Inhibition of cap-dependent gene expression induced by protein 2A of hepatitis A virus

The viral protein 2A of hepatitis A virus (HAV) Packs the conserved 18 aa s equence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760-1087) were linked to the 5'-UTR of poliovir us type 2 (Lansing strain) and inserted downstream of the gene encoding hum an growth hormone (GH). Following transfection of COS-1 cells, levels of GH (translation of which was entirely cap dependent) were determined in cultu re supernatants. Expression of HAV peptides extending from aa 764, 776 or 7 91 to 981 strongly inhibited cap-dependent translation of GH, whereas cap-i ndependent expression of a reporter gene (CAT) directed by the poliovirus R NA 5'-UTR was unaffected. The inhibitory effect was absent in constructs ex pressing either the short peptide encompassing aa 760-836 or proteins initi ated downstream of the putative cleavage site 836-837, suggesting that the boundaries of a functional HAV 2A may extend from the Gln/Ser junction 791- 792 to residue 981, while peptides initiated at the Gln/Ala pair 836-837 ma y result from alternative cleavage. Point mutations that substituted member s of the triad Ser(916), His(927) and Asp(931) abolished the inhibitory eff ect on cap-dependent translation, suggesting that the HAV-induced CPE may b e mediated by 2A protein.