AN INTEGRATIVE APPROACH DELINEATING THE CRITICAL FUNCTIONS OF ANTIVIRAL PROTEIN IFI16

Abstract

The human interferon-inducible PYHIN protein IFI16 binds directly to viral DNA during infection and potentiates intracellular signaling that ultimately abates viral replication. While IFI16 is thought to function through engaging important antiviral pathways related to transcription, apoptosis, and cell-to-cell signaling, its modes of action are largely undefined. While several other sensors of viral DNA have been characterized, IFI16 is the only to predominantly localize and function within the nucleus. Thus, for my Ph.D. research, I integrated proteomic, microscopy, molecular virology, mutagenesis, and biochemical analyses to delineate these poorly understood roles of IFI16, the molecular mechanisms that regulate it, and its interactions with DNA viruses and other critical cellular factors.

I demonstrate that IFI16 possesses a bi-partite nuclear localization signal (NLS), which is required for its binding and signaling in response to DNA of nuclear-replicating herpes simplex virus-1 (HSV-1). This is the first demonstration that innate immunity can be elicited by viral DNA within the nucleus. Upon investigating IFI16 protein interactions during HSV-1 infection, I determined that the viral ubiquitin ligase ICP0 binds to and promotes the proteasomal degradation of IFI16. Consequently, ICP0 effectively attenuates IFI16-dependent potentiation of immune signaling in response to HSV-1 infection. I further probe the IFI16 signaling pathway and demonstrate that it functions either downstream of or parallel to the central cytoplasmic STING-TBK-1-IRF3 DNA sensing axis. Using live cell imaging studies, I show unique IFI16 aggregation behaviors at discrete sub-nuclear foci in response to infection with DNA viruses. Furthermore, using CRISPR-mediated knockout studies, I show that IFI16 can additionally function as a potent transcriptional repressor of DNA virus gene expression. Therefore, IFI16 has the ability to work in antiviral defense by both inducing cytokine expression and by suppressing viral gene expression. Finally, I present the first interactome analysis of the entire family of human PYHIN proteins (IFI16, AIM2, MNDA, and IFIX), identifying their common and unique functions in transcription and chromatin modulation, cell cycle regulation, and DNA damage responses. From these studies, I conclude that IFIX is also an antiviral factor, also able to detect and respond to viral DNA.