CLEAVAGE SPECIFICITY OF PURIFIED RECOMBINANT HEPATITIS-A VIRUS 3C PROTEINASE ON NATURAL SUBSTRATES

Hepatitis A virus (HSV) 3C proteinase expressed in Escherichia call wa s purified to homogeneity, and its cleavage specificity towards variou s parts of the viral polyprotein was analyzed, Intermolecular cleavage of the P2 P3 domain of the HAV polyprotein gave rise to proteins 2A, 2B, 2C, 3ABC, and 3D, suggesting that in addition to the primary cleav age site, all secondary sites within P2 as well as the 3C/3D junction are cleaved by 3C, 3C-mediated processing of the P1-P2 precursor liber ated 2A and 2BC, in addition to the structural proteins VP0, VP3, and VP1-2A and the respective intermediate products. A clear dependence on proteinase concentration was found for most cleavage sites, possibly reflecting the cleavage site preference of 3C. The most efficient clea vage occurred at the 2A/2B and 2C/3A junctions. The electrophoretic mo bility of processing product 2B, as well as cleavage of the synthetic peptide KGLFSQ(double dagger)AKISLFYT, suggests that the 2A/2B junctio n is located at amino acid position 836/837 of the HAV polyprotein, Fu rthermore, using suitable substrates we obtained evidence that sites V P3/VP1 and VP1/2A are alternatively processed by 3C, leading to either VP1-2A or to P1 and 2k The results with regard to intermolecular clea vage by purified 3C were confirmed by the product pattern derived from cell-free expression and intramolecular processing of the entire poly protein, We therefore propose that polyprotein processing of HAV relie s on 3C as the single proteinase, possibly assisted by as-yet-undeterm ined viral or host cell factors and presumably controlled in a concent ration-dependent fashion.