-Morales C, Seelmeir S, et al. (2010) A capsid-encoded PPxY-motif facilitates adenovirus entry. PLoS Pathog 6: e1000808. 20. Kelkar SA, Pfister KK, Crystal RG, Leopold PL (2004) Cytoplasmic dynein mediates adenovirus binding to microtubules. J Virol 78: 10122–10132. 21. Leopold PL, Kreitzer G, Miyazawa N, Rempel S, Pfister KK, et al. (2000) Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum Gene Ther 11: 151–165. 22. Greber UF, Suomalainen M, Stidwill RP, Boucke K, Ebersold MW, et al. (1997) The role of the nuclear pore complex in adenovirus DNA entry. EMBO J 16: 5998–6007. Cellular Trafficking of FX-Ad5fiber35 Complex electron microscopy and flow cytometry analyses, to Daphna Fenel and Guy Schoehn for the quality control of adenovirions and hexon capsomeres by transmission EM. We are also grateful to Nicole Thielens and Isabelle Bally (IBS platform of the Partnership for Structural Biology and Institut de Biologie Structurale in Grenoble) for assistance and access to the Biacore facility, and to Sylvie Farget and Cathy Berthet for their efficient secretarial aid. We thank INSERM (Institut National de la Sante´ et de la Recherche Me´dicale) for supporting this project, which received a negative evaluation from the Scientific Committee of the French AERES (Agence d’Evaluation de la Recherche et de l’Enseignement Supe´rieur). Author Contributions Conceived and designed the experiments: SSH PB PF. Performed the experiments: SC GG AG SSH PF. Analyzed the data: SC GG AG PB PF SSH. Contributed reagents/materials/analysis tools: PH LL. Wrote the paper: PB PF SSH. 23. Trotman LC, Mosberger N, Fornerod M, Stidwill RP, Greber UF (2001) Import of adenovirus DNA involves the nuclear pore complex receptor CAN/ Nup214 and histone H1. Nat Cell Biol 3: 1092–1100. 24. Coughlan L, Alba R, Parker AL, Bradshaw AC, McNeish IA, et al. (2010) Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses 2: 2290–2355. 25. Alba R, Bradshaw AC, Parker AL, Bhella D, Waddington SN, et al. (2009) Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer. Blood 114: 965–971. 26. Kalyuzhniy O, Di Paolo NC, Silvestry M, Hofherr SE, Barry MA, et al. (2008) Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo. Proc Natl Acad Sci USA 105: 5483–5488. 27. Parker AL, McVey JH, Doctor JH, Lopez-Franco O, Waddington SN, et al. (2007) Influence of coagulation factor zymogens on the infectivity of adenoviruses pseudotyped with fibers from subgroup D. J Virol 81: 3627–3631. 28. Parker AL, Nicklin SA, Baker AH (2008) Interactions of adenovirus vectors with blood: implications for intravascular gene therapy applications. Curr Opin Mol Ther. pp 439–448. 29. Parker AL, Waddington SN, Buckley SM, Custers J, Havenga MJ, et al. (2009) Effect of neutralizing sera on factor x-mediated adenovirus serotype 5 gene transfer. J Virol 83. 30. Parker AL, Waddington SN, Nicol CG, Shayakhmetov DM, Buckley SM, et al. (2006) Multiple vitamin K-dependent coagulation zymogens promote adeno- virus-mediated gene delivery to hepatocytes. Blood 108: 2554–2561. 31. Shayakhmetov DM, Gaggar A, Ni S, Li ZY, Lieber A (2005) Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity. J Virol 79: 7478–7491. 32. Vigant F, Descamps D, Jullienne B, Esselin S, Connault E, et al. (2008) Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver. Mol Ther 16: 1474–1480. 33. Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, et al. (2008) Adenovirus serotype 5 hexon mediates liver gene transfer. Cell 132: 397–409. 34. Waddington SN, Parker AL, Havenga MJ, Nicklin SA, Buckley SM, et al. (2007) Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: fundamental involvement of coagulation factors and redundancy of CAR binding by Ad5. J Virol 81: 9568–9571. 35. Bradshaw AC, Parker AL, Duffy MR, Coughlan L, van Rooijen N, et al. (2010) Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X. PLoS Pathog 6: e1001142. 36. Sakurai F (2008) Development and evaluation of a novel gene delivery vehicle composed of adenovirus serotype 35. Biol Pharm Bull 31: 1819–1825. 37. Greig JA, Buckley SM, Waddington SN, Parker AL, Bhella D, et al. (2009) Influence of coagulation factor X on in vitro and in vivo gene delivery by adenovirus (Ad) 5, Ad35, and chimeric Ad5/Ad35 vectors. Mol Ther 17: 1683–1691. 38. Ganesh S, Gonzalez-Edick M, Gibbons D, Waugh J, Vanroey M, et al. (2009) Evaluation of biodistribution of a fiber chimeric, conditionally replication- competent (oncolytic) adenovirus in CD46 receptor transgenic mice. Hum Gene Ther 20: 1201–1213. 39. Roge´e S, Grellier E, Bernard C, Jouy N, Loyens A, et al. (2010) Influence of chimeric human-bovine fibers on adenoviral uptake by liver cells and the antiviral immune response. Gene Ther 17: 880–891. 40. Wang H, Li ZY, Liu Y, Persson J, Beyer I, et al. (2010) Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14. Nat Med;2010 Dec 12 [Epub ahead of print]. 41. Gaggar A, Shayakhmetov DM, Lieber A (2003) CD46 is a cellular receptor for group B adenoviruses. Nature Med 8: 746–755. PLoS ONE | www.plosone.org 17 May 2011 | Volume 6 | Issue 5 | e18205