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

Abstract

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and subsequent 40S recruitment. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 interacts rapidly and strongly with IFIT3 forming a stable heterotetramer. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that associates with IFIT1, forming an IFIT1:IFIT2:IFIT3 trimer. Site-directed mutagenesis revealed a C-terminal YxxxL motif in IFIT1 that mediates its association with IFIT3. Using various reporter mRNAs, we demonstrate for the first time that IFIT3 stabilises IFIT1 binding to cap0-mRNA and enhances its translation inhibition activity. Disrupting the binding interface between IFIT1 and IFIT3 abrogated this enhancement. This work reveals molecular aspects of IFIT assembly and provides an important missing link between IFIT interaction and function.