Fig.1. Identification of receptor X using Ad3 virions and Ad3 PtDd.

a) Competition of ³H-labeled Ad3 and Ad5 virus attachment to HeLa cells after pre-incubation with Ad3 BsDd, PtDd, or Ad fiber knobs. Attachment in PBS-treated cells was taken as 100%. N=5. Data are represented as mean +/− SEM). Ad3-PtDd vs. Ad3 knob: p=0.0033. b) Competition of Ad3-GFP and Ad35-GFP virus infection. HeLa cells were pretreated with Ad3 fiber knob or PtDd at increasing concentrations and then exposed to Ad3-GFP (left panel) or Ad35-GFP virus (right panel) at an MOI of 100pfu/cell. GFP expression was measured 18 hours later by flow cytometry. Data are represented as mean. Standard deviation was less than 10% for all data points. c) Attachment of ³H-labeled Ad3 and Ad35 viruses to human and non-human cell lines. Y79 and Ramos are human retinoblastoma and lymphoma cells, respectively. CHO cells are Chinese Hamster ovary cells. MMC and TC1 cells are mouse mammary carcinoma and lung carcinoma cells, respectively. TC1-CD46 cells express human CD46. Shown are the average number of viral particels attached per cells. N=5. d and e) Identification of receptor X by affinity capture and MS/MS. Membrane protein fractions were prepared from HeLa and Ramos cells. Protein blots were hybridized with Ad5/35++ virions (d) and Ad3 virions or Ad3 PtDd (e). Binding was visualized with polyclonal antibodies against Ad35++ knob (d) or Ad3 knob (e) (see also Supplementary Figs. 1f and g). Solubilized HeLa cell membrane lysates were also immunoprecipitated with DSG2 mAb 6D8 crosslinked with protein A/G plus agarose. Western blot of immunoprecipitates was performed with DSG2 monoclonal antibody AH12.2 (see antiDSG2-IP). f) MS/MS analysis of the 160 kDa band. Upper panel: Structure of DSG2. EC: extracellular domain, EA: juxtamembrane extracellular anchor domain, TM: transmembrane domain. Lower panel: amino acid sequence of DSG2. Highlighted are the peptide sequences captured by MS/MS analysis of the 160 kDa band. The triangles in the DSG2 scheme (top panel) indicate the localization of the identified peptides with regards to the different domains. MS/MS analysis detected 14 peptides DSG2 with a high confidence factor (20.8% protein coverage and Sequest cross correlation coefficient scores ranging from 2.6 to 5.5 for individual peptides). g–i) Biacore plasmon surface resonance studies with recombinant human DSG2 immobilized on sensorchips. Ad2, Ad3 and Ad5 at 5.10⁹ vp per ml (g), different concentrations of PtDd (h) or PtDd and Ad3 fiber knob (i) were injected over the activated surface and response signals were collected over the indicated time periods.