IFIT1 inhibits viral RNA and protein synthesis in cells infected with 2′O methyltransferase-deficient coronavirus.

<p>(<b>a–b</b>) HeLa cells were cotransfected for 48 h with an expression construct for the HCoV-229E receptor, human aminopeptidase N, and siRNAs targeting IFIT1 or the green fluorescent protein (GFP). Cells were then treated with 20 U IFN-α and infected with wild-type HCoV-229E (229E-WT; grey bars) or the 2′O methyltransferase-deficient HCoV-229E (D129A) mutant (229E-DA; red bars). Total RNA and protein were harvested 24 h post infection and analysed by quantitative RT-PCR (<b>a</b>) and western blotting (<b>b</b>), respectively. Quantitative RT-PCR data are from one of three representative experiments showing means ±SD for HCoV-229E nucleoprotein (229E-N) RNA after normalization to cyclin B (CycB) mRNA. (<b>c–d</b>) Bone marrow-derived macrophages (Mφ) derived from C57BL/6 (Ifit1^+/+) and Ifit1-deficient (Ifit1^−/−) mice were treated or not with 50 U of IFN-α for 2 h and infected with wild-type MHV (WT; grey bars) or 2′O methyltransferase-deficient MHV (DA; red bars). RNA and protein were harvested 8 h post infection and analysed by quantitative RT-PCR (<b>c</b>) and western blotting (<b>d</b>). Quantitative RT-PCR results are from one of three representative experiments, showing means ±SD for MHV nucleoprotein (MHV-N) RNA after normalization to the TATA-binding protein (TBP) mRNA. (<b>e</b>) Ifit1^+/+ and Ifit1^−/− mice were infected intraperitoneally with 5,000 plaque-forming units of MHV WT (grey bars) or DA (red bars). Viral titers in the spleens of 12 mice per condition were measured 48 h after infection. Data are shown as Tukey box-whisker plots (ND, not detectable; outlier indicated as black dot).