Fig 1.

Coinfection assay to identify host-directed pan-myxovirus inhibitors. (A) Schematic of the dual-target antiviral screen. Anticipated outcomes are specified. (B) Expression profiles of the different luciferase reporter constructs. Vero cells were independently infected with recMeV-ren and recRSV-ren (MOI = 0.2) or transfected with the IAV-firefly luciferase reporter and superinfected with IAV-WSN (MOI = 0.1). At the indicated times postinfection (PI), relative luciferase units (RLU) were determined. The shaded area shows the experimental window (28 to 34 hpi). Values were normalized to peak activities of the respective data series and represent means ± SD for three experiments. (C) Progeny titers of released (IAV-WSN) or cell-associated (recMeV) particles after infection of Vero cells singly or in combination. Values represent means and SD for three experiments. (D) Microscopic examination of Vero cells coinfected with recMeV-GFP and IAV-WSN. Where indicated, cells were incubated in the presence of 100 μM fusion inhibitory peptide (FIP). Recording was done at a magnification of ×200. (E) Cytometric analysis of doubly infected cells as described for panel B, with cells incubated in the presence of 100 μM FIP until harvest. Cells were immunostained with specific anti-IAV M2 antibodies (with APC conjugate), and APC fluorescence and EGFP autofluorescence were determined. Numbers show % distributions of singly and doubly fluorescent cells.