Comparative Functional Analysis of 12 Mammalian IFN-?4 Orthologs

Paquin, Ashley; Onabajo, Olusegun O.; Tang, Wei; Prokunina-Olsson, Ludmila

Despite being only poorly secreted, human IFN-l4 can efficiently activate IFN signaling (Prokunina-Olsson and others 2013; Onabajo and others 2015). We evaluated biological activity of the IFN-l4 orthologs after transient expression in HepG2 cells stably expressing an interferonstimulated response element (ISRE) coupled with luciferase reporter (ISRE-Luc); this system was previously used to characterize human IFN-l4. We observed differential biological activity of the IFN-l4 orthologs: compared with human IFN-l4, proteins from chimpanzee, orangutan, marmoset, and cynomolgus showed similar levels of biological activity (ISRE-Luc activation), while the dog protein showed significantly higher activity (by 40%). Orthologs from rhesus, panda, and elephant were significantly less active (25% compared with the human IFN-l4, Fig. 2 ). Biological activities of the IFN-l4 orthologs measured in this experiment are likely to be affected by differential affinity of these proteins to human receptors and do not represent quantitative comparisons. However, the fact that all of the IFN-l4 orthologs were able to activate ISRE-Luc reporter above the background level (control-Halo) indicates that these proteins are biologically active as IFNs, even in human cells. Because all of the IFN-l4 orthologs are predicted to have leader peptides and showed biological activity for induction of IFN signaling, which is typical for exogenously acting IFNs, it is likely that at least a portion of these proteins is secreted. At the same time, if these orthologs behave like the human IFN-l4, they will also be retained in the cytoplasm. To test this, we evaluated protein expression and cellular localization of all orthologs transiently expressed in HepG2 cells. Western blot analysis of protein lysates (Fig. 3) , flow cytometry analysis ( Supplementary Fig. S1 ), and confocal imaging ( Supplementary Fig. S2 ) confirmed that all IFN-l4 orthologs are detectable by an antibody or a fluorescent ligand for the Halo-tag, which is present on all these proteins.