The Duck Hepatitis B Virus Polymerase Is Activated by Its RNA Packaging Signal, ɛ

The hepatitis B viruses (hepadnaviruses) are small DNA-containing viruses that replicate by reverse transcription (reviewed in reference 3). The interaction between the hepadnavirus polymerase and a stem-loop (ɛ) at the 5′ end of the viral pregenomic RNA is a key event early in reverse transcription. ɛ is the primary element of the hepadnaviral RNA packaging signal (6, 7, 11) and is the origin of reverse transcription (10, 14, 19, 21). Binding between the polymerase and ɛ is required for RNA packaging. If the complex does not form, neither the polymerase nor the pregenomic RNA is incorporated into viral cores (1, 5). The polymerase initiates reverse transcription within a bulge in ɛ and synthesizes a 3- to 4-nucleotide (nt) minus-strand DNA which is covalently linked to the polymerase by virtue of the protein-priming mechanism of the hepadnaviruses (4, 20). This nascent minus-strand DNA is then transferred from ɛ at the 5′ end of the RNA pregenome to a short region of homology near the 3′ end of the RNA prior to further chain elongation (10, 14, 19, 21). Using recombinant duck hepatitis B virus (DHBV) polymerase expressed both in Saccharomyces cerevisiae (TYDP [16]) and translated in rabbit reticulocyte lysates (DP [20]), we have presented two lines of evidence implying that the interaction between the polymerase and ɛ has another key role early in hepadnavirus reverse transcription; this interaction also appears to be needed for the polymerase to mature to an enzymatically active form (18). The first evidence for a role of ɛ in the maturation of the polymerase is genetic and was obtained with TYDP expressed in S. cerevisiae. The key observation was made with a newly discovered activity of TYDP that synthesizes DNA at the 3′ end of exogenously added RNAs independently of the hepadnavirus protein priming reaction (the trans activity). TYDP could perform the trans reaction only when it had been translated in the presence of ɛ sequences competent to support protein-primed reverse transcription, despite the fact that the trans reaction does not employ protein-primed initiation (18). This indicated that ɛ has an additional role in DNA synthesis beyond being the origin of reverse transcription. The second line of evidence for a role for ɛ in the maturation of the polymerase is structural. Translation of DP in the presence of ɛ sequences competent to support RNA packaging and DNA synthesis resulted in significantly increased resistance to proteolysis relative to DP translated in the absence of a biologically functional ɛ (18). This demonstrated that the interaction with ɛ induces a structural alteration to the polymerase prior to its development of enzymatic activity. These data led to a model in which the polymerase binds to ɛ, undergoes a reversible alteration, and then becomes active (18). Here, we present three additional lines of evidence supporting this maturation model. First, we show that the trans activity is reverse transcription of the exogenous RNA and is an authentic property of the DHBV polymerase. Second, the maturation model predicts that the ɛ-dependent alteration to the polymerase would somehow affect the reverse transcriptase domain to activate it. This prediction is fulfilled by mapping the region of the polymerase that is altered following binding of ɛ to the reverse transcriptase domain itself. Finally, the correlation between adoption of the ɛ-dependent altered state and the development of enzymatic activity of the polymerase is expanded through analysis of 15 mutant polymerases.