The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA

Abstract

The human PYHIN proteins, AIM2, IFI16, IFIX, and MNDA, are critical regulators of immune response, transcription, apoptosis, and cell cycle. However, their protein interactions and underlying mechanisms remain largely uncharacterized. Here, we provide the interaction network for all PYHIN proteins and define a function in sensing of viral DNA for the previously uncharacterized IFIX protein. By designing a cell-based inducible system and integrating microscopy, immunoaffinity capture, quantitative mass spectrometry, and bioinformatics, we identify over 300 PYHIN interactions reflective of diverse functions, including DNA damage response, transcription regulation, intracellular signaling, and antiviral response. In view of the IFIX interaction with antiviral factors, including nuclear PML bodies, we further characterize IFIX and demonstrate its function in restricting herpesvirus replication. We discover that IFIX detects viral DNA in both the nucleus and cytoplasm, binding foreign DNA via its HIN domain in a sequence-non-specific manner. Furthermore, IFIX contributes to the induction of interferon response. Our results highlight the value of integrative proteomics in deducing protein function and establish IFIX as an antiviral DNA sensor important for mounting immune responses.

The Emerging Role of Nuclear Viral DNA Sensors

“…Originally, IFI16 was shown to bind to cytosolic viral DNA, physically engage STING, and induce IFN production in macrophage-like THP-1 cells (33)(34)(35). However, across non-immune cell types, IFI16 predominantly localizes to the nucleus (29,31,36,37). We defined a multi-partite nuclear localization signal on IFI16 and observed that its deletion triggers the localization of IFI16 exclusively to the cytoplasm (36).…”

“…A member of the interferon-inducible human PYHIN protein family (28,29), IFI16 possesses two C-terminal HIN200 domains that bind to DNA in a sequence-independent manner (30) and an N-terminal pyrin (PY) domain that mediates intramolecular homotypic interactions and cooperative assembly of IFI16 oligomers (31,32) (Fig. 1, A and B).…”

“…In agreement, IFI16 was not observed to associate with ASC, a requirement for the recruitment and maturation of caspase-1 (31, 53). More recently, comprehensive proteomic analyses of IFI16 interaction networks did not detect interactions between endogenous IFI16 and ASC during HSV-1 infections in primary fibroblasts (29,54). Several studies that probed IFI16 functions during early stages of herpesvirus infections have observed neither IFI16 inflammasome assembly nor caspase-1 maturation (31,36,37,40,41,54).…”

“…The other PYHIN members, IFIX and MNDA, although also localized predominantly to the nucleus, had few characterized immunological functions. Our recent proteomic investigation of the PYHIN family protein interaction networks revealed that IFIX associates with antiviral factors and that its expression is inversely correlated with herpesvirus replicative capacity (29). IFIX was further shown to bind DNA substrates in a sequence-independent manner and to contribute to type I IFN production (29).…”

“…Our recent proteomic investigation of the PYHIN family protein interaction networks revealed that IFIX associates with antiviral factors and that its expression is inversely correlated with herpesvirus replicative capacity (29). IFIX was further shown to bind DNA substrates in a sequence-independent manner and to contribute to type I IFN production (29). Moreover, during HSV-1 infection, IFIX associates with viral genomic DNA and remains localized to the nucleus (29).…”