Fig. 3: Interaction between NS1 and the CPSF complex is diminished by variation of amino acids at positions 103/106, but only completely abrogated by variation at position 184.
a Schematic representation of HA-tagged constructs used in AP-MS experiments. b Schematic representation of RBD- and ED-dependent interaction partners of NS1 proteins depending on amino acids at positions 103/106. c Normalized IBAQ-based log2 enrichment (ΔIBAQN) of depicted preys in comparison between R/SI/G (y-axis) and R/FG/G (x-axis) with control bait (CTR – ThoV M). Red dots show all significant interactors of the two proteins, with components of CPSF complex further highlighted. Further related data is presented in Supplementary Fig. 3a, b. d Western blot depicting detection of indicated proteins upon immunoprecipitation of HA-tagged R/SI/G or R/SI/R from RNase and DNase treated lysates of cells transiently expressing baits and GFP-tagged CPSF4. Depicted data are representative of 3 independent repeats. e HeLa cells were transfected with siRNA targeting CPSF4, non-targeting control siRNA (NTC) or transfection vehicle only (no-siRNA). 48 h post-transfection, RT-qPCR was used for quantification of expression levels of CPSF4 and APA of selected genes. ΔPDUI as a measure of APA status is shown alongside mean +/− sd for 4 separately processed wells. ΔCt values were calculated relative to the housekeeping gene RPLP0. Statistics were calculated using two-sided equal variance t-test. n.s. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.
