Our studies are directed toward understanding the structure and function of human interferon-alphas and their receptors. The objectives 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. 22 components of human IFN-alpha derived from Sendai virus-induced human lymphoblastoid cells (Namalwa) were isolated by sequential monoclonal antibody affinity chromatography, ultrafiltration and reverse-phase HPLC. Many biological properties of the human lymphoblastoid derived interferon have been examined. One component of interest, 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. Amino acid sequencing of IFN-alpha o revealed that it is 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 of IFN-alpha 21a is responsible for its distinctive antiproliferative and binding activities, we have developed a three IFN-alpha hybrids between IFN-alpha 2c and IFN-alpha 21a , HY-1 [IFN-alpha 21a (1-75)/alpha 2c (76-165)], HY-2 [IFN-alpha 21a (1-95)/alpha 2c (96-165)] and HY-3 [IFN-alpha 2c (1-95)/alpha 21a (96-166)]. All the hybrids have a similar antiviral specific activity on bovine cells and different antiproliferative and binding properties on Daudi cells. Data shows that if the N-terminal region is IFN-alpha 2, that the hybrid competes well for the IFN-alpha 2b binding site. If however, the N-terminal region is IFN-alpha 21a, there is very poor competition for the IFN-alpha 2b binding. This suggests that the N-terminal region may be important in the binding activities of IFNs. A domain affecting the antiproliferatife activity was found within the C-terminal region from amino acids 75-166. IFN-alpha 2c (75-95)/alpha 21a (96-166) has higher antiproliferative activity than any of the other hybrids on Daudi and WISH cells. The signal transduction properties of HY-2 and HY-3 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 pathway in a non-hematopoietic and a hematopoietic cell line and in primary human lymphocytes. The above hybrid work has recently been filed as a provisional patent application (NIH Reference# E-068-98/0). Two additional hybrids have been constructed within the past month which will be used to help specifically determine the part of the IFN molecule responsible for the above mentioned biological activity. Also, all hybrids are being analyzed for their immunomodulatory activities (natural killer cell activity and MHC Class I and II expression). Chemical characterization has always been a major program in our laboratory eg. N-terminal amino acid sequences. Currently, we are continuing our studies of the carbohydrate structure of the three major glycosylated human lymphoblastoid interferon alpha components . In addition, we are using the IFN-alpha components in collaborabive studies with other CBER scientists to examine; 1) their inhibition of HIV1 replication in primary monocytes and T cells and signal transduction activities
