Sullivan, O.M., Nesbitt, D.J., Schaack, G.A., Feltman, E.M., Nipper, T., Kongsomros, S., Reed, S.G., Nelson, S.L., King, C.R., Shishkova, E., Coon, J.J., Mehle, A., 2025. IFIT3 RNA-binding activity promotes influenza A virus infection and translation efficiency. Journal of Virology. https://doi.org/10.1128/jvi.00286-25

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-stimulated gene products, establishing a model for its broad antiviral activity. However, IFIT2 also binds viral mRNAs and enhances their translation, resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here, we validate direct interactions between IFIT3 and RNA using electrophoretic mobility shift assays. RNA-binding site identification experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability of IFIT3 to promote IAV gene expression and translation efficiency compared to wild-type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers; however, the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA binding and its ability to modulate translation of viral mRNAs during IAV infection.