Cross-Species Genome-Wide Analysis Reveals Molecular and Functional Diversity of the Unconventional Interferon-? Subtype

Shields, Lauren E.; Jennings, Jordan; Liu, Qinfang; Lee, Jinhwa; Ma, Wenjun; Blecha, Frank; Miller, Laura C.; Sang, Yongming

Innate immune IFNs confer antiviral and immunomodulatory roles through induction of hundreds of IFN stimulated genes (ISGs), which are generally classified into robust or tunable ISGs relative to their responsive intensity to IFN stimulation (10, 35, 36) . Whereas, most robust ISGs are involved in antiviral responses, tunable ISGs are more broadly modulatory for immune and developmental regulation (35, 36) . To test the differential potency of porcine IFN-ω in induction of ISGs, we measured the expression of six typical ISGs (three robust and three tunable) (10, 35, 36) in animal and human cells treated with the overexpressed peptides (IFN-α1, IFN-β, IFN-ω1, and IFN-ω5) for 24 h. The three robust ISGs are caspase 1, phospholipid scramblase 1 (PLSCR1), and ubiquitin-like ISG15 (ISG15); and the three tunable ISGs include interleukin 11 (IL11), IFN-regulatory factor 1 (IRF1), and tumor necrosis factor alpha receptor superfamily 10A (TNFRSF10A). Data show that, IFN-ω1 and IFN-ω5 stimulated the robust ISGs (esp. ISG15) to an extent similar to or higher than IFN-β and IFN-α1, respectively, in porcine or non-porcine cells. However, IFN-β and IFN-α1 are generally less active than IFN-ω5 in stimulation of the three tunable ISGs in the cells of nonporcine origin (Figure 7) . Interferon-regulatory factor (IRF) 3, IRF7 and Myxovirus resistance protein 1 (Mx1) genes are other representative ISGs that play key roles in IFN auto-regulation (such as IRF3 and IRF7 in further potentiation of IFN-β and IFN-α production in macrophages and pDCs, respectively) and in anti-Myxovirus (such as influenza) activity (10, 35, 36) . Using a promoter-reporter based bioassay (13, 23) , we analyzed the stimulation of IRF3, IRF7, and Mx1 expression by treatment with different concentration of IFN-α1, IFN-ω1, and IFN-ω5. Porcine IFN-ωs, particularly IFN-ω5 exerted higher activity in FIGURE 4 | Pairwise identity (%) plots among protein sequences of IFN-ω orthologs. Comparison and plot drawing were performed using a SDT program. IFN-ω molecules from individual Order or Genus might share a common progenitor, as also shown in the Figure 2 for the clustered phylogenic clades that share >86% of identity. It was also common to observe that IFN-ω molecule from a species is phylogenically closer to the orthologs from other species of the same Family/Genus than those from the same species. For the mammalian genus/species that have multiple genes of IFN-ω subtype, we observed that IFN-ω peptides such as in bats, moles, shrews and elephants are formed into two major sub-clusters (Upper); however, it is primarily only one sub-cluster (with one to several "outliers," labeled with red arrow or brackets) such as in swine and bovine species (Bottom). In addition, several clusters of IFN-ω peptides contain IFN-ω peptides from animals of different Genus/Family, such as that of the ungulate-mix (Alpaca, Camel, and Bioson) and the RBH-mix (Rhino, Bat, and Horse) clusters (Upper stimulation of IRF7 and Mx1 expression, but are similar to IFN-α1 for the effect on IRF3 promoter (Figure 8) . In summary, porcine IFN-ω subtypes, especially the highly antiviral IFN-ω5, potentially signal ISG expression to exert antiviral immunity differently from the classical IFN-α1 and IFN-β subtypes in both porcine and human cells (13, 23) .