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. 2015 Aug 19;89(18):9465–9476. doi: 10.1128/JVI.00996-15

FIG 3.

FIG 3

Ectopic expression of human IFIT1 does not affect IAV infection. (A) Impact of luciferase, IFITM3, or IFIT1 expression in HEK293T cells infected with IAV-PR8. HEK293T cells were transfected with Flag-tagged luciferase, IFITM3, or IFIT1 and then, 16 h later, infected with IAV-PR8 at an MOI of 5. Cells were harvested at 6 h postinfection and stained with MAbs specific for IAV-NP and Flag. Representative flow cytometry plots are shown for luciferase, IFITM3, and IFIT1. The relative infection level was calculated as the percent infection of transfected cells divided by the percent infection of nontransfected cells for each sample and then normalized to that of control (luciferase) transfected cells. The data represent the mean relative infection levels ± SD for three independent experiments with three or four technical replicates. (B) Expression of IFIT1 preferentially inhibits WNV-E218A. HEK293T cells were transiently transfected with Flag-tagged IFIT1 or a Flag-tagged luciferase control vector. After 48 h, cells were infected with WNV-WT or WNV-E218A (MOI of 10). The cells were harvested 24 h after infection and stained with MAbs specific for WNV E and Flag. Representative flow cytometry plots are shown for IFIT1-transfected cells infected with either WNV-WT or WNV-E218A. The relative infection level was calculated as the percent infection of transfected cells divided by the percent infection of untransfected cells. The percent infection of transfected cells was determined by dividing the percentage of the viral antigen+ Flag+ population by the percentage of the Flag+ population. The percent infection of the untransfected cells was determined by dividing the percentage of the viral antigen+ Flag− population by the percentage of the Flag− population. The calculation was done for each sample and then normalized to that for luciferase control-transfected cells to adjust for interassay variation. The data represent the mean relative infection levels ± SD for two independent experiments performed in triplicate. ***, P < 0.001; ****, P < 0.0001; ns, no significant difference.