Our studies are directed toward understanding the structure of the members of the human interferon (IFN)-alpha family and how they elicit their biological (antiviral, antiproliferative and immunomodulatory) activities. Previously, it was shown that specific regions of the IFN-alphas are associated with specific types of biological functions using IFN alpha hybrid and mutant molecules. To determine the domains of the IFN-alphas that are important for antiviral and antiproliferative activities we genetically engineered, expressed and purified 14 hybrid IFN-alpha species derived from natural human IFN-alpha 21a and IFN-alpha 2c. The secondary/tertiary structures of the human IFN hybrids were examined using a broad range of monoclonal antibodies (mAbs). Results have shown structural differences among our constructs as well as the parent molecules, IFN alphas 2c and 21a. Based on these data, we extended our studies on the interactions of the IFN hybrids with the IFN receptor subunit IFNAR2-ECD (Interferon alpha receptor 2-extracellular domain). ELISA results showed that helix E (138-154) is the most critical region of the interferon molecule for interaction with the IFNAR2-ECD. Our data suggests that the epitope 112-132 (helix D), which is recognized by several C-terminal anti-IFN-??2 mAbs, is not involved in the interaction of our constructs with IFNAR2-ECD. Assays done using two antigenically different IFNs which can bind independently of one another suggest that there may be more than one IFN-binding site on the IFNAR2-ECD. In addition, we are examining the interaction between IFN-alpha 2 subvariants (2a, 2b and 2c) and the extracellular domain of human IFN receptor (IFNAR2-EC) using a sandwich ELISA and anti-IFN monoclonal antibodies. Since the subvariants differ by only one or two amino acids, it is possible to determine if these amino acids are critical for structure/function of human IFN-alphas and for IFN/receptor interaction. In an effort to better understand the mechanisms of action and signaling pathways of the IFN alphas using Daudi (Burkitt!|s Lymphoma) cells, we have initiated gene expression microarray and proteomics analyses. It is anticipated that these two technologies will provide insight into the different levels of antiproliferative and antiviral activities observed with the various IFN-alpha hybrids and mutants. Oligonucleotide microarray analysis was used to compare gene expression profiles of IFN-alpha 2c, IFN- alpha 21a and two IFN-alpha hybrids (HY-4 and HY-5). Data showed that there are distinct expression patterns corresponding to the IFN groups (parental and hybrid). These diversities in gene regulation may contribute to different biological activities observed in the studied IFNs. Comparative analysis of cell lysates produced from IFN-alpha treated and untreated Daudi cells printed on protein microarrays and interrogated with antibodies against major phosphorylated forms of signaling proteins (e.g. pSTAT1, pSTAT2, pJak1, pTyk2) has been performed. In addition, we examined the relative abundance of proteins observed after treatment of Daudi cells with different interferon-alphas (IFN-alpha 2c and IFN-alpha 21a) using Isotope-Coded Affinity Tags (ICAT) technology. Using pathway analysis software, we are studying the effects of up-regulation and down-regulation of specific proteins in a variety of signal transduction pathways following IFN treatment. Finally we have studied the antiviral properties of our human interferon alpha constructs against the SARS virus in Vero cells. Preliminary results suggest that three constructs have very high antiviral activity.
