Gaussia luciferase reporter virus is suitable to measure inhibitory effects by IFITM proteins.

<p>(A) A549, MDCK and 293T cells were transduced with vectors encoding IFITM1, 2 or 3 or <i>cat</i> as control gene and subsequently infected with PR8-NS1-Gluc at MOI 0.1. Gaussia luciferase activity was determined from supernatants at 24 h post infection. The average of two (MDCK, 293T) or three (A549) separate experiments performed with triplicate samples is shown. Infection of cat expressing control cells was set as 100%. Error bars indicate SEM. (B) 293T cells transduced to express IFITM1, 2 or 3 or <i>cat</i> were transduced with reporter pseudoparticles bearing the surface proteins of VSV, MLV, LASV or influenza A virus (FLUAV). Firefly luciferase activity encoded by pseudovirus genomes was determined in cell extracts harvested at 72 hours after transduction. The average of three separate experiments performed with triplicate samples is shown. Infection of <i>cat</i> expressing control cells was set as 100%. Error bars indicate SEM. (C) A549 cells were infected with PR8-NS1-Gluc at MOI 0.1 in the presence of the indicated concentrations of cholesterol modulating agent U18666A. Gaussia luciferase activity in culture supernatants was determined at 24 hours post infection. (D) A549 cells were incubated with the indicated concentrations of U18666A and then transduced with pseudoparticles bearing influenza A virus HA and NA proteins on the surface. Firefly luciferase activity encoded by pseudovirus genomes was determined in cell extracts harvested at 72 h post transduction. (E) Viability of A549 cells treated with U18666A at the indicated concentrations or diluent DMSO was measured using MTT test. Panels C-E show the results of a representative experiment performed with triplicate samples; error bars indicate SD. Similar results were obtained in at least two separate experiments. All results were assessed for statistically significant differences using a two-tailed t test: *, P≤0.05; **, P≤0.01; ***, P≤0.001. cps, counts per second; VSV, vesicular stomatitis virus; MLV, murine leukemia virus; LASV, Lassa virus; FLUAV, influenza A virus.