IDENTIFICATION AND SITE-DIRECTED MUTAGENESIS OF THE PRIMARY (2A 2B) CLEAVAGE SITE OF THE HEPATITIS-A VIRUS POLYPROTEIN - FUNCTIONAL IMPACT ON THE INFECTIVITY OF HAV RNA TRANSCRIPTS/

The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remai n poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukary otic cells via recombinant vaccinia viruses. A substrate polyprotein s panning the putative HAV 2A/2B site was fused at its C-terminus to a p oliovirus VP1 reporter sequence. This substrate was cleaved efficientl y in trans by protease 3C derived from another recombinant vaccinia vi rus expressing a 3C precursor protein. N-terminal sequencing of the 2B -poliovirus VP1 fusion product identified the site of cleavage as the Gln(836)/Ala(837) dipeptide, 144 residues upstream of the originally p redicted site. Two mutations were introduced at the P1 position of the 2A/2B site: Gln(836), Asn, and Gln(836), Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia express ion system and resulted in a small replication focus phenotype of viru s rescued from infectious HAV RNA transcripts. Arg substitution abolis hed cleavage and was lethal to HAV replication. In addition to identif ying the site of the primary HAV polyprotein cleavage, these results s hed light on the in vivo specificities of the HAV 3C protease. (C) 199 5 Academic Press, Inc.