Esteban and Hutchinson BMC Genomics 2011, 12:261 http://www.biomedcentral.com/1471-2164/12/261 Page 8 of 12 Figure 4 Posterior probability of site classes for amino acids in two host response modifier genes . Shown for each site is the posterior probability of falling into the purifying (white), neutral (grey), or diversifying (black) class. Posterior probabilities were determined by Bayes empirical Bayes analysis. Dashed line at 0.5 shows the threshold for a site to be classified as diversifying. A) IL-18 binding protein. B) mitochondrial associated apoptosis inhibitor. Only the conserved C-terminal domain is shown, and the locations of the BH domains are indicated below the axis. Like highly conserved amino acids, variable positions under diversifying selection may indicate functionally or structurally important positions. Computational identifi- cation of amino acids under diversifying selection has revealed important functional or antigenic sites in sev- eral studies [32,33]. One of the genes identified in this study was the Interleukin-18 binding protein (IL-18BP), which was previously shown to attenuate the immune response in mice [22]. The crystal structure of the Ectromelia virus (ECTV) IL-18 binding protein was recently determined [34]. Bayes empirical Bayes analysis demonstrated that most residues are under purifying selection, while some are neutral and a few are under diversifying selection (Figure 4). The crystal structure and previous mutagenesis studies [35] identified contact residues important in binding the ligand, IL-18. Most of these residues were found to be under purifying selec- tion, supporting a requirement for conservation of these residues to maintain function. Interestingly, 2 residues shown through crystallography to be contacts in the binding interface were also identified as being under diversifying selection (D48 and I115 in CPXV-BR, E48 and L115 in ECTV). Both of these residues interact with binding site C on human IL-18 [34]. Evidence of diversi- fying selection in these positions may suggest a role for these residues in adaptation to IL-18 of the specific host species of the virus. Another important host response modifying gene that we identified is the mitochondrial associated apoptosis inhibitor (F1L in VACV-Cop). Apoptosis is an impor- tant cellular response to infection that serves to limit viral replication through removal of infected cells. F1L inhibits apoptosis through binding to the pro-apoptotic protein Bak [36,37] thereby inhibiting the permeabiliza- tion of the mitochondrial membrane, a critical step in apoptosis. Interaction with Bak is via Bcl2-like homology (BH) domains that are highly divergent but nonetheless form characteristic BH domain folds [37]. Among the