Interferons are a type of cytokine. That is, they are small proteins involved in cell signaling, of which there are two types. Interferon-y (IFN-y) is a Type II, or immune, interferon, with roles crucial to the ability of mammals to respond to pathogens.1
Orthopoxvirus (OPV) is a genus of closely-related, large DNA viruses. OPV pathogens affecting various host organisms with strict host restriction, such as human variola virus (VARV) and mouse ectromelia virus (ECTV), the causative agents of smallpox and mousepox, respectively.2,3 Orthopoxviruses express IFN-y-binding proteins (IFN-yBPs) that, by efficiently binding IFN-y, block IFN-y-mediated signaling cascades4 responsible for activating crucial antiviral defense mechanisms in the host. IFN-yBPs are thus important virulence factors, and are highly conserved in all orthopoxviruses, including VARV.3
Ectromelia virus IFN-yBP, or IFN-yBPECTV, is similar to other IFN-yBPs in structure. The protein consists of an IFN-y receptor ligand-binding domain, as well as a helix-turn-helix (HTH) motif that is structurally related to the transcription factor TFIIA.3,5 The structure of the ligand-binding domain is similar to that of the extracellular region of cellular protein IFN-yR1, a membrane-spanning cytokine receptor. This extracellular region is thus called a cytokine receptor homology region (CRHR). Efficient IFN-y antagonism is achieved when the HTH motif forms a tetramerization domain, resulting in an IFN-yBPECTV/IFN-y complex containing four IFN-yBPECTV chains and two IFN-y dimers.3
The crystalline structure of IFN-yBPECTV consists of a cytokine receptor homology region (CRHR) composed of two fibronectin type III (FBNIII) domains containing seven conserved beta strands each. The two FBNIII domains are connected to an HTH motif containing alpha helices H2 and H3 by an extended linker.3
To form the dimer, two IFN-yBPECTV monomers are linked by an interchain between two Cis-216 residues, located in the extended linker region. The dimer is stabilized by that occur between aspartic acid residues, Asp-165 and Asp-167, located on the FBNIII domains, and the Lys-230 and Arg-237 residues, located on helix H2 in the two-fold-related HTH motif. This extensive dimer interface locks the two-fold-related IFN-yBPECTV monomers into an extended, .3
H-shaped formation occurs exclusively through contacts made between residues in H3 of the HTH motifs. Most of the contacts within the tetramer domain are interactions involving residues Tyr-246, Phe-250, Phe-261 , and Met-264. There are also specific essential to tetramerization between Asp-257 and Ser-253 in adjacent H3 helices at the center of the tetramer. An IFN-yBPECTV Phe250Ala mutant forms dimers rather than tetramers, suggesting that is critical for the hydrophobic interactions involved in IFN-yBPECTV tetramer formation.3
Each IFN-yBPECTV tetramer binds two IFN-y homodimers. The complex thus contains four IFN-yBPECTV chains and four IFN-y chains. The complex itself is not planar, as is clear when it is viewed from the . Rather, the two IFN-yBPECTV dimers are oriented at an angle of approximately 63 degrees, with respect to one another. Thus, it is the tetramerization achieved by the binding of two IFN-yBPECTV dimers that forms a bivalent and high-affinity binding site for IFN-y, and this binding site is crucial for efficient IFN-y antagonism.3
An important recognition site for IFN-y within IFN-yBPECTV is located at the interface of FBNIII(1) and FBNIII(2) within the monomer. This interface contains six receptor loops (L2 to L7), two of which, L2 and L3, contain important for binding. Specifically, residues located in L2 and L3 participate in two conserved , which occur between L2 residue Tyr-47IFN-yBP(ECTV) and Glu-112IFN-y, and L3 residue Trp-78IFN-yBP(ECTV) and Gly-18IFN-y, for a total of 8 hydrogen bonds.3
33 total IFN-y residues bury accessible surface area into each IFN-yBPECTV/IFN-y complex , with nearly half of this area being attributed to the C terminus of IFN-y. IFN-yBPECTV makes fewer contacts with the helical domain of IFN-y than does cellular protein IFN-yR1, but far more extensive contacts with this C terminal domain. C terminal residues in IFN-y form an extensive network of with the IFN-yBPECTV, with this binding site being primarily made up of aspartic acid on the L6 receptor loop. The IFN-yBPECTV residues that make up this binding site, with the exception of D162, are conserved in all known IFN-yBP sequences. This indicates evolution of a unique method across all orthopoxviruses for binding and sequestering the conserved IFN-y C terminal tail, which is critical for IFN-y antagonism.3
(2) Sigal, Luis J. The pathogenesis and immunobiology of mousepox. Advances in Immunology 129 (2016): 251-276.
(3) Nuara, Anthony, et al. Structure and mechanism of IFN-y antagonism by an orthopoxvirus IFN-y-binding protein. Proceedings of the National Academy of Sciences 105.6 (2008): 1861-1866.
(4) Zaidi, M. Raza and Glenn Merlino. The Two Faces of Interferon-y in Cancer. Clinical Cancer Research 17.19 (2011): 6118-6124.
(5) Gupta, Kapil, et al. Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID. eLife 6 (2017): e30395.