Regulation of the Antiviral Innate Immune Response by Ufmylation

Abstract

Cellular detection of viral infection activates an antiviral program through the induction of interferons (IFN), which ultimately limit viral replication and reduce viral spread. This induction of IFNs is initiated by proteins that sense viral RNA, such as RIG-I, and proteins that coordinate the resulting signaling, like its adaptor protein MAVS. These proteins, along with others, form a signaling pathway that is highly controlled to provide a fine balance between clearance of viral infection, while preventing prolonged or excessive IFN induction which can lead to autoimmunity. As such, the RIG-I signaling cascade is carefully regulated by multiple mechanisms, including post-translational modifications (PTMs), protein-protein interactions, and protein localization. For example, following sensing of viral RNA, RIG-I is ubiquitinated, leading to its activation and subsequent interaction with the molecular trafficking protein 14-3-3epsilon, forming a RIG-I signaling complex. This interaction with 14-3-3epsilon facilitates the relocalization of activated RIG-I from the cytosol to intracellular membranes where it interacts with the adaptor protein MAVS leading to the propagation of various signals that induce IFN. Importantly, while some post-translational modifications are well studied in regulation of the RIG-I signaling pathway and are known control many aspects of IFN induction, our current knowledge of the full set of regulatory mechanisms that govern the RIG-I activation cascade is incomplete. Previously, to determine novel regulators of RIG-I signaling, we identified proteins that relocalize to the RIG-I/MAVS signaling platform at ER-mitochondrial membrane contact sites. These relocalized proteins include UFL1, the E3 ligase of the small ubiquitin-like modification called UFM1. Similar to ubiquitination, the ufmylation process conjugates a small protein (UFM1) to lysine residues of target proteins using an E1, E2, and E3 ligase machinery system. Further, a protease specific to ufmylation can remove the modification, allowing for dynamic regulation of protein function. However, in contrast to ubiquitin, the ways in which ufmylation regulates protein function is still unclear. While the role of well-described PTMs such as ubiquitination and phosphorylation have a well-respected role in the regulation of the RIG-I signaling pathway, little is known about how emerging ubiquitin-like modifications, such as ufmylation, regulate this antiviral signaling pathway. Indeed, how these newly discovered PTMs regulate protein function has not yet been elucidated, requiring further study. Through investigating how ufmylation may regulate RIG-I signaling, I found that UFL1 is essential for the induction of IFN following RNA virus infection using a combination of IFN reporter assays, RT-qPCR to assess transcription, and immunoblots and ELISA to investigate protein expression. This regulatory role of UFL1 relies on its ability to conjugate UFM1 onto target proteins, leading me to hypothesize that ufmylation of proteins themselves may be driving signaling regulation. Indeed, altered expression of UFM1, or any of the proteins involved in its conjugation also positively regulated IFN. Utilizing co-immunoprecipitation assays, I found that following RNA virus infection, UFL1 is recruited to the membrane targeting protein 14-3-3epsilon. Further, I found that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling utilizing a series of RIG-I activation mutants. As I hypothesized that the primary role of UFL1 in RIG-I signaling regulation was through ufmylation of an interacting protein, I assessed the ufmylation status of 14-3-3epsilon, and I found that 14-3-3epsilon has an increase in covalent UFM1-conjugation when RIG-I activation is induced by viral infection. Additionally, loss of either cellular UFM1 or UFL1 expression prevents the interaction of 14-3-3epsilon with RIG-I. The consequences of which are impaired RIG-I recruitment to MAVS sites at ER-mitochondrial membranes. Indeed, I found that loss of UFM1 abrogates the interaction of RIG-I with MAVS via co-immunoprecipitation, and MAVS activation by oligomerization using semi-denaturing detergent agarose gel electrophoresis, and thus downstream signal transduction which induces IFN. These results reveal ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a novel post-translational control for IFN induction. Having discovered a role for ufmylation in regulating the interaction of 14-3-3epsilon with RIG-I, I hypothesized that there might be other proteins regulated by ufmylation during RIG-I signaling, as PTMs play a crucial role in the regulation of a multitude of proteins in the antiviral response pathways. Utilizing an unbiased proteomics approach, I profiled all the UFM1-interacting proteins during viral infection. These data revealed multiple proteins that display differential UFM1-interaction, including TBK1, the serine-threonine kinase that plays a key role in the induction of IFNs. Follow-up work revealed that five of the top 8 proteins identified modulate IFN induction. Further studies to identify how ufmylation influences these proteins functions, and how their ufmylation status regulates their role in RIG-I signaling is needed, but this reveals a broad role for ufmylation as an important RIG-I pathway regulatory PTM. Further, as many intracellular pathogen sensing pathways share common signaling proteins, ufmylation may also regulate these signaling processes as well. Overall, my work has revealed a previously undiscovered node of RIG-I regulation and uncovered a new class of RIG-I regulatory proteins that become ufmylated during the antiviral response.