Fig. 3. Kinetic and thermodynamic insights into cation-dependent reovirus-integrin interactions.

Studies were conducted using AFM on model surfaces (a–e) and living cells (f–h) and using BLI (i). a Schematic of experimental set up to probe cation-dependent reovirus interactions with α2β1 integrin-coated model surface. b, c Dynamic force spectroscopy (DFS) plots show distribution of average rupture forces determined at eight distinct loading rate (LR) ranges for interactions between T3SA+ virions and β1 integrin-coated model surface in the presence of Mn²⁺ (for further details, see methods section) (b) or Mg²⁺ (c). Data corresponding to single interactions were fit with the Bell–Evans (BE) model (I, black curve), providing average k_off and x_u values. Dashed lines represent predicted binding forces for two (II) and three (III) simultaneous uncorrelated interactions (Williams-Evans [WE] prediction). d, e The binding probability is plotted as a function of the contact time. Least-squares fit of the data to a mono-exponential decay curve (line) provides average kinetic on-rate (k_on) of the probed interaction. Comparison of K_D (k_off/k_on) values shows that Mn²⁺ increases the affinity of T3SA+ virions for β1 integrins. f–h Assessment of cation-dependent reovirus interaction with integrins expressed on living cells. DFS plots of data obtained using model surfaces (gray circles and living cells (red dots) in the presence of either Mn²⁺ (g) or Mg²⁺ (h). Histogram of the force distribution observed on cells and a multi-peak Gaussian fit of data (n = 900 data points) are shown at the side. Error bars indicate s.d. of the mean value. All data are representative of N = 5 independent experiments. i Sensorgrams obtained using biolayer interferometry (BLI) show interaction of T3SA+ with β1 integrins immobilized on amine-reactive biosensors under conditions shown.