IFITs in Context: Investigating Pro- and Anti-Viral Mechanisms of IFIT2 and IFIT3

• Main Author: Schaack, Grace A
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2023
• Subjects: Cellular biology; Molecular biology; Virology
• ISBN: 9798379727772

The IFITs (interferon-induced proteins with tetratricopeptide repeats) are important innate immune effectors with broad-spectrum antiviral activities. Previous studies by our group revealed that IFIT2 and IFIT3 exert both anti- and pro-viral activities during influenza A virus (IAV) infection. IFIT2 enhances translation of bound transcripts by preventing or relieving ribosomal pausing, but the exact mechanism by which IFIT2 achieves this effect is unknown. Here we investigate IFIT2 and IFIT3 mechanisms of action during a type I interferon (IFN) response, exploring RNA-protein and protein-protein interactions with these IFITs. We find that IFIT2 binds to and enhances translation of AU-rich mRNAs, including transcripts for many interferon-stimulated genes and cytokines. IFIT2 affects a similar repertoire of host RNAs during a generic type I IFN response and during IAV infection, revealing that the pro-translational activity of IFIT2 is not restricted to the setting of IAV infection but is rather a characteristic function of IFIT2 generalizable to any type I IFN-inducing process that explains how IFIT2 can promote establishment of an antiviral environment in the host. We also characterize IFIT3 as an RNA-binding protein with structural and functional similarities to IFIT2. To identify other cellular co-factors that might mediate the translation-enhancing effect of IFIT2, we employed a proximity labeling approach using AirID and a split-complementation version of AirID, which we term splitAirID, to identify protein interactors of IFIT2 and IFIT3 at large and in specific dimeric configurations. We identified a broad set of interactors for IFIT2 and IFIT3 using full-length AirID and a more specific subset of interactors for homodimers and heterodimers of IFIT2 and IFIT3. We identified IFIT1B as an interactor of homo- and heterodimeric IFIT3 and found that IFIT1B exhibits pro-viral activity during influenza virus infection. Combining our datasets with previously described interactors of IFIT2 and IFIT3, we reveal protein folding as a novel facet of the IFIT2/IFIT3 functional interaction network, leading to a new hypothesis that the pro-translational effect of IFIT2 might be mediated by recruitment of chaperones that promote co-translational protein folding, thereby enhancing translational efficiency. This work expands our experimental toolkit for studying host-virus interactions and deepens our mechanistic understanding of IFIT2 and IFIT3, placing them in the broader context of IFIT protein-protein interactions and defining their dichotomous roles as broadly acting antiviral host factors with pro-influenza viral activities.