In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed epsilon (H epsilon) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and H epsilon as the obligatory template. We have purified HP from human cells that retained H epsilon binding activity in vitro. Furthermore, HP purified as a complex with H epsilon, but not HP alone, displayed in vitro protein priming activity. While the HP-H epsilon interaction in vitro and in vivo required the H epsilon internal bulge, but not its apical loop, and was not significantly affected by the cap-H epsilon distance, protein priming required both the H epsilon apical loop and internal bulge, as well as a short distance between the cap and H epsilon, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-H epsilon interactions, RNA packaging, and protein priming.