IDENTIFICATION OF ACTIVE-SITE RESIDUES IN PROTEASE 3C OF HEPATITIS-A VIRUS BY SITE-DIRECTED MUTAGENESIS

Picornavirus 3C proteases (3C(pro)) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3 C(pro) of hepatitis A virus (HAV) to those of other picornaviruses hav e resulted in prediction of active-site residues: histidine at positio n 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonuc leotide-directed mutagenesis was targeted to 3C(pro) in the contest of natural poly peptide precursor P3. Autocatalytic processing of the po ly-protein containing wild-type or variant 3C(pro) was tested by in vi vo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybr id system and by in vitro translation of corresponding RNAs. Compariso n with proteins present in HAV-infected cells showed that both express ion systems mimicked authentic polyprotein processing. Individual subs titutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contra st, a P3 polyprotein in which D98 was substituted by asparagine underw ent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more p ronounced loss of processing. Therefore, apparently H44 and C172 are a ctive-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprot ein cleavage sites may reduce proteolytic activity, presumably by alte ring substrate conformation.