IN-VITRO MUTATIONAL AND INHIBITORY ANALYSIS OF THE CIS-ACTING TRANSLATIONAL ELEMENTS WITHIN THE 5'-UNTRANSLATED REGION OF COXSACKIEVIRUS B3- POTENTIAL TARGETS FOR ANTIVIRAL ACTION OF ANTISENSE OLIGOMERS

The 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) RNA for ms a highly ordered secondary structure that has been implicated in co ntrolling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5'UTR in the intercistronic space, were translated in rabbit r eticulocyte lysates. In the presence of an upstream cistron, the chlor amphenicol acetyltransferase gene, designed to block ribosomal scannin g, the CVB3 5'UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the prese nce of an internal ribosomal entry site (IRES). This finding was furth er supported by the data on predicted secondary structures within the 5'UTR. Of special note, analysis of various deletion mutants demonstra ted that the IRES of CVB3 is located roughly at stem-loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (st em-loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings, in v itro translation inhibition assays using RNA fragments of the 5'UTR as inhibitor were performed. Both antisense and sense RNA segments trans cribed from these two cis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking rib osome and initiation factor binding within the 5'UTR via specific hybr idization to their viral RNA target sequences, while sense sequences m ay function by competing with viral RNA for ribosomes and/or translati on initiation factors. These cis-acting translational elements may ser ve as potential targets for the antiviral action of oligomers. (C) 199 7 Academic Press.