IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

To investigate the impact of hetero-oligomerisation on the ability of IFIT1 to inhibit cap0 dependent translation we used a similar approach to that previously described by Young et al. (22) . In vitro transcribed mRNAs comprising a firefly luciferase (Fluc) reporter flanked by either the human globin or zika virus (ZV) 5 and 3 untranslated regions (UTRs) were post-transcriptionally capped as described in the Materials and Methods. The -globin and ZV constructs represent mRNAs with weak or strong secondary structures in the 5 UTR, respectively. Schematics of the two constructs are shown in Figure 3A . These mRNAs were incubated with different IFIT heterocomplexes before addition of rabbit reticulocyte lysate (RRL). Translation was quantified by measuring the luminescence from the Fluc reporter. The amount of IFIT1 added was equalised by western blotting against the His-tag on the purified IFIT1 in each complex. The upper panel of Figure 3B shows an SDS-PAGE analysis of the protein complexes used in these experiments while the lower panel shows an anti-His-tag western blot of the same complexes. The linearity of the western blot signal to protein concentration was confirmed as shown in Figure S3 .