Ectromelia virus (ECTV) encodes an IFN-γ-binding protein (IFN-γBPECTV) that disrupts IFN-γ signaling and its ability to induce an antiviral state within cells. IFN-γBPECTV is an important virulence factor that is highly conserved (>90%) in all orthopoxviruses, including variola virus, the causative agent of smallpox. The 2.2-Å crystal structure of the IFNγ-BPECTV/IFN-γ complex reveals IFN-γBPECTV consists of an IFN-γR1 ligand-binding domain and a 57-aa helix-turn-helix (HTH) motif that is structurally related to the transcription factor TFIIA. The HTH motif forms a tetramerization domain that results in an IFN-γBPECTV/IFN- γ complex containing four IFN-γBPECTV chains and two IFN-γ dimers. The structure, combined with biochemical and cell-based assays, demonstrates that IFN-γBPECTV tetramers are required for efficient IFN-γ antagonism. © 2008 by The National Academy of Sciences of the USA.