KISSING OF THE 2 PREDOMINANT HAIRPIN LOOPS IN THE COXSACKIE-B VIRUS 3'-UNTRANSLATED REGION IS THE ESSENTIAL STRUCTURAL FEATURE OF THE ORIGIN OF REPLICATION REQUIRED FOR NEGATIVE-STRAND RNA-SYNTHESIS

Higher-order RNA structures in the 3' untranslated region (3'UTR) of e nteroviruses are thought to play a pivotal role in viral negative-stra nd RNA synthesis. The structure of the 3'UTR was predicted by thermody namic calculations using the STAR (structural analysis of RNA) compute r program and experimentally verified using chemical and enzymatic pro bing of in vitro-synthesized RNA. A possible pseudoknot interaction be tween the 3D polymerase coding sequence and domain Y and a ''kissing'' interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain d esignation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyak ov, and V. I. Agol (Nucleic Acids Res. 20:1739-1745, 1992), The higher -order RNA structure of the 3'UTR appeared to be maintained by an intr amolecular kissing interaction between the loops of the two predominan t hairpin structures (X and Y) within the 3'UTR. Disturbing this inter action had no effect on viral translation and processing of the polypr otein but exerted a primary effect on viral replication, as was demons trated in a subgenomic coxsackie B3 viral replicon, in which the capsi d P1 region was replaced by the luciferase gene. Mutational analysis d id not support the existence of the pseudoknot interaction between hai rpin loop Y and the 3D polymerase coding sequence. Based on these expe riments, we constructed a three-dimensional model of the 3'UTR of coxs ackie B virus that shows the kissing interaction as the essential stru ctural feature of the origin of replication required for its functiona l competence.