Fig. 4.

ITAF₄₅ is required for viruses containing a Type II IRES. (A) Left: eHAP1 control and ITAF₄₅^KO cells were infected with FMDV (O1 Kaufbeuren) and cultured in an Incucyte to monitor the rate of cytopathic effect (CPE) development via measurement of cell confluency. The average confluency of three biological replicates was plotted against time using GraphPad Prism (GraphPad Software). Right: Representative image of cell monolayers at 12 h postinfection. (B) Replication of FMDV in eHAP1 control and ITAF₄₅^KO cells was assessed via transfection of 90 ng of an FMDV subgenomic replicon wherein the capsid protein coding regions were replaced with a GFP reporter. Replication was assessed using the Incucyte S3 2019B ver2 software to quantify the total integrated green intensity per well using a cutoff value of 0.5. (C) eHAP1 control and ITAF₄₅^KO cells were infected with TMEV (GDVII strain) at an MOI of 50 and lysed at 24 h postinfection to quantify viral RNA using RT-qPCR. (D) Top: eHAP1 control and ITAF₄₅^KO cells were used to titrate ERAV stock where the end-point titers were calculated using the Spearman–Kärber method. Bottom: Crystal violet staining of cells inoculated with indicated virus. TMEV and ERAV dataset represent means ± s.d (n = 3 independent biological replicates). P values were determined by a paired T-test using GraphPad Prism (GraphPad Software) ***P < 0.0002 and *P < 0.0332.