ISG15-USP18 signaling restrains viperin-dependent metabolic antiviral restriction

Type I interferon (IFN-I) responses are tightly regulated to balance antiviral defense with cellular homeostasis. In humans, interferon-stimulated gene 15 (ISG15) functions as a critical negative regulator of IFN-I signaling by stabilizing the IFN negative regulator USP18, yet the functional consequences of ISG15 deficiency remain elusive. Here, we show that the loss of ISG15 exaggerates the JAK-STAT activation and, downstream, amplifies multiple ISGs including the nucleotide-modifying enzyme RSAD2 (viperin). Our quantitative proteomics, genetic reconstitution, and signaling analyses establish that defective USP18 stabilization skews the IFN response towards viperin expression. This amplified ISG network promotes viperin-catalyzed accumulation of the antiviral nucleotide analog ddhCTP, resulting in enhanced inhibition of viral RNA synthesis and the replication of Crimean-Congo hemorrhagic fever virus and SARS-CoV-2. Together, these findings demonstrate an ISG15-USP18-viperin axis that can be targeted to boost the metabolic antiviral restriction.