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. 2011 Mar 31;7(3):e1001329. doi: 10.1371/journal.ppat.1001329

Figure 2. Serum induces a Dynasore resistant entry route.

Figure 2

(A) The effect of increasing concentrations of FCS (in PBS) on IAV entry. HeLa cells were infected in the absence (grey bars) or presence (black bars) of 80 µM dynasore with IAV (strain WSN; MOI 0.5) using the Gluc-entry assay. Entry was determined in presence of increasing concentrations of FCS (plotted on the x-axis). Luciferase activity was determined 16 hrs p.i. (plotted on the y-axis relative to the activity obtained in absence of dynasore (grey bars, 100%) for each concentration of FCS. Entry of IAV became completely dynasore-resistant in the presence of 10% FCS. (B) Inhibition of VSV entry by 80 µM dynasore (DY) in presence of 10% FCS. HeLa cells were infected with VSV in PBS or PBS+10% FCS (labeled 10% FCS) in absence (grey bars) or presence (black bars) of 80 µM dynasore (DY). In presence of 10% FCS VSV infection appears still to be fully sensitive to dynasore. (C) IAV entry via the dynamin-dependent (DYNA-DEP) or dynamin-independent (DYNA-IND) route depends on the presence of sialic acid receptors. Hela cells were treated with V. cholerae neuraminidase (NEU; black bars) or mock-treated (-; grey bars) prior to infection in order to remove surface exposed sialic acids. Cells were infected (strain WSN; MOI 0,5) using the Gluc-entry assay. DYNA-DEP entry was determined in PBS and DYNA-IND entry in PBS containing 10% FCS and 80 µM dynasore. Both pathways appear to be equally sensitive to de-sialylation of cells. (D) Comparison of the kinetics of DYNA-DEP IAV entry and DYNA-IND IAV entry. DYNA-DEP entry was determined in PBS and DYNA-IND entry in PBS containing 10% FCS and 80 µM dynasore (80 DY) using the Gluc-entry assay as described for panel C. Entry was allowed to proceed for 15 to 240 minutes (x-axis) by the addition of full growth medium containing 10 nM BafA1 at the indicated timepoints p.i. Luciferase activity was plotted in absolute RLU (y-axis), demonstrating that similar entry efficiencies were obtained via the two pathways after 4 hrs of entry. The experiment was repeated several times with different batches of serum yielding similar results.