Figure 1. A luciferase reporter assay for quantitative analysis of IAV entry.

(A, and B) HeLa cells were transfected with the IAV reporter plasmid pHH-Gluc 24 hrs before infection. Inhibitors were added at 1 hr before infection with IAV (strain WSN; MOI of 0.1). Cells were kept at 37°C in DMEM supplemented with 10% FCS at all times. Luciferase activity (RLU, Relative Light Units) was measured 16 hrs p.i. and plotted on the Y-axes relative to the control (infection in presence of 0.2% DMSO, the solvent of both inhibitors) (error bars represent S.D. derived from triplicates). (A) Inhibitory effect of BafA1 (concentration range 0.625 nM to 10 nM on the x-axis) (B) Inhibitory effect of Dynasore (concentration range 20 to 160 µM on the x-axis) (C) The effect of the presence of 10 nM BafA1 during different periods (−1 hr to 16 hrs p.i.; 1 hr to 16 hrs p.i.; 2 hrs to 16 hrs p.i. and −1 hr to 2 hrs p.i as plotted on the x-axis) of a multi-cycle infection with IAV (strain WSN; MOI 0.5). Y-axis and control as in panel A and B. BafA1 was only strongly inhibitory when already present before inoculation with IAV. Reversibility of inhibition was shown upon withdrawal of BafA1 at 2 hrs p.i. (−1 hr to 2 hrs). (D) Examination of the effect of 10 nM BafA1 on subcellular localization of IAV particles by confocal fluorescence microscopy. HeLa cells were grown on glass cover slips and infected with IAV (strain WSN; MOI of 10) and fixated after 30 min, 3 hrs or 6 hrs (column 1, 2 or 3 respectively). Infection was performed in 0.2% DMSO (upper row panels) or in the presence of 10 nM BafA1 (lower row panels). The nucleus was visualized by DNA staining with TOPRO-3 (red). IAV infection was visualized by staining with monoclonal antiserum directed against NP (green). In the absence of inhibitor, IAV localized to the nucleus after 3 hrs, while new virus particles spread to the cytoplasm after 6 hrs. BafA1 (lower row panels) caused accumulation of incoming virus particles at a peri-nuclear location. (E) Quantitative determination of IAV entry by a single-cycle Gluc-entry assay. HeLa cells (10,000 cells/well in DMEM supplemented with 10% FCS) were transfected with pHH-Gluc 24 hrs prior to infection with a serial dilution of infectious IAV particles (plotted on the x-axis). Two hours after infection 10 nM BafA1 was added to block any further entry. Cells were incubated for a further 14 hrs to allow expression of luciferase activity (y-axis; Relative Light Units, RLU). (F) Effect of Dynasore and BafA1 on IAV entry in the Gluc-entry assay. Dynasore (DY, dark grey bars; 20, 40 or 80 µM) or BafA1 (light grey bars; 1.25, 2.5 or 5 nM) were present from 1 hr prior to infection (strain WSN; MOI 0.5) to 2 hrs p.i. after which the inhibitor-containing medium was replaced with medium containing 10 nM BafA1 to block any further entry. Cells were incubated for a further 14 hrs to allow the quantitative expression of luciferase activity (y-axes; RLU relative to the control infection without inhibitor). Whereas BafA1 displayed dose-dependent inhibition of IAV entry, dynasore did not significantly inhibit IAV entry. Addition of Dynasore (DY, 20, 40 or 80 µM) at 2 hrs p.i. (black bars; MOI 0.5; no inhibitors present from −1 to 2 hrs) demonstrated that Dynasore does not have a post-entry effect.