@article{mbs:/content/journal/jgv/10.1099/0022-1317-79-5-1121, author = "Urban, M and McMillan, D J and Canning, G and Newell, A and Brown, E and Mills, J S and Jupp, R", title = "In vitro activity of hepatitis B virus polymerase: requirement for distinct metal ions and the viral epsilon stem-loop.", journal= "Journal of General Virology", year = "1998", volume = "79", number = "5", pages = "1121-1131", doi = "https://doi.org/10.1099/0022-1317-79-5-1121", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-79-5-1121", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", abstract = "Hepadnaviruses have a complex replication cycle which includes reverse transcription of the pregenomic RNA. The initial step in this process in hepatitis B virus (HBV) requires the viral polymerase to engage a highly stable region of secondary structure within the pregenomic RNA termed the epsilon stem-loop. While reverse transcriptases belonging to the retrovirus family use a specific cellular tRNA as primer, HBV polymerase utilizes a tyrosine residue located within its own N terminus. Therefore, the first deoxyribonucleotide is covalently coupled to HBV polymerase prior to extension of the DNA strand by conventional reverse transcription. We have expressed HBV polymerase in a baculovirus and following purification have found it to be active with respect to protein-priming and reverse transcription of copurified RNA. Importantly, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV polymerase were also investigated for both the protein-priming and reverse transcription activities of this enzyme. Reverse transcription was dependent on magnesium, with an optimal concentration of 5 mM. However, protein-priming was strongly favoured by manganese ions and was optimal at a concentration of 1 mM. Thus, using manganese as sole source of metal ions our activity assay is restricted to the protein-priming event and will allow the search for novel antivirals specifically blocking this unique mechanism.", }