Crystal structure of an inhibitor complex of the 3C proteinase from hepatitis a virus (HAV) and implications for the polyprotein processing in HAV

The proteolytic processing of the viral polyprotein is an essential step du ring the life cycle of hepatitis A virus (HAV), as it is in all positive-se nse, single-stranded RNA Viruses or animals. In HAV the 3C proteinase is th e only proteolytic activity involved in the polyprotein processing. The spe cific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ra y crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited w ith the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulti ng complex had an acetyl-Val-Phe-amide group covalently attached to the S-g amma atom of the nucleophilic Cys 172 of the enzyme. Crystals of the comple x of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffractin g to 1.9-Angstrom resolution. The final refined structure consists of 4 mol ecules of HAV 3C (C24S,F82A) acetyl-val-Phe-amide, 1 molecule of DMSO, 1 mo lecule of glycerol, and 514 water molecules. There are considerable conform ational differences among the four molecules in the asymmetric unit. The fi nal R-factor is 20.4% for all observed reflections between 15.0- and 1.9-An gstrom resolution and the corresponding R-tree is 29.8%. The dipeptide inhi bitor is bound to the S-1' and S-2' specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well- defined S-2' specificity pocket and suggests that the P-2' residue could be an important determinant for the selection of the primary cleavage site du ring the polyprotein processing in HAV. (C) 1999 Academic Press.