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 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.
Urban
M
M
Roche Discovery Welwyn, Welwyn Garden City, Herts, UK.
McMillan
D J
DJ
Canning
G
G
Newell
A
A
Brown
E
E
Mills
J S
JS
Jupp
R
R
D000818:Animals; D002413:Cations, Divalent; D002460:Cell Line; D015684:Gene Products, pol / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D008670:Metals; D012367:RNA, Viral / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D015684:Gene Products, pol; D008670:Metals; C066027:P protein, Hepatitis B virus; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins