Fig. 4

SINV infection is reduced in DDX5-depleted HCT116 cells. A Western blot on HCT116 mock or SINV-GFP infected cells upon siCTRL and siDDX5 transfection. DDX5 and viral capsid were detected by specific primary antibodies. GAPDH was used as loading control. Position and molecular weight (KDa) of protein size markers are shown on the left. B RT-qPCR of SINV genomic (g) RNA in SINV-GFP infected HCT116 cells (MOI 0.1, 24 hpi), upon siDDX5 and siCTRL transfection. Results are expressed as relative to GAPDH and normalized to the siCTRL condition. C SINV-GFP viral titers from infected HCT116 cells transfected with siCTRL or siDDX5 were quantified by plaque assay. Results in B-C represent the mean ± standard deviation (SD) of three biological replicates (n = 3). * p ≤ 0.05, B one- sample t test or C paired Student’s t test. D Schematic diagram of the DDX5 CRIPR/Cas9 KO strategy. Two gRNAs targeting DDX5 exon 2 and intron 2 respectively were used to delete the 5’ splice site (ss) region (5’ss is shown in green, #1 target region in blue and #2 target region in magenta). The purple arrow represents an indel in DDX5 exon 2 for the KO allele 1. The grey rectangle represents a 378 nucleotide (nt) deletion in KO allele 2. E Agarose gel showing the PCR fragments amplified from cellular genomic DNA, corresponding to the WT and KO alleles, respectively, which were gel purified and sequenced. Amplicon size is shown: the upper band corresponds to KO allele 1, the lower band corresponds to the KO allele 2. F Western blot on mock or SINV-GFP infected cells in WT or DDX5 KO HCT116 cells. Antibodies against DDX5 and viral capsid were used. GAPDH was used as loading normalizer. G Level of SINV genomic RNA relative to GAPDH monitored by RT-qPCR in WT or DDX5 KO HCT116 cells upon SINV-GFP infection at MOI 0.1 for 24 h. Results are plotted as fold change relative to WT cells. H Viral particles of SINV-GFP from the supernatant of SINV-GFP infected WT or DDX5 KO HCT116 cells were quantified on Vero E6 cells by plaque assay. The results from three independent experiments are presented here. I Representative plaque assay images after crystal violet staining of Vero E6 cells infected with SINV-GFP supernatant at the indicates dilutions (10⁻³ and 10⁻⁴) from infected WT or DDX5 KO HCT116 cells. J SINV-GFP infection kinetics in WT and DDX5 KO HCT116 cells. The relative GFP fluorescence area (expressed in percentage) of WT and DDX5 KO HCT116 cells as a function of time was measured after SINV GFP infection at an MOI of 0.01 (left panel), 0.1 (middle panel) and 1 (right panel) every 6 h for 72 h with a CellcyteX automated cell counter and analyser. WT HCT116 cells, black; DDX5 KO HCT116 cells, red. Error bars in G-H and I represent the mean ± standard deviation (SD) of three independent biological experiments. * p ≤ 0.05, **p ≤ 0.01, G one- sample t test or H paired Student’s t test. One representative experiment out of three is shown in (A, F and I)