Characterizing the mechanisms of cytoplasmic sensing by the DNA sensor IFIX

Abstract

Sensors of foreign DNA within the mammalian cell are essential to the intrinsic and innate immune defense against viral infection. Of these, the Interferon-Inducible Protein X (IFIX) is the most recently discovered and least understood. Previous findings show that IFIX can serve as a cytoplasmic DNA sensor despite being predominantly nuclear, but the mechanism by which IFIX’s subcellular localization is regulated remains uncharacterized. This project aims to elucidate how IFIX subcellular localization is regulated. Specifically, I performed nuclear export inhibition and mutational analyses to show that active nuclear export plays a role in the recruitment of IFIX to viral DNA during cytoplasmic DNA sensing. Two NES motifs predicted by bioinformatics were now experimentally validated. Additionally, I investigated three putative nuclear localization (NLS) motifs within IFIX. We find that single deletions of NLS motifs 2 and 3 individually impact the localization of IFIX. Furthermore, substitution of lysine K138 on NLS motif 2 for the acetyl-mimic glutamine promotes cytoplasmic localization by inhibiting nuclear import. These findings suggest that NLS motifs 2 and 3 contribute to the nuclear import of IFIX and that post-translation modification through acetylation plays a role in retaining IFIX in the cytoplasm during cytoplasmic DNA sensing. Lastly, we further characterized the involvement of different IFIX domains in immune signaling, and showed that the induction of an interferon response is dependent on IFIX oligomerization via its PY domain. Altogether, by defining the mechanisms regulating IFIX subcellular localization and oligomerization, these studies provide insight into the mechanisms involved in viral DNA sensing by IFIX, which may ultimately provide new targets for antiviral therapies.