Structure-Function Analysis of Mouse Interferon Alpha Species: MuIFN- 10, a Subspecies with Low Antiviral Activity

Abstract

A mouse interferon alpha gene (MuIFN-.alpha.10) was isolated from a BALB/c cosmid genomic library. The gene was located on a 1.8 kb HindII fragment and a 5.1 kb EcoRI fragment. The coding region and parts of the 5'' and 3'' non-coding regions were sequenced. The results showed that the MuIFN-.alpha.10 gene encoded a protein of 167 amino acids. Like most other muIFN-.alpha. species it contained a putative N-glycosylation site at amino acid positions 78 to 80. It also possessed cysteine residues at positions 1, 29, 86, 99 and 129. In the signal peptide, in addition to cysteine 21, which is present in all MuIFN-.alpha. species sequenced so far, a cysteine was found at position 22. At the amino acid level MuIFN-.alpha.10 showed strong homology to MuIFN-.alpha.1 (only 15 out of 167 amino acids were different). The MuIFN-.alpha.10 gene was transiently expressed in monkey COS cells under the direction of the simian virus 40 early promoter. The protein product secreted by COS cells was equally active on mouse (L929) and hamster (CHO) cells. However, as compared to MuIFN-.alpha.1 and MuIFN-.alpha.4 the specific activity on mouse cells of the protein was 10- to 100-fold lower. To find out which region of its structure was responsible for this low activity, hybrids of the genes encoding MuIFN-.alpha.10 and MuIFN-.alpha.1 were constructed using the two common XmnI sites which correspond to positions between amino acids 67 and 68 and 123 and 124, respectively. The data showed that hybrid constructs which were MuIFN-.alpha.-like from amino acid 68 or MuIFN-.alpha.10-like from position 124 to the C terminus possessed high antiviral activity. Other hybrid constructs were hardly active at all. This implied that the amino acid 68 to 123 region was mainly responsible for the low antiviral activity of MuIFN-.alpha.10. In this part of the molecule MuIFN-.alpha.1 and MuIFN-.alpha.10 differed in only five amino acids. A serine at position 110 and a valine at 85 were unique to MuIFN-.alpha.10 as compared to all known MuIFN-.alpha. and human IFN-.alpha. subspecies.