FORMATION OF A FUNCTIONAL HEPATITIS-B VIRUS-REPLICATION INITIATION COMPLEX INVOLVES A MAJOR STRUCTURAL ALTERATION IN THE RNA TEMPLATE

The DNA genome of a hepatitis B virus is generated by reverse transcri ption of the RNA pregenome. Replication initiation does not involve a nucleic acid primer; instead, the hepadnavirus P protein binds to the structured RNA encapsidation signal epsilon, from which it copies a sh ort DNA primer that becomes covalently linked to the enzyme. Using in vitro-translated duck hepatitis B virus (DHBV) P protein, we probed th e secondary structure of the protein-bound DHBV epsilon RNA (D epsilon ) and observed a marked conformational change compared to free D epsil on RNA. Several initiation-competent mutant RNAs with a different free -state structure were similarly altered, whereas a binding-competent b ut initiation-deficient variant was not, indicating the importance of the rearrangement for replication initiation and suggesting a mechanis tic coupling to encapsidation.