MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3

The Membrane-Associated RING-CH (MARCH) family of E3 ubiquitin ligases modulates membrane protein stability and contributes to host antiviral immune responses. Their functions largely depend on the nature of protein substrates. In this study, we identified interferon-induced transmembrane protein 3 (IFITM3), a critical antiviral effector protein, as a novel substrate of MARCH8 by coimmunoprecipitation coupled with LC-MS/MS analysis. Mechanistically, MARCH8 promotes the lysosomal degradation of IFITM3 primarily through K63-linked polyubiquitination at lysine 24, which facilitates its trafficking and turnover from the plasma membrane to endosomes and lysosomes. MARCH8-KO cells exhibit plasma membrane accumulation of IFITM3 compared with WT controls after interferon treatment. Functionally, MARCH8 expression attenuated the IFITM3-mediated restriction of vesicular stomatitis virus and influenza A virus entry, thereby increasing cell susceptibility to viral infection. Together, these findings establish a novel regulatory mechanism whereby MARCH8 modulates innate immunity through regulating the trafficking and turnover of antiviral protein IFITM3.