Multiple phosphorylation combinatorially regulates IFI16 antiviral immune signaling

In mammalian cells, DNA sensors are critical players in intrinsic and innate immunity, serving to detect the presence of pathogenic DNA and activate immune responses. Among DNA sensors, the interferon inducible protein 16 (IFI16) has emerged as a key sensor of foreign DNA in the nucleus. Its function is critical in host defense against a number of viruses, including herpesviruses, papillomavirus, and the retrovirus HIV. In the context of infection with the nuclear-replicating herpes simplex virus type 1 (HSV-1), IFI16 rapidly localizes to the nuclear periphery, where it binds to incoming viral DNA. Upon binding to viral DNA, IFI16 forms dynamic on/off puncta-like aggregates via oligomerization. IFI16 then functions in two ways to inhibit virus replication by both inducing cytokine expression and suppressing viral gene expressions. However, little is known about how IFI16 antiviral functions are regulated at the nuclear periphery. It is not clear what regulates the dynamic puncta formation and how it translates to downstream functions, such as the IFI16-mediated suppression of viral gene expression.
Here, we integrate molecular virology, microscopy, proteomics, and high-throughput next-generation DNA sequencing to gain insights into mechanisms governing IFI16 regulation and its transcriptional regulatory functions. We demonstrate that IFI16 undergoes localization-specific phosphorylation and that several phosphorylation sites act in a combinatorial manner to modulate dynamic puncta formation. IFI16 phosphorylation is critical for regulating cytokine induction, but not for suppressing viral gene expression. This provides the first example of decoupling the two antiviral functions of IFI16. We further validated that CDK2 and GSK3B are capable of phosphorylating IFI16 at the nuclear periphery during infection. Overall, our results provide a novel mechanism by which the immune signaling initiation and maintenance of IFI16 is achieved with high temporal resolution.