ISG15 Differentially Modulates Clade Ib and II MPXV Infection

Abstract The unprecedented human-to-human transmission of Clade IIb monkeypox virus (MPXV) during the 2022 outbreak has renewed focus on host determinants of viral fitness. Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein with broad immunomodulatory functions, yet its role in MPXV infection remains unclear. Using representative strains from recent and historical outbreaks spanning Clades I and II, we show that ISG15 deficiency enhances viral replication and protein production in murine cells. Notably, the 2024 Democratic Republic of Congo strain belonging to Clade Ib displays reduced sensitivity to ISG15, demonstrating differences in host antiviral susceptibility among MPXV isolates and suggesting clade-specific adaptation. ISG15 also influences viral immune evasion, as knockout cells infected with Clade II viruses expressed fewer immunomodulatory proteins and exhibited marked reductions in host protein phosphorylation than their wild type counterparts. These results identify ISG15 as a determinant of MPXV infection and underscore evolutionary differences between clades.