Double-ring assembly of mpox virus I3L reveals an unconventional mechanism for ssDNA engagement.

Poxviruses, including variola and mpox virus (MPXV), are large dsDNA viruses that replicate their genomes in the host cytoplasm via virally encoded proteins. The single-stranded DNA-binding protein (SSB) I3L is an essential component of this replication machinery, yet its structural mechanism remains to be fully elucidated. Here, we determined the cryo-EM structure of MPXV I3L and constructed a structural model of its complex with ssDNA. Unlike canonical SSBs, I3L forms an architectural double-ring assembly. The individual I3L protomer adopts an OB-fold variant with specialized elements beyond the classic pattern. Furthermore, biochemical assays and structural modeling suggest an assembly-dependent ssDNA-engagement mode, while monomeric binding features remain conserved. These distinctive structural features suggest a specialized molecular mechanism for poxviral DNA replication. Our findings advance the mechanistic understanding of poxvirus genome maintenance and provide perspectives for antiviral development against MPXV.