Fig. 2.
A single amino acid substitution (T425A) abrogates FX biding to HAdv5. (A) Kinetic response data and dissociation constant (Kd) for FX binding to the indicated mutant viruses obtained by using surface plasmon resonance analysis (Biacore, GE Healthcare Biosciences, Pittsburgh, PA). Black indicates experimentally obtained data. Orange indicates global fits of these data to 1:1 single-site interaction model. Representative data obtained from four independent experiments are shown. (B to D) In vivo analysis of hexon-mutated viruses. (B) Histological analysis of virus-encoded GFP expression in mouse hepatocytes 48 hours after intravenous infection of mice with WT HAdv5 (WT) or mutated viruses. Representative fields are shown (n = 5 biological replicates). GFP expression is observed as green fluorescence on fixed liver sections. Corresponding fields in 4′,6-diamidino-2-phenylindole channel (blue, nuclei-specific staining) are shown. Scale bar, 100 μm. (C) Western blotting analysis of GFP expression in the livers of mice shown in (B). The biological duplicate samples for each virus are shown. (D) Colocalization of virus particles (red) with splenic CD169+ and MARCO+ marginal zone macrophages (green) observed 1 hour after infection for indicated viruses analyzed by means of confocal microscopy. Scale bar, 10 μm. Representative fields are shown. n = 5 biological replicates.