ZNFX1 is an interferon-stimulated RNA helicase with unprecedented bifurcating E3 ubiquitin ligase activity that combines allosteric and transthiolating mechanisms to generate distinct K63- and K48-linked ubiquitin chains, enabling ATP- and RNA-dependent formation of reversible aggregates that entrap and silence viral RNA through a deubiquitination-sensitive mechanism that is essential for antiviral immunity and impaired in patient variants associated with severe immunodeficiency.

The detection of viral RNA inside cells triggers a diverse range of antiviral responses, including global translation inhibition, interferon secretion and RNA sequestration. Mutations in the gene ZNFX1 cause severe paediatric immunodeficiencies, including chronic viral infection and autoinflammation. Here, we show that ZNFX1 is an RNA helicase with cryptic and unusual bifurcating E3 ubiquitin ligase activity. Nucleotide-dependent RNA binding stimulates ZNFX1 to generate complex ubiquitin chains via a two-component ubiquitin circuit wired in parallel, with ubiquitin flux occurring via either of two competing paths. One route produces K63-linked polyubiquitin that drives ZNFX1 aggregation and RNA entrapment; the other route produces K48-linked polyubiquitin that drives ZNFX1 turnover. RNA entrapment restricts RNA virus replication, and is reversible by deubiquitination. Patient ZNFX1 variants are defective for viral restriction, linking RNA entrapment to antiviral immunity in vivo.