ABSTRACT
Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in-frame AUG codons initiate the long open reading frame (positions 734-736 and 740-742). The extra-cistronic region extending between the uncapped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5'-terminal extra-cistronic region of HAV RNA were inserted in the intergenic region of the bi-cistronic plasmid pSV-GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS-1 cells, the transient expression of both genes was quantified. The importance of the 3'-PRT appeared to be strongly influenced by the length of the 'spacer' sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the 'spacer' back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3'-PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5'-PRT fluctuated: while integrity of domain 100-106 was always stringently required for initiation to occur, the activity of domain 113-118 paralleled that of the 3'-PRT, and the opposite applied to domain 121-126, whose contribution became relevant only after switching off the 3'-PRT. Systematic mutations introduced in the 'spacer' sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.