African swine fever virus A151R protein antagonizes the antiviral activity of barrier-to-autointegration factor (BAF) by targeting its dsDNA-binding activity

Barrier-to-autointegration factor (BAF) is a ubiquitous double-stranded DNA-binding protein that compacts DNA and can restrict poxvirus replication in the cytoplasm. BAF antiviral DNA-binding activity is tightly regulated by dynamic phosphorylation mediated by viral and cellular enzymes. For example, vaccinia virus counteracts BAF by encoding the B1 kinase, which phosphorylates BAF and abrogates its DNA-binding activity. Some DNA viruses, such as African swine fever virus (ASFV), undergo cytoplasmic replication but appear to lack a B1-like kinase. Interestingly, ASFV encodes A151R, a viral protein recently found to stably interact with BAF. Here, we demonstrate that A151R is capable of counteracting the antiviral properties of BAF. Structural modeling indicates that A151R is not a protein kinase and does not phosphorylate BAF but instead directly targets its double-stranded DNA-binding interface. This interaction enhances genome replication and progeny production of a B1-deficient virus. Mechanistically, A151R markedly impairs BAF DNA binding and disrupts its dimerization, a key requirement for high-affinity DNA association. Importantly, disruption of the A151R-BAF interaction abolishes these effects and restores BAF antiviral function. In addition, expression of the unphosphorylatable BAF mutant, which normally exhibits strong chromatin association, was redistributed to the cytoplasm in the presence of A151R, further supporting phosphorylation-independent regulation of BAF-DNA association. In conclusion, our findings support a previously unrecognized mechanism by which ASFV A151R disables BAF antiviral activity by obscuring its DNA-binding interface and inhibiting DNA binding in a phosphorylation-independent manner.IMPORTANCEDNA viruses replicating in the cytoplasm must overcome host intrinsic defenses to ensure productive replication, yet the mechanisms underlying their antagonism of the DNA-binding antiviral factor BAF remain incompletely understood. Here, we identify African swine fever virus (ASFV) A151R as a novel viral regulator that disables BAF by targeting its double-stranded DNA-binding interface rather than altering its phosphorylation state. We demonstrate that A151R impairs BAF DNA binding, disrupts its dimerization, and promotes viral DNA accumulation and progeny production in a BAF-dependent manner. Importantly, this activity requires A151R-BAF interaction and is independent of BAF phosphorylation status. Our findings reveal a previously unrecognized strategy employed by ASFV to neutralize host DNA-binding restriction factors and expand the molecular framework of BAF-mediated antiviral defense.