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 generated a panel of IFIT1 mutants based on the IFIT1 dimer crystal structure (PDB: 5W5H). The point mutants Y460E and L464E expressed and purified similarly to the wild type protein. Both mutants eluted as monomeric species during SEC ( Figure 5C ) consistent with disruption of IFIT1 homodimerisation as previously reported (BioRxiv: https://doi.org/10.1101/152850). However, the L464E substitution only had a modest effect on the interaction of IFIT1 with IFIT3 ( Figure 5D) . While a small portion of IFIT1-L464E eluted as a monomer, a stable tetramer with IFIT3 was the predominant species, like the wild type IFIT1 shown in Figure 2A . The Y460E substitution destabilises IFIT1:IFIT3 oligomerisation to a greater extent than the L464E substitution, with less IFIT1-Y460E sequestered to the IFIT1:IFIT3 complex ( Figure 5E ) but, as is clear in the SDS-PAGE analysis of this experiment (gel inset), IFIT1-Y460E can still associate with IFIT3. We therefore generated a Y460E/L464E double mutant of IFIT1 (IFIT1-YL). IFIT1-YL eluted as a monomeric species on SEC, similarly to the Y460E and L464E single mutants ( Figure 5C ). However, in contrast to these two single point mutants, IFIT1-YL did not interact with IFIT3 during SEC ( Figure 5F ).