SEQUENCE-SPECIFIC AND STRUCTURE-SPECIFIC DETERMINANTS IN THE INTERACTION BETWEEN THE RNA ENCAPSIDATION SIGNAL AND REVERSE-TRANSCRIPTASE OF AVIAN HEPATITIS-B VIRUSES

Hepatitis B viruses (HBVs) replicate by reverse transcription of an RN A intermediate, Packaging of this RNA pregenome into nucleocapsids and replication initiation depend crucially on the interaction of the rev erse transcriptase, P protein, with the cis-acting, 5' end-proximal en capsidation signal epsilon, The overall secondary structure is similar in all of the hepadnaviral epsilon signals, with a lower and an upper stem, separated by a bulge, and an apical loop, However, while epsilo n is almost perfectly conserved in all mammalian viruses, the epsilon signals of duck HBV (DHBV) and heron HBV (D epsilon and H epsilon, res pectively) differ substantially in their upper stem regions, both in p rimary sequence and in secondary structure; nonetheless, H epsilon int eracts productively with DHBV P protein, as shown by its ability to st imulate priming, i.e., the covalent attachment of a deoxynucleoside mo nophosphate to the protein, In this study, we extensively mutated the variable and the conserved positions in the upper stern of D epsilon a nd correlated the functional activities of the variant RNAs in a primi ng assay with secondary structure and physical P protein binding, Thes e data revealed a proper overall structure, with the bulge and certain key residues, e.g., in the loop, being important constraints in prote in binding, Many mutations at the evolutionarily variable positions co mplied,vith these criteria and yielded priming-competent RNAs, However , most mutants at the conserved positions outside the loop were defect ive in priming even though they had epsilon-like structures and bound to P protein; conversely, one point mutant in the loop with an apical structure different from those of De and HE was priming competent, The se results suggest that P protein binding can induce differently struc tured epsilon RNAs to adopt a new, common conformation, and they suppo rt an induced-fit model of the epsilon-P interaction in which both com ponents undergo extensive structural alterations during formation of a priming-competent ribonucleoprotein complex.