Structure-function study of the region encompassing residues 26-40 of human interferon-alpha 4: identification of residues important for antiviral and antiproliferative activities

The highly conserved amino acid residues Leu-30 and Arg-33 of human interferon-alpha 4 (IFN-alpha 4) have previously been identified as important for biological activity. In this study, the sequence around Arg-33 was targeted to determine the importance of other residues in this region. A library of analogues containing amino acid substitutions spanning residues 26-37 was generated using site-directed and random mutagenesis. Analogues were expressed in vitro and assayed for antiviral and antiproliferative activity on human cells. No significant separation between the antiviral and antiproliferative activities was observed for any of the analogues tested. Substitutions at positions 26, 27, 31, 32, 34, 35, and 37, did not substantially affect biological activity. However, substitution of Phe-36 with arginine resulted in a greater than 100-fold decrease in biological activity. Thus, together with previous data, the residues in this region identified as most important for biological activity include Leu-30, Arg-33, and Phe-36. Recently published models for the three-dimensional structure of human IFN-alpha and the X-ray crystallographic structure of murine IFN-beta, suggest that the region investigated in this study forms a loop at the surface of the protein. Thus, residues Leu-30, Arg-33, and Phe-36, could be involved in binding to the Type-I IFN receptor, or in interactions with signal-transducing molecules.