Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells

Figure 1.. Ifit1 decreases MNV infection in RAW264.7 cells. ( A) Ifit1 knockout RAW264.7 cells were generated by CRISPR-Cas9 gene editing. Cells were stimulated with IFNβ for 12 hours then analysed by western blotting against Ifit1 and Ifit2/Ifit3. GAPDH was included as a loading control for each membrane. ( B, C) Infection of wild-type (WT) and Ifit1 knockout (KO) RAW264.7 cells at ( B) high or ( C) low multiplicity of infection (MOI) with murine norovirus (MNV-1). Viral titres were determined by 50% tissue culture infectious dose (TCID 50) in BV2 cells and expressed as log 10-transformed values. At late time points, indicated, severe cytopathic effect (cpe) was visible. Graphs show the mean and the standard error of three biological replicates. Titres were compared between WT and KO cells for each time point by two-tailed Student’s t-test. Asterisks indicate that a statistically significant difference (p < 0.05) was observed for both KO cell lines.

Figure 2.. Calicivirus translation is resistant to Ifit1 inhibition. ( A) Schematic representations of in vitro transcribed cap0 genomic RNA or VPg-linked genomic and subgenomic RNAs, purified from infected cells, used for in vitro translation and toeprint assays. ( B) In vitro translation of cap0 or VPg-linked RNA from murine norovirus (MNV), porcine sapovirus (PSaV) and feline calicivirus (FCV). VP1, the dominant protein product produced from the VPg-linked subgenomic RNA, is indicated. ( C). Toeprint analysis of MNV, PSaV and FCV VPg-linked and cap0 RNA. Ifit1 binding is indicated by a cDNA product 6-7 nt shorter than the full-length signal (FL), indicated by black arrowheads. Red arrowheads indicate a 1-2 nt shorter full-length signal on VPg-linked RNAs.

Figure 3.. Ifit1 promotes type I IFN expression following cytoplasmic RNA sensing. ( A– F) Wild-type (WT) and Ifit1 knockout (KO) RAW264.7 cells were stimulated with ( A, B) 2 μg transfected polyI:C (polyI:C t), or ( C, D) 1 μg/mL polyI:C or ( E, F) 10 ng/mL lipopolysaccharide (LPS) in the cell culture medium. RNA was extracted and analysed by RT-qPCR for IFNβ ( A, C, E) and TNFα ( B, D, F) mRNA, expressed as fold induction over untreated cells, normalised against GAPDH (2 -ΔΔCq). Graphs show the mean and the standard error of three biological replicates. Fold induction was compared between WT and KO cells for each time point by two-tailed Student’s t-test. Asterisks indicate that a statistically significant difference (p < 0.05) was observed for both KO cell lines.