Identification of residues in the ectromelia virus gamma interferon-binding protein involved in expanded species specificity

Gamma interferon (IFN-gamma) production is important in the host response to, and recovery from, infection with Ectromelia virus (ECTV) and Vaccinia virus (VACV). The orthopoxviruses have evolved several mechanisms to subvert the IFN-gamma response. IFN-gamma-binding protein (IFN-gamma BP) is a virally encoded homologue of the host IFN-gamma receptor that blocks the effects of IFN-gamma in the infected host. Unlike the cellular receptors, whose ligand specificity is restricted to their own species, the orthopoxvirus IFN-gamma BPs bind IFN-gamma from several species. The reason for this relaxed specificity has yet to be explained. ECTV, a mouse pathogen, encodes an IFN-gamma BP that has been shown to inhibit the activity of both human and murine IFN-gamma (hIFN-gamma and mIFN-gamma respectively). In contrast, the IFN-gamma BP from VACV is unable to inhibit mIFN-gamma, but retains activity against hIFN-gamma. To determine which region(s) in the ECTV sequence is responsible for its ability to bind to mIFN-gamma with high affinity, a series of chimeric IFN-gamma BPs, as well as individual point mutants in the ECTV sequence corresponding to the amino acid changes from the VACV sequence, were constructed. The affinities of the chimeric and point mutant IFN-gamma BPs for mIFN-gamma were tested by using surface plasmon resonance and bioassay. By using this strategy, several key residues in the ligand-binding domains of the ECTV sequence have been identified that are responsible for high-affinity binding to mIFN-gamma. Substitution of the ECTV residue at these positions in VACV resulted in a dramatic increase in the affinity of the VACV IFN-gamma BP for mIFN-gamma.