The 100-kDa 2 ',5 '-oligoadenylate synthetase catalyzing preferentially the synthesis of dimeric pppA2 ' p5 ' A molecules is composed of three homologous domains

The 2-5A synthetases represent a family of proteins implicated in the mecha nism of the antiviral action of interferon. When activated by double-strand ed RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)(n), n greater than or equal to 1. Three forms of human 2-5A synthetases have been described corresponding to proteins of 40/46 (p40/p46), 69/71 (p69/p71), and 100 kDa (p100). Here we describe the molecular cloning and characterization of p100. By screening a cDNA express ion library with a specific p100 polyclonal antibody, we first isolated 590 -nucleotide cDNA fragment which was subsequently used to isolate the full-l ength 6365-nucleotide cDNA. This cDNA recognizes a distinct interferon-indu ced messenger RNA of 7 kilobases. It has an open reading frame encoding a p rotein of 1087 amino acids including the sequence of seven peptides obtaine d by microsequencing of the natural p100 protein, which was purified from i nterferon-treated human cells. p100 is composed of three adjacent domains, each homologous to the previously defined catalytic unit of 350 amino acids , which is present as one unit in p40/p46 and as two units in p69/p71. The recombinant p100 synthesized preferentially dimeric 2',5'-oligoadenylate mo lecules and displayed parameters for maximum enzyme activity similar to the natural p100. These results confirm that the enzymatic activity of p100 is distinct compared with that of p40/p46 and p69/p71.