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 human IFIT family oligomerisation and examine the influence of interaction with IFIT2 and IFIT3 on IFIT1 mRNA cap0 binding activity we reconstituted the IFIT1:IFIT2:IFIT3 hetero-complex in vitro. To this end, His-tagged IFIT1, IFIT2 and IFIT3 were individually expressed in bacteria and purified as described in Materials and Methods. The His-tag was removed from IFIT2 and IFIT3 but retained on IFIT1 for later detection. Each protein was subjected to size exclusion chromatography with multi angle light scattering (SEC-MALS) to analyse IFIT assembly. SEC-MALS reveals the molecular mass of species eluting at different volumes from a size exclusion column providing information about the oligomeric state of these species. Varying concentrations of IFIT were analysed by SEC-MALS and, consistent with a recent report (BioRxiv: https://doi.org/10.1101/152850), IFIT1 oligomerised in a concentration dependent manner ( Figure S2A ). In contrast, IFIT2 eluted as two species corresponding to a stable dimer or tetramer ( Figure S2B) . Surprisingly, the IFIT2 dimer had a similar elution volume to the lowest concentration of IFIT examined. This demonstrates the importance of using the SEC-MALS technique which directly determines the mass of particles in solution from their Rayleigh scattering, instead of relying on the elution volumes of molecular weight standards during SEC to infer oligomeric state. When analysed alone, IFIT3 also eluted as a mostly dimeric species, with a smaller peak corresponding to a monomer ( Figure S2C ). We next examined the oligomeric status of complexes containing mixtures of the individually purified IFIT proteins. When incubated at 4 ï‚°C for 1 hour IFIT1 and IFIT3 formed a stable complex that eluted with a molecular mass of 221 kDa ( Figure 2A, peak a) . Analysis of the protein in peak a by SDS-PAGE (gel inset) shows that IFIT1 and IFIT3 are equimolar, indicating that this complex represents stable IFIT1:IFIT3 tetramers. A later eluting species likely corresponding to IFIT1:IFIT3 dimers (123 kDa) is also evident (Figure 2A , peak b). IFIT1 and IFIT2 were incubated at 30 ï‚°C for 1 hour and analysed by SEC-MALS. The IFIT1:IFIT2 complex was less defined and eluted as multiple species ranging from 148 to >234 kDa as measured by SEC-MALS ( Figure S2D ). While the most abundant IFIT1:IFIT2 complex form is likely tetrameric, the presence of several overlapping peaks precludes reliable determination of specific molecular mass and hence oligomeric state.