IN-VITRO ACTIVITY OF HEPATITIS-B VIRUS POLYMERASE - REQUIREMENT FOR DISTINCT METAL-IONS AND THE VIRAL EPSILON-STEM-LOOP

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 t o the retrovirus family use a specific cellular tRNA as primer, HBV po lymerase utilizes a tyrosine residue located within its own N terminus . Therefore, the first deoxyribonucleotide is covalently coupled to HB V polymerase prior to extension of the DNA strand by conventional reve rse transcription. We have expressed HBV polymerase in a baculovirus a nd following purification have found it to be active with respect to p rotein-priming and reverse transcription of copurified RNA. Importantl y, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV po lymerase were also investigated for both the protein-priming and rever se 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 opti mal at a concentration of 1 mM. Thus, using manganese as sole source o f metal ions our activity assay is restricted to the protein-priming e vent and will allow the search for novel antivirals specifically block ing this unique mechanism.