DESCRIPTION (provided by applicant): Influenza virus infection and its complications remains a leading cause of death in the US, and burdens the nation economically with costs of up to $87 billion annually. We, and others, have shown that the interferon- induced transmembrane protein 3 (IFITM3) inhibits influenza virus infections in vitro, and IFITM3 has also been shown to be essential for innate resistance to influenza virus infection in both mice and humans. Despite the importance of IFITM3 in broadly inhibiting influenza and other viruses, this knowledge has not yet inspired new therapeutics because it remains unclear precisely how IFITM3 hinders viral infection and how IFITM3 trafficking and function are regulated biochemically. Our overall hypothesis is that IFITM3 stability and localization are controlled by a series of post-translational modifications that ensure proper delivery and anchoring of IFITM3 to endolysosomes, where its intramembrane domains decrease membrane fluidity to prevent influenza virus fusion/infection. Aim 1 is based on our novel discovery that the E3 ubiquitin ligase NEDD4 is a negative regulator of IFITM3 cellular levels and activity. NEDD4 may represent a novel drug target for improv- ing IFITM3-mediated innate immunity against influenza virus, and our work examining IFITM3 ubiquitination by NEDD4, and the crosstalk between this and other modifications will provide molecular details regarding IFITM3 regulatory mechanisms that must be understood in order to ultimately devise such therapeutics based on IFITM3 biology. Aim 2 is built upon our discovery that a specific DHHC domain-containing palmitoyltransferase is able to palmitoylate IFITM3, which we previously demonstrated is an essential modification necessary for IFITM3 antiviral activity. We will analyze potential IFITM3 palmitoylation defects caused by polymorphisms reported for this enzyme, thus laying the groundwork for its study as an influenza virus susceptibility factor. Our preliminary data also suggest that IFITM3 palmitoylation must occur at a specific cellular compartment for positive effects on its activity to be observed. Thus we will investigate putative DHHC localization mechanisms that account for this effect. This series of experiments will identify critical DHHC trafficking and specificity determinants, and reveal which cellular compartments support the proper palmitoylation/activation of IFITM3. In Aim 3 we will examine the function of IFITM3 intramembrane regions in altering cellular membranes to inhibit virus infections. We have provided evidence that IFITM3's predicted transmembrane domains act instead as intramembrane domains. Intramembrane domains often alter membrane structure, and our preliminary data indicates that IFITM3 indeed decreases membrane fluidity. Thus, we will examine the necessity and sufficiency of IFITM3 intramembrane domains in decreasing membrane fluidity and inhibiting virus infection. In the course of all of these studies we will appy the knowledge gained toward the development of an optimally active but minimally sized antiviral poly- peptide construct. Overall, these research aims should reveal molecular strategies for controlling and exploiting IFITM3 in fighting influenza virus and viral diseases.

PUBLIC HEALTH RELEVANCE: The antiviral effector protein, IFITM3, is essential for innate resistance to influenza virus and other infections. We will identify and characterize molecular determinants of IFITM3 antiviral activity. Insights gained will be critical for developing novel IFITM3-based strategies for preventing and combating viral disease.