Fig. S1. MSD plot and movie, movie of CD81 dynamics and MSD analysis. Example of CD81 single-molecule tracking using Atto647N-labelled Fab fragments (A). A neural network (see Experimental procedures) is used to determine CD81 behaviour within a trajectory. Each identified segment is then analysed using MSD analysis and a plot of Brownian (B) and confined (C) segments. The first four points are used to determine the apparent diffusion coefficient of the molecule. The fit of the confined trajectory has been performed using 50% of the dots.


Fig. S2. CD81 single-molecule tracking profiles. The relative frequency of CD81 trajectories with Brownian (B), mixed (M) or confined (C) behaviour observed with anti-CD81 Fabs 2s66 and 2s155 (A). A minimum of 10 cells and 250 trajectories were measured per parameter as predetermined by BMC sampling. Scatter plots for Brownian CD81 diffusion coefficients measured with anti-CD81 Fabs 2s66 or 2s155 in non-polarized HepG2 cells, where the median Brownian DC values for 2s66 and 2s155 are 0.17 ± 0.2 μm2 s−1 and 0.16 ± 0.2 μm2 s−1 respectively (B). Each point represents one trajectory with the vertical line indicating the median.


Fig. S3. Anti-HCV E2 specificity. Representative TIRF images of GFP.VPR HCVpp, Env-pp or VSV Gpp bound to non-polarized HepG2 cells costained with anti-HCV E2 11/20 Fab-Atto647N. Seventy-nine per cent of GFP.VPR expressing HCVpp costained with anti-HCV E2 11/20. The threshold for the green and red signals was defined as twice the mean background plus 3 standard deviations of uninfected HepG2 cells. Colocalization was established following the analysis of 40 cells. Scale bar represents 10 μm.

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