MITD1 restricts HIV-1 release by targeting ESCRT-III proteins.
HIV-1 release from infected cells requires coordinated action of the viral Gag protein and the host endosomal sorting complexes required for transport (ESCRT) machinery. How innate immunity regulates ESCRT function to limit viral egress remains unclear. Here, we show that MITD1, a cell type-specific interferon-stimulated gene, is a potent inhibitor of ESCRT-dependent HIV-1 budding. MITD1 engages ESCRT-III subunits CHMP1B and CHMP4B, driving their incorporation into aggregates and impairing ESCRT-III assembly during HIV-1 budding. This disrupts the final steps of virion release, causing an accumulation of immature particles at the plasma membrane. MITD1 overexpression reduces HIV-1 release from cell lines, primary peripheral blood mononuclear cells (PBMCs), and human microglia, while sparing the release of ESCRT-independent viruses. These findings establish MITD1 as an interferon-stimulated restriction factor that limits HIV-1 egress by perturbing ESCRT-III assembly.