Fig. 6.

PCIF1 does not regulate VSV mRNA sensing or IFIT1/3 binding. (A) The indicated A549 cells were pretreated with vehicle (0.1% BSA) or 500 U ⋅ ml⁻¹ IFN-β for 5 h prior to infection with VSV at a MOI of 5 for 3 h. Cells were lysed and IFN-β mRNA levels determined by qRT-PCR (n = 4, ±SD, NS: 0.08 < P < 0.80, Student’s t test). (B) Schematic for testing IFIT1/3 affinity for differentially methylated cap structures [structure shown from Protein Data Bank 6C6K (32)]. IFIT1-IFIT3 complexes (1 µM) were bound to 1 nM radiolabeled RNA with a m⁷GpppA cap in the presence of unlabeled competitor RNAs at the indicated concentration with the indicated cap-proximal nucleotide. A high affinity of the competitor for IFIT1/3 will result in displacement of the radiolabeled substrate and loss of signal. A low affinity will allow the radiolabeled substrate to remain bound. (C) Radiolabeled RNA bound to IFIT1/3 was measured by filter-binding assay and phosphorimaging and normalized to a control without competitor RNA (n = 3, ± SD, least squares regression line shown).