Identification of an E3 ubiquitin ligase that regulates cellular resistance to influenza virus infection by controlling levels of the antiviral protein IFITM3 (INM3P.363)

Abstract

Abstract Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cellular antiviral restriction factor that is able to inhibit infection by a wide variety of viruses, including influenza and Ebola. We previously showed that IFITM3 turnover is promoted by its ubiquitination, though the ubiquitin ligase responsible for this modification was not identified. Here we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in vitro and in cells. Accordingly, we found that NEDD4 knockout mouse fibroblasts accumulate IFITM3, and that this accumulation is lost upon reconstitution of these cells with NEDD4. Importantly, IFITM3 accumulation results in enhanced resistance of NEDD4 knockout cells to influenza virus strains. Similarly, knockdown of NEDD4 in several human lung cell lines resulted in a buildup of IFITM3 and resistance to infection, demonstrating an evolutionary conservation of this regulatory mechanism in mice and humans. Further supporting a role for NEDD4 as a regulator of IFITM3 turnover, overexpression of the NEDD4 inhibitory protein, interferon stimulated gene 15 (ISG15), led to increased IFITM3 levels in human lung cells but decreased IFITM3 ubiquitination, demonstrating a new synergistic link between ISG15 and IFITM3. This work implicates NEDD4 as a therapeutic target for the prevention or treatment of influenza virus infections, and introduces a new paradigm for up-regulating the cellular level of an IFN-inducible antiviral protein independently of IFNs.