Fig. 5

Inhibition of TRIM25-mediated RIG-I ubiquitination and signalling by NS1. a Effect of NS1 and mutants on the ubiquitination of 3FLAG-RIG-I-2CARD by TRIM25 in HEK293T cells. 3FLAG-RIG-I-2CARD, TRIM25 and NS1 were co-transfected into HEK293T cells and WCLs were subjected to IP with anti-FLAG beads and immunoblotted with α-ubiquitin and α-FLAG antibodies. The asterisk indicates an unspecific band. b In vitro ubiquitination assays with UBE2N/UBE2V1 and TRIM25-FL in the absence or presence of increasing amounts of NS1 supplemented with fluorescent AttoUb. Assays were carried out with NS1-ED or NS1-FL (R38A/K41A/W187A or W187A) and the reaction was monitored over 60 min. Gels were scanned with a Typhoon FLA 9500 scanner and the fluorescence converted to black and white. c 3FLAG-RIG-I-2CARD, TRIM25, and increasing amounts of NS1 WT and mutants (25, 50 and 100 ng) were co-transfected into HEK293T cells. Interferon induction by 3FLAG-RIG-I-2CARD was measured in a Luciferase-based reporter assay, where Luciferase activity is controlled by the IFN-β promoter. The mCherry plasmid was used as a control. The error bars shown are the standard deviation (s.d.) of mean from two independent experiments, each with triplicates