Characterizing phosphorylation-driven IFI16 liquid-liquid phase separation and innate immune signaling during HSV-1 infection

Abstract

The detection of pathogenic DNA by host sensors is a key step in activating host immune responses against microbial infection. Interferon-inducible protein 16 (IFI16) is a nuclear DNA sensor responsible for recognizing the genomes of nuclear-replicating DNA viruses such as herpes simplex virus type 1 (HSV-1). Subsequently, IFI16 activates intrinsic innate immune responses via the induction of antiviral cytokines and suppression of virus gene expression. Recently, our group has established that IFI16 undergoes liquid-liquid phase separation (LLPS), nucleated by DNA. However, little is known about how IFI16 LLPS is regulated, or how it contributes to IFI16 antiviral immune signaling; furthermore, the mechanism by which IFI16 transmits an immune signal also remains unclear. Here, we use molecular virology, microscopy, molecular genetics, and proteomics to investigate these questions. We demonstrate that multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS. We find that this phosphorylation is regulated by CDK2 and is required for IFI16 induction of antiviral cytokines. Additionally, we identify candidates for IFI16 downstream signaling molecules using a proximity labeling-mass spectrometry approach, resulting in a detailed view of LLPS-dependent IFI16 interactions during HSV-1 infection. Overall, these findings significantly expand the knowledge regarding IFI16 interactions and provide insight into the regulation of this critical antiviral factor by LLPS.