Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase

摘要: Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of first deoxyribonucleotide to polymerase polypeptide. We recently expressed human hepatitis B virus (HBV) transcriptase (pol) insect cells by using recombinant baculovirus system. The purified protein is active nucleotide priming and reactions. In this report, we demonstrate tyrosine residue at amino acid number 63 within TP (terminal protein) domain site minus-strand DNA. Analysis pol polypeptides with mutations RT (reverse transcriptase) domains indicated both were required for vitro activity. Polymerase proteins not capable complementing each other reaction, suggesting transcomplementation between full-length was possible. However, when as separate polypeptides, they formed highly stable complex transcription. presence an epsilon stem-loop dramatically increased activity assays, even though displayed similar activities absence epsilon. These data raise possibility assay, may play role formation functional RT, rather than being only template priming. results indicate system, it possible dissect protein-protein protein-RNA interactions HBV genome replication.