THE DISTANCE BETWEEN THE 3'-PYRIMIDINE-RICH TRACT AND THE AUG CODON MODULATES INTERNAL INITIATION OF TRANSLATION OF HEPATITIS-A VIRUS-RNA

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 unc apped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): o ne 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 rel ative contribution of these elements to the process of internal initia tion of translation, cDNA representations of the 5'-terminal extra-cis tronic 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 (GPT) and the bacterial enzyme chloramphenicol acetylt ransferase (CAT), and following transfection of COS-1 cells, the trans ient 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 initiati on site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation t o occur. Bringing the length of the 'spacer' back to its actual size i n HAY RNA (i,e, 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to t his process of the 3'-PRT became marginal, Concomitantly, the importan ce of the functional domains previously identified in the 5'-PRT fluct uated: while integrity of domain 100-106 was always stringently requir ed 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, Syst ematic mutations introduced in the 'spacer' sequences suggest that the length of this region may be responsible for the down regulation of t ranslation of HAV RNA and, possibly, for its lengthy replication cycle . (C) 1998 Federation of European Biochemical Societies.