Ad35 knob interaction with CD46. (A) Partial amino acid sequence of wild-type fiber knob (Ad35K) and the Ad35 knob mutants Ad35K-279 (ablated for CD46 binding) and Ad35K⁺⁺. The corresponding affinities are indicated. The localization of the critical amino acid residues within a 3-dimensional model of the Ad35 fiber knob domain has been reported recently.^26,27  (B) CD46 surface levels. Raji cells were incubated with 20 μg/mL Ad35K-279, Ad35K, Ad35⁺⁺, or with anti-CD46 mAb. At the indicated time points, surface CD46 levels of cells were analyzed by flow cytometry. Shown is the percentage of CD46 levels (mean values and standard deviations), compared with CD46 mean fluorescence intensity of PBS-treated cells; n > 6; P = .021 for Ad35K versus Ad35K⁺⁺ samples (15 minutes, 6 hours, 24 hours, and 48 hours combined). (C) Ad35 knob surface levels. Raji cells were incubated with Ad35K or Ad35K⁺⁺ at room temperature for 1 hour. After washing with PBS, cells were incubated with fresh medium at 37°C for 15 minutes (■) or 6 hours (□). The levels of cell-bound Ad35 knob were analyzed by flow cytometry with the use of a mouse anti-His tag antibody followed by an anti–mouse antibody conjugated with Alexa Fluor 488. Shown is the fluorescence intensity (mean values and SDs); n > 6. (D-I) Immunofluorescence microscopy analysis of CD46, Cy3-Ad35K⁺⁺, cathepsin B, and caveolin. For better clarity of cytoplasmic signals, the microphotographs shown are from HeLa cells. Similar results were obtained with Raji cells. (J) Inhibition of Ad35-GFP infection. Raji cells were treated with 20 μg/mL Ad35K-279, Ad35K, or Ad35K⁺⁺ for 24 hours and, after washing, infected with Ad35-GFP at the indicated MOIs (multiplicities of infection). GFP expression was analyzed 24 hours later. Shown are the mean values and SDs; n = 3.