Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ε) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific ε binding, we have deiined the protein and RNA sequences required for specific HBV RT-ε interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for ε binding. With respect to the ε RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of ε, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-ε interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-ε interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some ε sequences by the RT may be required specifically for viral DNA synthesis.
All Science Journal Classification (ASJC) codes
- Insect Science