A Role for IFN-ɛ: Type I interferons (IFNs) are critical cytokines involved in host defense against pathogens, particularly viruses. IFN-ɛ is an IFN-like gene encoded within the type I IFN locus in mice and humans whose function has not been characterized. Fung et al. (p. 1088 ) created mice with a genetic deletion in Ifn -ɛ and found that, like other type I IFNs, IFN-ɛ signals through the IFN-α receptors 1 and 2. However, unlike these other cytokines, which are primarily expressed by immune cells and are induced upon immune cell triggering, IFN-ɛ was expressed exclusively by epithelial cells of the female reproductive tract in both mice and humans and its expression was hormonally regulated. IFN-ɛ–deficient mice were more susceptible to infection with herpes simplex virus 2 and Chlamydia muridarum , two common sexually transmitted pathogens.

Alpha and beta interferons (IFN-α and IFN-β) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-α2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-β. Compared to wild-type (wt) IFN-α2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-β, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-β, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-β and higher levels of gene induction or repression than those for wt IFN-α2. Thus, we show that the differential activities of IFN-β are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-α subtypes suggests that IFN-α has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-β.

Type I interferons bind to two cell surface receptors, ifnar1 and ifnar2, as the first step in the activation of several signal transduction pathways that elicit an anti-viral state and an anti-proliferative response. Here, we quantitatively mapped the complete binding region of ifnar2 on interferon (IFN)α2 by 35 individual mutations to alanine and isosteric residues. Of the six “hot-spot” residues identified (Leu-30, Arg-33, Arg-144, Ala-145, Met-148, and Arg-149), four are located on the E-helix, which is located at the center of the binding site flanked by residues on the A-helix and the AB-loop. The contribution of residues of the D-helix, which have been previously implicated in binding, proved to be marginal for the interaction with the extracellular domain of ifnar2. Interestingly, the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFNα2. Thus, hydrophobic interactions seem to play a significant role stabilizing this interaction, with the charged residues contributing toward the rapid association of the complex. Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated, suggesting that ifnar2 binding is the rate-determining step in cellular activation. Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses. Considering the strict dependence of the cellular response on affinity, these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential.