ANKLE2 (Ankyrin repeat and LEM domain-containing protein 2) is an emerging host factor with previously undefined roles in antiviral defense. Here, we show that ANKLE2 can exert robust antiviral activity against vaccinia virus by regulating the phosphorylation status of barrier-to-autointegration factor (BAF), a ubiquitous DNA-binding protein that compacts DNA and restricts viral replication. We first demonstrate that depletion of endogenous ANKLE2 increased BAF phosphorylation and rescued replication of B1-knockout vaccinia virus, whereas reconstitution restored restriction. We then perform domain-mapping experiments of ANKLE2, revealing that its LEM domain and Caulimovirus domain (CD domain) are essential for BAF dephosphorylation, ANKLE2-BAF association, and/or antipoxviral activity, whereas the transmembrane (TM) domain restricts cytoplasmic redistribution and functions as a negative regulator. Together, these findings uncover a previously unrecognized host defense pathway against poxviruses, provide new insight into how host ANKLE2 proteins coordinate antiviral responses, and reveal a novel antiviral role for ANKLE2 in limiting vaccinia virus DNA replication and progeny release through regulation of BAF phosphorylation.IMPORTANCEVaccinia virus relies on disabling host defenses to replicate efficiently, with the host DNA-binding protein BAF representing a key target for viral kinases. Here, we uncover ANKLE2 as a critical host factor that counteracts vaccinia virus by sustaining the antiviral activity of BAF. ANKLE2 promotes BAF dephosphorylation, thereby preventing viral escape from BAF-mediated restriction. Our results reveal that distinct domains of ANKLE2 differentially regulate its antiviral activity, with the LEM and CD domains promoting BAF dephosphorylation and antiviral activity, and the transmembrane domain acting as a negative regulator by limiting cytoplasmic redistribution. These findings highlight ANKLE2 as a domain-dependent regulator of host defense and expand our understanding of the molecular circuitry that controls poxvirus replication.
Keywords: ANKLE2; B1; BAF; antiviral activity; phosphorylation; poxvirus; vaccinia virus.