Regulation of RIG-I signaling and viral immune evasion by ufmylation

ABSTRACT Type I and III interferons (IFN) restrict RNA virus infection. Infected cells produce IFN through signaling activated by pattern recognition receptors such as RIG-I. Both RIG-I and downstream signaling must be highly coordinated for efficient antiviral responses. As such, these processes are regulated by several post-translational modifications (PTMs). These PTMs are essential for both the activation and eventual termination of RIG-I- signaling. While RIG-I-signaling is coordinated by ubiquitination and phosphorylation, the mechanisms by which other PTMs, such as ubiquitin-like modifiers, may regulate RIG-I-signaling are largely unknown. Our preliminary data reveal that the ubiquitin-like modifier called ufmylation regulates multiple proteins involved in RIG-I signaling for optimal IFN induction in response to viral infection. Further, our data suggest that ufmylation is utilized by viruses to evade the host intracellular innate immune response. Therefore, the goal of this proposal is to determine how ufmylation regulates the intracellular innate immune response to virus infection and viral evasion. Based on our preliminary data, the central hypothesis is that ufmylation modulates the function of host and viral proteins to regulate antiviral innate immunity and viral evasion. Guided by our preliminary data, this hypothesis will be tested by pursuing the following three specific aims: 1) Define how UFL1, the ufmylation E3 ligase, promotes the activation of RIG-I; 2) Understand the molecular mechanism by which ufmylation of a key protein in the RIG-I signaling pathway downregulates its function and signaling; 3) Determine how dengue virus co-opts the ufmylation conjugation system for immune evasion. In Aim 1, the molecular mechanisms by which UFL1 induces the activation of RIG-I in response to RNA virus sensing will be defined. In Aim 2, the mechanism and dynamics of how ufmylation regulates the function of a signaling protein in RIG-I pathway will be determined. In Aim 3, the function by which ufmylation of a DENV protein promotes DENV immune evasion, both in human cell lines and in primary cells, will be determined. Taken together, the work proposed in this application will be significant and innovative because it will attribute a novel immune regulatory function to ufmylation, contribute to our understanding of its basic functions, and uncover a novel control (ufmylation) of antiviral innate immunity. Additionally, this work will provide understanding of a host-directed process that is utilized by viruses for immune evasion to facilitate viral replication. Overall, this work will define a new PTM that coordinates the RIG-I signaling pathway, which will improve our knowledge of both antiviral immunity and regulation of innate immune pathways that will lead to increased understanding of the mechanistic causes of dysregulated IFN that can ultimately result in autoimmune disease. It will also define a new mechanism of immune evasion by flaviviruses that will have implications for therapeutic and vaccine strategies to limit their infection.