ISG15 differentially modulates Clade Ib and II MPXV infection

The sustained human-to-human transmission of Clade IIb monkeypox virus (MPXV) during the 2022 outbreak underscores the need to define host determinants of viral fitness across species. ISG15 encodes a ubiquitin-like modifier with antiviral activity, yet its role in MPXV infection and host adaptation remains unclear. Using representative strains from Clades Ib and II, we identify a striking species-specific difference in ISG15-mediated restriction. In murine cells, ISG15 deficiency primarily increases viral protein accumulation with minimal effects on infectious viral production, indicating limited antiviral restriction. In contrast, in human cells, ISG15 restricts replication of recent epidemic strains since ISG15 deficiency enhances viral growth, establishing ISG15 as a restriction factor in humans. ISGylome profiling identifies candidate viral and host substrates underlying this effect. Together, these findings reveal species-specific ISG15-dependent control of MPXV infection and support a model in which rodents serve as permissive reservoirs while humans represent a more restrictive host due at least in part to the antiviral activity of human ISG15. We study ISG15 during MPXV infection in human and murine cells. ISG15 deficiency increases infectious virus production in human cells but alters viral protein accumulation in murine cells, and ISGylome analysis identifies ISGylated viral proteins.