18: Characterization of human interferon-alpha subtypes and mutants

Abstract

IFN- α subtypes and mutants have been cloned in our laboratory in a yeast expression system. The mutants were constructed with point mutations at the C-termini working under the hypothesis that so doing would increase binding affinity of the IFN to its receptor and hence its activity. The following subtypes, IFN- α 1a, IFN- α 2c, IFN- α 6, IFN- α 8b and IFN- α 14c were purified by HPLC and characterized. Their antiproliferative activities were tested on Daudi (Burkitt’s lymphoma) cells and their antiviral activities were tested on A549 (human adenocarcinoma epithelial) cells, OVCAR-3 (ovarian cancer) cells and HUH7 (hepatocellular carcinoma) cells. IFN- α 8b and IFN- α 2c had the highest antiproliferative activities. IFN- α 8b had the highest antiviral activity while IFN- α 6 had the lowest on the three cell lines tested. In addition, in both A549 and HUH7 cells, the antiviral activities of IFN- α 8b were seen to be a log greater than that of IFN- α 2c. To determine relationships of these IFNs to the interferon-alpha receptor 2 (IFNAR2), soluble, extracellular IFNAR2 (IFNAR2-EC) neutralization assays were done in which the IFNs were exposed to IFNAR2-EC in the antiviral assays. Addition of the soluble IFNAR2-EC decreased the antiviral activities of all of the IFN subtypes ten-fold except for IFN- α 1a whose antiviral activity was decreased two fold. Western blot analyses of STAT pathways in A549 cells did not show any apparent correlation with phosphorylation of STAT 1, 2 or 3 (1 h post IFN treatment) and either antiviral or antiproliferative activities. Western blot analyses done to detect selected Interferon Stimulated Genes (ISGs), at both 6 and 15 h timepoints, show that the ISGs RIG-G, UBE2l6 and Mx1 were most up regulated in cells treated with IFN- α 1a, - α 2c and - α 14c, while PKR and IRF9 were equally up regulated by all of the IFN subtypes tested. We plan to more broadly characterize both the subtypes and mutants.