Our studies are directed toward understanding the structure and function of human interferon-alphas and their receptors. The objective of these studies is to delineate the rationale for the existence of this family of structurally-related proteins and to understand the mechanism by which they elicit their pleiotropic biological activities. We have isolated and characterized 22 natural human lymphoblastoid interferons. One Interferon alpha component we have isolated, IFN-alpha o, exhibits high antiproliferative activity on Daudi and AU937 cells, but has low affinity for the IFN alpha 2b binding site on both cell lines. Partial amino acid sequencing of IFN-alpha o revealed that it is essentially indistinguishable from the amino acid sequence derived from the DNA sequence of IFNA21. In order to study the relationship between component o and IFN-alpha 21a, we cloned and expressed the IFNA21 gene in a pQE-30 expression system. The expressed protein was purified by metal chelate and 4F2 monoclonal antibody affinity chromatography. Biological functions of IFN-alpha 21a suggest that recombinant IFN-alpha 21a is indistinguishable from human lymphoblastoid IFN-alpha component o. In an effort to determine what domain(s) of IFN-alpha 21a is responsible for its distinctive antiproliferative and binding activities, we have developed six IFN-alpha hybrids between IFN-alpha 2c and IFN-alpha 21a (HY-1 through HY-6). All the hybrids have a similar antiviral specific activity on bovine cells but different antiproliferative and binding properties on Daudi cells. If the 76-166 amino acid residue region is IFN- a2c (76-95)/ IFN-a 21a (96-166), antiproliferative activity is highest (HY-3). If the 81-95 amino acid residue region is IFN-a2c, antiproliferative activity is greater than seen when the same region is IFN-a 21a. There are only three amino acids which differ between IFN-a2c and IFN-a 21a in the 81-95 region. Construction of 4 mutants in this region has shown that a change in one amino acid residue results in increased antiproliferative. Data from competitive binding assays show that the N-terminal region may be important in the binding activities of IFNs. The signal transduction properties of HY-2 [IFN- a 21a (1-95)/a2c (96-165)] and HY-3 [IFN- a 2c (1-95)/a21a(96-166)] were evaluated by Electrophoretic Mobility Shift Analysis (EMSA) and RNase Protection Assays. Both HY-2 and HY-3 induced activation of STAT 1 and 2. Additionally, at concentrations where no AP activity was seen, HY-2 induced a variety of IFN responsive genes to the same degree as HY-3. RNase Protection Assays also indicate that, at concentrations where no AP activity was seen for HY-2, this construct retained the ability to induce a variety of IFN-inducible genes. These data suggest that the antiproliferative response may not be solely directed by the STAT 1 and 2 pathways in a non-hematopoietic and a hematopoietic cell line and in primary human lymphocytes. The above hybrid work has been filed as a patent application (NIH Reference# E-068-98/1) on June 29, 1999. Also, the work on hydrids 1-3 (HY-1 through HY-3) is the subject of a paper recently published in the Journal of Immunology (July 15, 1999 edition). All hybrids are being analyzed for their immunomodulatory activities (natural killer cell activity and MHC Class I and II expression). In addition, we are using the IFN-alpha hybrids in collaborative studies with both CBER and National Cancer Institute scientists to examine their inhibition of HIV1 replication in primary monocytes and T cells and signal transduction activities.
