Our studies are directed toward understanding 1) the structure and function of human interferon-alphas and their receptors and 2) the mechanisms by which they elicit their pleiotropic biological activities. In an effort to determine the domain(s) of IFN-alpha 21a that are responsible for its distinctive antiproliferative and binding activities, we have employed genetic engineering to construct a number of interferon hybrids using as parents IFN-a21a and IFN-a2c. They exhibit distinct biological and binding properties. The hybrids have a similar antiviral specific activity on bovine cells but different antiproliferative and binding properties on human 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-a21a in the 81-95 region. Construction of 8 mutants in this region by site-directed mutagenesis and cassette mutagenesis has shown that tyrosine at positions 86 and 90 are very important in the antiproliferative activity of IFN-a. In addition, substitution of tyrosine at position 86 with a hydrophobic amino acid (isoleucine) results in comparable levels of antiproliferative activity. Data from competitive binding assays show that the N-terminal region of the IFN molecule may be important in the binding activities of IFN. If the N-terminal region of the IFN has the IFN-a2c sequence it competes well with 125I-IFN-a2b. Each of the hybrids and mutants which competes well with 125I-IFN-a21a have the IFN-a21a sequence at their N-termini and have either tyrosine or isoleucine at positions 86 or 90. 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 ProtectionAssays. 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. Comparison of the secondary structures of one of the site directed mutant, SDM-1 (HY-4 [86 S-->Y]) and two of the cassette mutants, CM-2 (SDM-1 [86 Y-->I]) and CM-4 (SDM-1 [86 Y-->A])by circular dichroism (CD) yielded very similar spectrums. These results suggest that the amino acid replacements at position 86 did not change the secondary structure and that the differences in biological activity seen among these mutants is not due to conformational change. 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 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.
