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

We initially confirmed that IFIT1, IFIT2 and IFIT3 interact in lysates from HEK293T cells using proteomics. In contrast to previous studies (12, 14) we used nuclease treated samples confirming that this interaction occurs in cells in an RNA-independent manner. Moreover, we confirmed that IFIT1 could interact directly with IFIT2 or IFIT3 by SEC. We then employed SEC-MALS to examine IFIT heterocomplex assembly for the first time. Unlike standard SEC, when coupled with protein gel analysis SEC-MALS enables reliable identification of oligomeric status. Figure 6 shows schematic representations of each of the IFIT complexes reconstituted in vitro in this study. Consistent with previous reports ((11) and BioRxiv: https://doi.org/10.1101/152850), IFIT1 was found to reversibly dimerise in a concentration dependent manner ( Figure S2A ). While both IFIT2 and IFIT3 individually form stable homodimers as previously reported (13, 7) , an unexpected tetrameric form of IFIT2 was also detected ( Figure S2B and C) . Surprisingly, we found that the IFIT1:IFIT3 interaction is more stable than the IFIT1:IFIT1 complex (compare Figures 2A and S2A) . When analysed by SEC-MALS the predominant IFIT1:IFIT3 oligomeric state was a stable tetramer containing equimolar amounts of both proteins. The IFIT2 dimer eluted from the SEC at a volume expected for a monomeric species ( Figure S2B ) and as such had a similar elution volume as IFIT1. Very weak interaction of IFIT1 and IFIT2 was detected by SEC when these proteins were incubated at 4 ï‚°C (RF and TS unpublished data). author/funder. All rights reserved. No reuse allowed without permission.