Journal of General Virology, Vol 79, 1121-1131, Copyright © 1998 by Society for General Microbiology

ARTICLES

In vitro activity of hepatitis B virus polymerase: requirement for distinct metal ions and the viral epsilon stem-loop

M Urban, DJ McMillan, G Canning, A Newell, E Brown, JS Mills and R Jupp
Roche Discovery Welwyn, Welwyn Garden City, Herts, UK.

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.

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