Evidence for activation of the hepatitis B virus polymerase by binding of its RNA template

The hepatitis B viruses replicate by reverse transcription of an RNA pregenome by using a virally encoded polymerase. A key early step in replication is binding of the polymerase to an RNA stem-loop (epsilon) of the pregenome; epsilon is both the RNA encapsidation signal and the origin of reverse transcription. Here we provide evidence that this interaction is also key to the development of enzymatic activity during biosynthesis of the polymerase. Duck hepatitis B virus polymerase expressed in Saccharomyces cerevisiae can synthesize DNA from epsilon-containing RNAs and can also end label other small RNAs. Expression of functional polymerase in S. cerevisiae requires interaction between the polymerase and epsilon during or shortly after translation for it to develop any enzymatic activity; if epsilon is absent during expression, the polymerase is inactive on RNAs both with and without epsilon. Functional duck polymerase can also be produced by in vitro translation, and synthesis of the polymerase in the presence of epsilon induces resistance in the polymerase to proteolysis by papain, trypsin, and bromelain. Induction of the resistance is specific for epsilon sequences that can support RNA encapsidation and initiation of DNA synthesis. Induction of the resistance precedes initiation of DNA synthesis and is reversible by degradation of epsilon. These two sets of data (i) support a model in which binding of epsilon to the polymerase induces a structural alteration of the polymerase prior to the development of enzymatic activity and (ii) suggest that this alteration may be required for the polymerase to mature to an active form.