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

Together, our results demonstrate the first in vitro reconstitution of the human IFIT2:IFIT3 and IFIT1:IFIT2:IFIT3 heterocomplexes, provide novel details about IFIT complex assembly and reveal for the first time that IFIT3 enhances IFIT1 cap0-mRNA binding and translation inhibition. Our reconstituted complexes provide a solid foundation for future molecular analysis of IFIT assembly and function. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/261776 doi: bioRxiv preprint IFIT1+IFIT3 is shown in grey shadow for reference. (G) Luciferase activity from RRL incubated with cap0--globin Fluc RNA and WT or mutant IFIT1 with or without IFIT3 as indicated. Data are normalised to the luciferase activity in the absence of IFIT1 and shown as the mean  the standard error of three separate experiments. Statistical analysis was performed comparing conditions joined by dotted lines using an unpaired, two-tailed Students T-test. P values are indicated, and  denotes statistical significance. Figure 6 . Schematic representation of the IFIT complexes analysed in this study. Cartoons depicting IFIT1 (yellow), IFIT2 (green) and IFIT3 (blue) complexes reconstituted in vitro from individually purified proteins. Weak interactions are indicated by reversible arrows. IFIT1 and IFIT2 structures and dimerisation interactions have been characterised by X-ray crystallography (BioRxiv: https://doi.org/10.1101/152850 and (30)). No experimentally derived structure for IFIT3 homo-or heterodimers are currently available. Therefore, IFIT3 and IFIT2:IFIT3 dimerisation, and IFIT1 heterooligomerisation are modelled on the IFIT1 dimer (PDB: 5W5H) and IFIT2 dimer (PDB: 4G1T) crystal structures and supported by experimental evidence as discussed in the text. The association of IFIT1 and IFIT2 is less well defined as indicated by the question marks.