Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction

Hepatitis A virus (HAV) differs from other members of the family Picornavir idae in that the cleavage of the polyprotein at the 2A/2B junction, commonl y considered to be the primary polyprotein cleavage by analogy with other p icornaviruses, is mediated by 3C(pro), the only proteinase encoded by the v irus. However, it has never been formally demonstrated that the 2A/2B junct ion is the site of primary cleavage, and the actual function of the 2A sequ ence, which lacks homolog with sequence of other picornaviruses, remains un known. To determine whether 2A functions in cis as a precursor with the non structural proteins, we constructed dicistronic HAV genomes in which a hete rologous picornaviral internal ribosome entry site was inserted at the 2A/2 B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possi ble cis replication function spanning the 2A/2B junction. However, infectio us virus was recovered from recombinant HAV genomes containing exogenous pr otein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not d ependent upon the length of the inserted sequence. An HAV recombinant conta ining a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were del eted from replicating viruses, but this did not result from homologous reco mbination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segme nts of the inserted sequence were retained in the deletion mutants. These r esults indicate that the HAV polyprotein can tolerate an insertion at the 2 A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding seque nces inserted at the 2A/2B junction are novel and potentially useful protei n expression vectors.