Proteinase 3C of hepatitis A virus (HAV) cleaves the HAV polyprotein P2-P3 at all sites including VP1/2A and 2A/2B.

Thus far, the only virus-encoded proteinase of hepatitis A virus (HAV) detected is 3C, which was shown to catalyze proteolysis of most of the suggested cleavage sites within the HAV precursor polyprotein. To elucidate whether or not HAV proteinase 3C and its precursors are involved in processing of the yet unidentified sites in the polyprotein P2-P3, the genomic region of 3C including flanking sequences were expressed in a bacterial system and by cell-free translation. In both systems 2A-reactive proteins of 10 (2A) and 16 kDa (delta VP1-2A) were processing products of a polyprotein representing delta VP1-P2-P3* (delta and * denote N- or C-terminally truncated proteins, respectively), thus providing evidence for cleavage at sites VP1/2A and 2A/2B by proteinase 3C. In the cell-free expression system, processing at the P2/P3 junction was rapid and complete, whereas sites 3A/3B, 3B/3C, and 3C/3D were inefficiently cleaved, as evidenced by the accumulation of the stable precursor polypeptides P3* and 3ABC. In contrast to the eukaryotic system, mature 3C was produced in Escherichia coli. Intermolecular cleavage by recombinant 3C occurred at all putative sites within the proteolytically inactive polyprotein P2-P3* mu. The results of this study indicate that proteinase 3C mediates the primary as well as the secondary cleavages of the HAV polyprotein and thus shows an activity profile broader than that of 3C proteinases of other picornaviruses.