Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 ( IFITM3) Dually Regulates Its Endocytosis and Ubiquitination

Background: IFITM3 restricts the fusion of viruses within endolysosomes. Results: Phosphorylation of IFITM3 on Tyr(20) blocks IFITM3 endocytosis and ubiquitination. Conclusion: Tyr(20) of IFITM3 is part of a YXX endocytosis signal and has a dual role in regulating IFITM3 ubiquitination. Significance: Identifying mechanisms regulating IFITM3 trafficking and activity is crucial for understanding and manipulating IFITM3 biology for combating virus infections. Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr(20)) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr(27). Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr(20) caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr(27) mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr(20) is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXX phi (where X is any amino acid and phi is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr(20) both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr(20) may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3.